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The influence of gamma radiation on catheptic activity and on ultrastructure of lysosomes and postmortem… Ali, Mumtaz 1975

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THE  INFLUENCE OF GAMMA RADIATION ON CATHEPTIC  ACTIVITY AND ON ULTRASTRUCTURE OF AND POSTMORTEM  LYSOSOMES  SKELETAL MUSCLE OF  POULTRY ( G a l l u s  domesticus)  BY MUMTAZ A L I B.Sc,  West P a k i s t a n  Agricultural University,  M.Sc., West P a k i s t a n  1963  A g r i c u l t u r a l U n i v e r s i t y , 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  t h e Department o f Food  Science  We a c c e p t t h i s t h e s i s required  THE  as conforming t o t h e  standard  UNIVERSITY OF BRITISH May, 1975  COLUMBIA  In  presenting  an  advanced  the  Library  I  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  that  it  purposes  for  may  be  It  financial  FOOD  of  for  June  27,  1975-  of  of  Columbia,  British  by  the  is understood gain  Columbia  for  extensive  granted  SCIENCE  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  fulfilment  available  permission.  Department  Date  freely  permission  representatives. thesis  partial  shall  Head  be  requirements  reference copying  that  not  the  of  copying  agree  and  of my  I  this  that  study. thesis  Department or  for  or  publication  allowed without  my  i.  ABSTRACT  A t h r e e p a r t study i s p r e s e n t e d  dealing with  r a d i a t i o n - i n d u c e d r e l e a s e o f c a t h e p s i n s from i s o l a t e d  lyso-  somes , i r r a d i a t i o n i n a c t i v a t i o n o f c a t h e p s i n s , and u l t r a s t r u c t u r a l changes i n i r r a d i a t e d lysosomes and s k e l e t a l muscle.  Chicken  l i v e r lysosomes were i s o l a t e d by d i f f e r e n -  t i a l centrifugation Isolated  and s u c r o s e d e n s i t y g r a d i e n t  technique.  lysosomes were i r r a d i a t e d w i t h doses i n t h e range  of 0.10 t o 1.0 Mrad o f gamma r a d i a t i o n .  Irradiation re-  s u l t e d i n a decrease i n absorbance (540 nm) o f l y s o s o m a l suspensions  i n c u b a t e d a t 37°C and a  nounced i n c r e a s e i n f r e e of c a t h e p s i n s . was  correspondingpro-  enzyme a c t i v i t y due t o r e l e a s e  Rate o f r e l e a s e o f c a t h e p s i n D from lysosomes  c o n s i d e r a b l y s l o w e r when i n c u b a t e d a t 4°C compared  w i t h 37°C. Cathepsins  A, B, C, and D showed a d i f f e r e n t i a l  r e l e a s e under t h e i n f l u e n c e o f gamma r a d i a t i o n .  Cathepsins  C and D were more r e a d i l y r e l e a s e d as compared w i t h s i n s A and B.  cathep-  A f t e r 72 hours o f i n c u b a t i o n a t 4°C, f r e e  a c t i v i t y o f c a t h e p s i n s A, B, C, and D i n 1.0 Mrad i r r a d i a t e d lysosomal  suspensions  reached 80.2,  p e r c e n t o f t o t a l enzyme c o n t e n t ; c o n t r o l samples was 14.0, respectively.  16.7,  70.5,  85.5,  and 81.6  f r e e enzyme a c t i v i t y o f 27.7,  and 26.6 p e r c e n t  T o t a l a c t i v i t y o f cathepsins  A, B, C, and D d e c l i n e d  as a r e s u l t o f i r r a d i a t i o n , due t o apparent p a r t i a l i n a c t i v a t i o n o f t h e enzymes. r e s i s t a n t and c a t h e p s i n  C a t h e p s i n A was most r a d i a t i o n  B was c o m p a r a t i v e l y  sensitive  t o r a d i a t i o n i n a c t i v a t i o n . Cathepsins e x h i b i t e d  higher  r a d i a t i o n r e s i s t a n c e when i r r a d i a t e d i n l y s o s o m a l suspens i o n compared w i t h s o l u b l e enzyme form. t i v i t y o f cathepsins  Radiation sensi-  was h i g h e r a t pH 4.0 and 5.5 compared  w i t h pH 7.0 and 8.5. I r r a d i a t i o n i n d u c e d changes i n hemoglobin s u b s t r a t e , rendering  i t resistant to catheptic digestion.  i n e l e c t r o p h o r e t i c p a t t e r n as w e l l as v i s i b l e  Changes spectra  o f i r r a d i a t e d hemoglobin were i n d i c a t i v e o f a l t e r a t i o n s i n the substrate. I s o l a t e d chicken  l i v e r lysosomes and c h i c k e n  pector-  a l i s muscle were s u b j e c t e d t o 1.0 Mrad o f gamma r a d i a t i o n . S e c t i o n s o f embedded samples were s t u d i e d by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y and s u r f a c e u l t r a s t r u c t u r e d e t a i l s were examined by s c a n n i n g e l e c t r o n m i c r o s c o p y .  Irradiation  enhanced t h e r e l e a s e o f i n n e r dense m a t e r i a l from lysosomes. A f t e r 4 8 and 72 hours o f i n c u b a t i o n most o f t h e i r r a d i a t e d p a r t i c l e s appeared as h o l l o w r i n g s o f l y s o s o m a l membrane. In some i r r a d i a t e d p a r t i c l e s , leakage o f l y s o s o m a l cont e n t s from "weak p o i n t s " i n t h e l y s o s o m a l membrane was observed.  There were i n d i c a t i o n s t h a t i r r a d i a t i o n weakened  the membrane s t r u c t u r e which caused leakage o f m a t e r i a l  from lysosomes and e v e n t u a l d i s r u p t i o n o f p a r t i c l e s on prolonged i n c u b a t i o n .  Scanning e l e c t r o n m i c r o s c o p y  pro-  vided f u r t h e r evidence that lysosomal m a t e r i a l leaked from "weak p o i n t s " i n t h e l y s o s o m a l membrane c a u s i n g l y sosomes t o appear as "empty s a c k s " r a t h e r than t o t a l l y disrupted particles. T r a n s m i s s i o n e l e c t r o n microscopy  of cryofractured  s k e l e t a l muscle r e v e a l e d t h a t i r r a d i a t i o n caused an i n c r e a s e i n i n t e r f i b r i l l a r spaces and some breaks i n t h e m y o f i b r e s e s p e c i a l l y a t t h e I band r e g i o n .  Scanning e l e c t r o n m i c r o -  graphs o f i r r a d i a t e d muscle showed f i s s u r e s between t h e myofibrils.  C o n t r o l samples had a smooth s u r f a c e a t t h e  t r a n s v e r s e b r e a k s , w h i l e i r r a d i a t e d t i s s u e had s p i k e - l i k e s t r u c t u r e s a t t h e surface o f these breaks. These s t u d i e s p r o v i d e e v i d e n c e t h a t i r r a d i a t i o n caused s t r u c t u r a l changes i n lysosomes r e s u l t i n g i n i n c r e a s e d leakage o f l y s o s o m a l c o n t e n t s and r e l e a s e o f l y sosomal enzymes.  S t r u c t u r a l changes a t t h e f i b r e and  f i b r i l l e v e l o f i r r a d i a t e d muscle a r e a l s o i n d i c a t e d . e f f e c t s a r e l i k e l y fundamental  to textural alterations  of muscle s u b j e c t e d t o post-mortem i r r a d i a t i o n .  The  iv. GENERAL TABLE OF CONTENTS  PAGE  l  ABSTRACT TABLE OF CONTENTS (GENERAL)  iv  TABLE OF CONTENTS CCHAPTER 1)  V  TABLE OF CONTENTS (CHAPTER 2)  vi  TABLE OF CONTENTS (CHAPTER 3)  vii  TABLE OF CONTENTS (CHAPTER 4)  ix  L I S T OF TABLES  xi  L I S T OF FIGURES ACKNOWLEDGEMENTS  xii xviii  CHAPTER 1  1  CHAPTER 2  46  CHAPTER 3  96  CHAPTER 4  135  LITERATURE  CITED  181  V.  CHAPTER 1.  GENERAL INTRODUCTION AND LITERATURE REVIEW PAGE  1.1,  General I n t r o d u c t i o n  1  1.2.  L i t e r a t u r e Review  3  1.2.1. 1.2.2. 1.2.3.  1.2.4.  Effect of ionizing radiation on meats  3  Effect of ionizing radiation on p r o t e i n s and amino a c i d s  6  E f f e c t of i o n i z i n g r a d i a t i o n on enzymes and enzyme a c t i v i t y  11  Lysosomal c a t h e p s i n s  21  1.2.4.1. 1.2.4.2. 1.2.4.3. 1.2.4.4. 1.2.4.5. 1.2.4.6.  Cathepsin A (EC 3.4.2.-) Cathepsin B (EC 3.4.4,-)  21 22  Cathepsin C (EC 3.4.4.9,)  22  Cathepsin D (EC 3.4.4.23)  23  Cathepsin E (EC 3.4.4.-)  23  Neutral proteinases (EC 3.4.4.-)  23  1.2.5.  Lysosomes and l y s o s o m a l sins i n tissue  1.2.6.  Lysosomal concept  28  1.2.7.  H e t e r o g e n e i t y o f lysosomes  29  1.2.8.  S t a b i l i t y o f lysosomes  30  1.2.9.  U l t r a s t r u c t u r e o f lysosomes  35  1.2.10. U l t r a s t r u c t u r e o f muscle  cathep-  24  42  vi. CHAPTER 2.  EFFECT OF GAMMA RADIATION ON RELEASE OF CATHEPSINS FROM LYSOSOMES PAGE  2.1.  Introduction  46  2.2.  Experimental  50  2.2.1.  Materials  50  2.2.2.  G e n e r a l sample p r e p a r a t i o n  50  2.2.2.1, 2.2.2.2.  2.3.  liver  I s o l a t i o n of l y s o somes  50 51  2.2.3.  Irradiation  51  2.2.4.  Light s c a t t e r i n g properties o f lysosomes  53  2.2.5.  R e l e a s e o f c a t h e p s i n s from l y s o somes  53  2.2.6.  Enzyme assays  54  2.2.6.1.  Cathepsin A  54  2.2.6.2.  C a t h e p s i n s B and C  54  2.2.6.3.  Cathepsin D  55  R e s u l t s and D i s c u s s i o n 2.3.1. 2.3.2. 2.3.3.  2.4.  Chicken tissue  56  Proteolytic a c t i v i t y of tissue as a f f e c t e d by gamma r a d i a t i o n  56  Light scattering properties o f l y s o s o m a l suspensions  60  Release o f l y s o s o m a l c a t h e p sins  63  2.3.3.1.  Cathepsin D  64  2.3.3.2,  C a t h e p s i n s A, B, C  74  Summary and C o n c l u s i o n s  93  vii. CHAPTER 3.  RADIATION SENSITIVITY OF LYSOSOMAL CATHEPSINS AND  HEMOGLOBIN SUBSTRATE PAGE  3.1.  Introduction  96  3.2.  Experimental  98  3.2.1.  Treatment o f samples f o r enzyme study  98  3.2.2.  Enzyme assay  98  3.2.3.  Enzyme a c t i v i t y i n suspensions  3.2.4.  lysosomal  98  3.2.3.1.  Residual a c t i v i t y  98  3.2.3.2.  Enzyme a v a i l a b i l i t y after irradiation of lysosomes i n the i n t a c t and r u p t u r e d s t a t e  99  3.2.3.3.  pH d u r i n g tion  99  3.2.3.4.  Sucrose c o n c e n t r a t i o n during i r r a d i a t i o n  101  3.2.3.5.  Temperature d u r i n g irradiation  101  3.2.3.6.  Irradiation combination ment  101  H y d r o l y s i s of hemoglobin  irradia-  - heat treat-  irradiated  102  3.2.4.1.  In s o l u t i o n  102  3.2.4.2.  I n dry s t a t e  102  3.2.5.  A b s o r p t i o n spectrum o f d i a t e d hemoglobin  irra-  3.2.6.  Agarose g e l e l e c t r o p h o r e s i s o f hemoglobin a f t e r h y d r o l y s i s by cathepsin D .  102  102  viii. CHAPTER 3. ( C o n t i n u e d ) PAGE 3.2.6.1. 3.3.  Electrophoresis procedure  R e s u l t s and D i s c u s s i o n 3.3.1.  3.3.3.  3.3.4.  3.3.5.  104  Lyso soma! suspension  104  S o l u b l e enzyme f r a c t i o n a t v a r i o u s pHs  107  A v a i l a b i l i t y o f c a t h e p s i n s A, B, and C a f t e r i r r a d i a t i o n o f i n t a c t and d i s r u p t e d lysosomes  113  Radiation i n a c t i v a t i o n of c a t h e p s i n D under v a r i o u s conditions  118  3.3.1.2. 3.3.2.  104  Radiation inactivation of c a t h e p s i n s A, B, C, and D 3.3.1.1.  103  3.3.3.1.  Sucrose concentration  118  3.3.3.2.  Temperature  120  R a d i a t i o n - heat combination treatment f o r i n a c t i v a t i o n o f lysosomal cathepsin D  120  Radiation-induced in substrate  12 3  3.3.5.1.  3.3.5.2.  3.3.5.3.  changes  Hydrolysis of i r r a d i a t e d hemoglobin a t d i f f e r e n t pHs  12 3  H y d r o l y s i s o f hemoglobin after i r r a diation i n soluble or dry s t a t e  125  Electrophoretic pattern of i r r a d i a t e d hemoglobin a f t e r h y d r o l y s i s by cathepsin D  127  ix.  CHAPTER 3.  (Continued) PAGE 3.3.5.4.  S p e c t r a l changes in irradiated hemoglobin  129  3.4.  Summary and C o n c l u s i o n s  132  CHAPTER 4.  EFFECT OF GAMMA RADIATION ON ULTRASTRUCTURE OF LYSOSOMES AND CHICKEN SKELETAL MUSCLE PAGE  4.1.  4.2.  Introduction  135  4.1.1.  Lysosomes  135  4.1.2.  Muscle  136  Experimental  137  4.2.1.  G e n e r a l sample p r e p a r a t i o n  137  4.2.1.1.  Lysosomes  137  4.2.1.2.  Tissue  137  4.2.2.  4.3.  Sample p r o c e s s i n g f o r E l e c t r o n microscopy 4.2.2.1.  Scanning e l e c t r o n microscopy  138  4.2.2.2,  Transmission e l e c t r o n microscopy  138  R e s u l t s and D i s c u s s i o n 4.3.1.  13 8  R a d i a t i o n - i n d u c e d changes i n u l t r a s t r u c t u r e o f lysosomes 4.3.1.1.  Internal ultrastruct u r e o f lysosomes  140 140 140  X.  CHAPTER 4.  (Continued) PAGE 4.3.1.2.  4.3.2.  4.4.  Surface u l t r a structure of lysosomes  E f f e c t o f i r r a d i a t i o n on ultrastructure of skeletal muscle  156  161  4.3.2.1.  Transmission e l e c t r o n microscopy  161  4.3.2.2.  Surface u l t r a s t r u c t u r e o f muscle  169  Summary and C o n c l u s i o n s  178  xi. LIST OF TABLES  Table 1  2  3 4  Page E f f e c t o f low doses o f gamma r a d i a t i o n on f r e e c a t h e p t i c a c t i v i t y o f c h i c k e n liver tissue  56  E f f e c t o f h i g h doses o f i r r a d i a t i o n on free catheptic a c t i v i t y of chicken l i v e r tissue  57  Release o f c a t h e p s i n s A, B, C, and D from lysosomes a t 4°C a f t e r i r r a d i a t i o n  85  S u b s t r a t e s and i n c u b a t i o n c o n d i t i o n s f o r c a t h e p s i n assays  100  E f f e c t o f pH on r a d i a t i o n s e n s i t i v i t y o f s o l u b l e l y s o s o m a l c a t h e p s i n s A, B, C, and D  108  6  E f f e c t of i r r a d i a t i o n - heat combination treatment on i n a c t i v a t i o n o f c a t h e p s i n D  121  7  H y d r o l y s i s o f hemoglobin by l y s o s o m a l c a t h e p s i n D a t v a r i o u s pHs a f t e r irradiation i n soluble state  124  E f f e c t o f i r r a d i a t i o n o f hemoglobin i n dry and s o l u b l e s t a t e on i t s h y d r o l y s i s by l y s o s o m a l c a t h e p s i n D a t pH 3.8  126  5  8  xii. LIST OF FIGURES Figure 1  Page Scheme f o r i s o l a t i o n o f l y s o s o m a l fraction  52  2  E f f e c t o f gamma r a d i a t i o n on l i g h t s c a t t e r i n g p r o p e r t i e s o f lysosomes  62  3  Free c a t h e p s i n D a c t i v i t y o f l y s o s o m a l s u s p e n s i o n as i n f l u e n c e d by gamma r a d i a t i o n and i n c u b a t i o n a t 37°C  65  Free c a t h e p s i n D a c t i v i t y o f l y s o s o m a l suspension as i n f l u e n c e d by gamma r a d i a t i o n and i n c u b a t i o n a t 4°C  67  R e l e a s e o f c a t h e p s i n D from lysosomes as i n f l u e n c e d by gamma r a d i a t i o n  69  Release o f c a t h e p s i n A from lysosomes as i n f l u e n c e d by gamma r a d i a t i o n  76  Release o f c a t h e p s i n B from lysosomes as i n f l u e n c e d by gamma r a d i a t i o n  77  R e l e a s e o f c a t h e p s i n C from lysosomes as i n f l u e n c e d by gamma r a d i a t i o n  79  Comparative e f f e c t o f gamma r a d i a t i o n (1.0 Mrad) on t h e r e l e a s e o f l y s o s o m a l c a t h e p s i n s A, B, C, and D  81  Comparative e f f e c t o f v a r i o u s r a d i a t i o n doses on r e l e a s e o f c a t h e p s i n s from lysosomes a f t e r 4 8 hours i n c u b a t i o n a t 4°C  82  Radiation i n a c t i v a t i o n of cathepsins A, B, C, and D i n i n t a c t lysosomes a t pH 7.0  105  Radiation inactivation of soluble l y s o s o m a l c a t h e p s i n s a t pH 5.5  109  Radiation i n a c t i v a t i o n of soluble l y s o s o m a l c a t h e p s i n s a t pH 7.0  110  4  5 6 7 8 9  10  11  12 13  xiii. LIST OF FIGURES ( C o n t i n u e d ) Figures 14  Page A v a i l a b i l i t y of cathepsin A a f t e r i r r a d i a t i o n o f d i s r u p t e d and i n t a c t lysosomes  115  A v a i l a b i l i t y of cathepsin B a f t e r i r r a d i a t i o n of d i s r u p t e d and i n t a c t lysosomes  116  A v a i l a b i l i t y of cathepsin C a f t e r i r r a d i a t i o n o f d i s r u p t e d and i n t a c t lysosomes  117  E f f e c t o f sucrose c o n e n t r a t i o n on r a d i a t i o n s e n s i t i v i t y of cathepsin D a f t e r 0.5 Mrad dose  119  E l e c t r o p h o r e t i c pattern of i r r a d i a t e d hemoglobin b e f o r e ( 1 , 3, and 5) and a f t e r ( 2 , 4, 6) c a t h e p t i c d i g e s t i o n a t pH 3.8  12 8  19  R a d i a t i o n - i n d u c e d s p e c t r a l changes i n hemoglobin s u b s t r a t e  130  20  Transmission e l e c t r o n micrographs of n o n - i r r a d i a t e d lysosomes w i t h o u t i n c u b a t i o n . LI = lysosomes w i t h dense m a t r i x ; L2 = lysosomes w i t h l i g h t m a t r i x ; MV = m u l t i v e s i c u l a r body. #75 ,000X  141  Transmission e l e c t r o n micrographs o f n o n - i r r a d i a t e d lysosomes w i t h o u t i n ^ cubation. LI = lysosomes w i t h dense m a t r i x ; L2 = lysosomes w i t h l i g h t m a t r i x ; A, 75,000X; B, 80,000X; C, 80,000X; D, 120,000X  142  Transmission e l e c t r o n micrographs of n o n - i r r a d i a t e d lysosomes a f t e r 48 hour i n c u b a t i o n a t 4°C; arrow = leakage o f l y s o s o m a l c o n t e n t ; dm = d i l u t e d m a t r i x ; mv = m u l t i v e s i c u l a r body. A, 50,000X; B and C, 75,000X  143  15  16  17  18  21  22  LIST OF FIGURES ( C o n t i n u e d ) Figure 23  24  25  26  27  28  29  X 1 V  *  Page Transmission e l e c t r o n micrographs o f n o n - i r r a d i a t e d lysosomes a f t e r 72 hours i n c u b a t i o n a t 4°C; arrow = d i s r u p t e d dense l y s o s o m e s ; dmv = d i s r u p t e d m u l t i v e s i c u l a r body; rm = r e s i d u a l m a t e r i a l . A, 30,000X; B, 50,000X; C, 80,000X  145  Transmission e l e c t r o n micrographs o f lysosomes d i s r u p t e d by T r i t o n X-100 t r e a t m e n t ; arrows = clumps o f l y s o s o m a l dense m a t r i x . A, 50,000X; B, 80,000X; C, 30,000X  146  Transmission e l e c t r o n micrographs o f i r r a d i a t e d (l.OMrad) lysosomes a f t e r 48 hours i n c u b a t i o n a t 4°C. A, 75,000X; B, 50,000X; C, 150,000X  147  Transmission e l e c t r o n micrographs of i r r a d i a t e d lysosomes (1.0 Mrad) a f t e r 72 hours i n c u b a t i o n a t 4°C. L r = l y s o some a f t e r r e l e a s e o f i t s c o n t e n t s ; rm = r e l e a s e d m a t e r i a l from lysosome; arrows = clumps o f dense l y s o s o m a l matrix. A, 45,000X; B, 120,000X; C and D, 50,000X  148  Transmission e l e c t r o n micrographs o f n o n - i r r a d i a t e d lysosomes a f t e r 1 hour i n c u b a t i o n a t 37°C; a = complete l e a k a g e o f l y s o s o m a l c o n t e n t ; b and c = p a r t i a l l e a k a g e ; cv = c a v i t y . A, B, and D, 50,000X; C, 30,000X  150  T r a n s m i s s i o n e l e c t r o n micrographs o f n o n - i r r a d i a t e d lysosomes a f t e r 1 hour i n c u b a t i o n a t 37°C showing v a r i o u s s t a g e s o f leakage o f l y s o s o m a l c o n t e n t s ( a , b, and c ) . A, 30,000X; B and C, 80,000X; D, 200,000X  151  Transmission e l e c t r o n micrographs o f i r r a d i a t e d lysosomes (1.0 Mrad) a f t e r 1 hour i n c u b a t i o n a t 37°C; arrow = weak p o i n t i n l y s o s o m a l membrane showing l e a k a g e . A, B, and C, 80,000X; D, 120,000X  152  XV.  LIST OF FIGURES  (Continued)  Figure 30  31  32  33  34  35  Page T r a n s m i s s i o n e l e c t r o n micrographs o f i r r a d i a t e d lysosomes (1.0 Mrad) a f t e r 1 hour i n c u b a t i o n a t 37°C; Lc = l e a k e d l y s o s o m a l c o n t e n t s ; arrow = weak p o i n t i n l y s o s o m a l membrane showing l e a k a g e . A, 80,000X; B and C, 50,000X; D, 60,000X.  153  T r a n s m i s s i o n e l e c t r o n micrographs o f i r r a d i a t e d lysosomes (1.0 Mrad) a f t e r 1 hour i n c u b a t i o n a t 37°C; dL = d i s r u p t e d lysosomes; mv = m u l t i v e s i c u l a r body; dm = damaged membrane; arrows = weak p o i n t s i n t h e membrane. A, 30,000X; B, 80,000X; C, 120,000X; D, 200,000X.  154  Scanning e l e c t r o n m i c r o g r a p h s o f noni r r a d i a t e d lysosomal p a r t i c l e s a f t e r 48 hours i n c u b a t i o n a t 4°C; Lc = l e a k a g e o f c o n t e n t s ; arrows = damaged p a r t i c l e s ; r s = r o u g h - s u r f a c e d p a r t i c l e . A and B, 24,000X; C, 60,000X; D, 50,000X.  157  Scanning e l e c t r o n m i c r o g r a p h s o f noni r r a d i a t e d lysosomal p a r t i c l e s a f t e r 48 hours i n c u b a t i o n a t 4°C; am = amorphous m a t e r i a l ; Lc = l e a k a g e o f c o n t e n t s ; dp = damaged p a r t i c l e s ; arrows = s i t e o f l e a k a g e . A and B, 24,000X; C, 90,000X; D, 360,000X.  158  Scanning e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) l y s o s o m a l p a r t i c l e s a f t e r 48 hour i n c u b a t i o n a t 4°C; am = amorphous m a t e r i a l ; dp = damaged p a r t i c l e s ; Lc = l e a k a g e o f c o n t e n t s . A, 60,000X; B, 90,000X; C and D, 50,OOOX.  159  Scanning e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) l y s o s o m a l p a r t i c l e s a f t e r 4 8 hour i n c u b a t i o n at 4°C; am = amorphous m a t e r i a l ; dp = damaged p a r t i c l e s ; Lc = l e a k a g e o f c o n t e n t s ; arrow = p o i n t o f l e a k a g e ; de = d e p r e s s i o n . A 90,000X; B, 240,000X o f p a r t i c l e enc i r c l e d i n A. ?  LIST OF FIGURES ( C o n t i n u e d ) Figure 36  Transmission e l e c t r o n micrographs o f non-irradiated chicken p e c t o r a l i s m u s c l e , 3 hours post-mortem; A = A band; 1 = 1 band; Z = Z l i n e . A, 30,000X; B, 20,000X; C, 80,000X; D, 20,000X.  37  Transmission e l e c t r o n micrographs o f non-irradiated chicken p e c t o r a l i s m u s c l e , 3 hours post-mortem; A = A band; 1 = 1 band; Z = Z l i n e . A, 18,000X; B, 75,000X.  38  Transmission e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) c h i c k e n pect o r a l i s m u s c l e , 3 hours post-mortem; b r = b r e a k s ; t f = t w i s t e d f r e e ends of f i b r i l s . A and B, 20,000X; C, 45,000X; D. 30,000X.  39  Transmission e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) c h i c k e n pect o r a l i s m u s c l e , 3 hours post-mortem; Z = Z l i n e ; dz = d i s i n t e g r a t e d Z l i n e . A, 30,000X; B, 120,O00X; ( a r e a from A ) .  40  T r a n s m i s s i o n e l e c t r o n micrographs o f i r r a d i a t e d (l.OMrad) c h i c k e n pect o r a l i s m u s c l e , 3 hours post-mortem; sf = separated f i b r i l s . 25,000X.  41  T r a n s m i s s i o n e l e c t r o n micrographs o f cytoplasmic p a r t i c l e s i n i r r a d i a t e d (1.0 Mrad) c h i c k e n p e c t o r a l i s muscle 3 hours post-mortem; dp = damaged particles. 75,000X.  42  Transmission e l e c t r o n micrographs o f cytoplasmic p a r t i c l e s i n non-irradiated c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem. A, 30,000X; B, 120,000X; C, 150,000X; D, 120,000X.  43  Scanning e l e c t r o n m i c r o g r a p h s o f noni r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s muscle. A, 480X; B, 1,600X.  LIST OF FIGURES ( C o n t i n u e d ) Figure 44  Scanning e l e c t r o n m i c r o g r a p h s o f noni r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; A, t r a n s v e r s e b r e a k , 2,000X; B, s a r colemma, 20 ,000X.  45  Scanning e l e c t r o n m i c r o g r a p h s o f i r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; c t = c o n n e c t i v e t i s s u e ; s f = space between f i b r e s . A, 16OX; B, 32OX; C, 800X.  46  Scanning e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) and c r y o f r a c tured chicken p e c t o r a l i s muscle, 3 hours post-mortem; A, arrow = sepa r a t i o n o f f i b r e s , 120X; B, p e r f o r a t e d sarcolemma, s f = s e p a r a t e d f i b r i l s , 16,000X.  47-A  Scanning e l e c t r o n m i c r o g r a p h o f i r r a d i a t e d (1.0 Mrad) c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; f = f i s s u r e s . 2,000X.  47-B  A r e a o f r e c t a n g l e i n f i g u r e 47-A at h i g h e r m a g n i f i c a t i o n (20,000X); f = f i s s u r e s ; arrows = p a r t o f s a r coplasmic r e t i c u l u m .  xvlll.  ACKNOWLEDGEMENTS The author  wl*he*  to expre**  gA.atA.tudz to Vr. J . F. Richard*, ol food Sc.te.ncz, Unlver*lty hi* *upervl*lon  Prole**or, Ve.pat1tm2.nt  ofa Brltl*h  and guidance  and p>iepa)tatton ofi thl* Appreciation  hi* thank* and  during  Columbia,  lor  the. l n v e * t l g a t l o n  dl**ertatlon.  I* expre**ed  to Vr. W. V. Powrle,  Vr. S. Hakal and Vr. J. V'ander*toep, Ve.pan.tme.nt ol Food Science, ol Poultry  and Vn.. R. C. Tltz*lmmon*,  Science,  The a**l*tance  lor *ervlng  o{ many other  Vepartment  on the re*earch  member*, *tall  committee.  and *tu.de.ntt>,  ol the Ve.pan.tme.nt oh Vood Science. I* alio  gratelully  acknowledged. The. a**l*tance Mlcro*cope  ol Mr*. P. K. Gill,  Laboratory,  Vacuity  and the u*e ol scanning Vepartment  The. authon  ol kgn.lcultun.al  election  ol Metallurgy  mlcro*cope  I* greatly  wl*he*  to expre**  thank*  Energy  Comml**lon  ion. relieving  dutle*  during  the course  ol thl*  International *upport  acknowledge.*  Vevelopment  lor thl*  *tudy  with  Science.*,  In the  appreciated.  Atomic  The author  Election  to the.  him Irom  Pakistan oHlclal  *tudy. thank*,  the  Agency lor providing  under the Colombo  Plan.  Canadian financial  1. CHAPTER 1.  1.1.  GENERAL INTRODUCTION AND LITERATURE REVIEW  GENERAL INTRODUCTION R a d i a t i o n - s t e r i l i z a t i o n has been s u c c e s s f u l l y  a p p l i e d t o o b t a i n m i c r o b i o l o g i c a l l y s t a b l e meats. problem i s p r e s e n t e d by r a d i a t i o n r e s i s t a n c e  A major  o f enzymes,  as r e s i d u a l p r o t e o l y t i c a c t i v i t y has been shown t o cause o f f - f l a v o r s , b i t t e r t a s t e , and t e x t u r a l changes i n r a d i a t i o n s t e r i l i z e d meats ( C a i n e t a l . , 1958; Coleby e t a l . , 1961;  B a i l e y and Rhodes, 1964). The  has  p r o t e o l y t i c a c t i v i t y i n post-mortem muscle  been a t t r i b u t e d t o l y s o s o m a l c a t h e p s i n s ( S u z u k i e t a l . ,  1967;  O k i t a n i e t a l . , 1973).  I t has been n o t e d t h a t  v i d u a l enzymes possess g r e a t l y d i f f e r i n g r a d i a t i o n tivity  (Vas,  1966); i n f o r m a t i o n  s i n s i s l a c k i n g and i s h i g h l y  indi-  sensi-  regarding i n d i v i d u a l cathepdesirable.  Another important f a c t o r i n f l u e n c i n g the p r o t e o l y sis  i n i r r a d i a t e d t i s s u e i s the a v a i l a b i l i t y of lysosomal  enzymes.  Increased p r o t e o l y t i c a c t i v i t y a f t e r i r r a d i a t i o n  of muscle t i s s u e has been r e p o r t e d by K l e i n and Altman (1972b) ; t h i s c o u l d be due t o r a d i a t i o n - i n d u c e d lysosomal structure  r e s u l t i n g i n r e l e a s e o f c a t h e p s i n s and  i n c r e a s i n g t h e a v a i l a b i l i t y o f t h e enzymes. extent o f release  The r a t e and  o f l y s o s o m a l c a t h e p s i n s w i l l markedly  a f f e c t t h e course o f p r o t e o l y s i s . information  changes i n  on t h e r e l e a s e  However, as y e t d e t a i l e d  o f c a t h e p s i n s has n o t been r e -  2.  p o r t e d and i t was  the aim o f t h i s study t o i n v e s t i g a t e  the e f f e c t o f i r r a d i a t i o n on the r e l e a s e o f  cathepsins  A, B, C, and D from lysosomes. I n f l u e n c e o f i r r a d i a t i o n oh u l t r a s t r u c t u r e o f l y sosomes would p r o v i d e f u r t h e r i n f o r m a t i o n r e g a r d i n g s t a b i l i t y as w e l l as r e l e a s e o f c a t h e p s i n s .  Ultrastructural  changes i n i r r a d i a t e d muscle might be p a r t i a l l y f o r i t s extensive p o s t - i r r a d i a t i o n degradation t i c enzymes.  their  responsible by p r o t e o l y -  U l t r a s t r u c t u r a l s t u d i e s o f lysosomes and muscle  would h e l p g r e a t l y i n u n d e r s t a n d i n g w i t h i r r a d i a t i o n process  the events a s s o c i a t e d  and p o s t - i r r a d i a t i o n  behaviour  o f meats. This t h e s i s describes a three p a r t study  dealing  w i t h t h e e f f e c t s o f gamma r a d i a t i o n on: 1) .  R e l e a s e o f c a t h e p s i n s A, B, C, and D from i s o l a t e d lysosomes.  2) .  I n a c t i v a t i o n of lysosomal cathepsins  under  v a r i o u s c o n d i t i o n s ; and changes i n hemoglobin substrate. 3) .  U l t r a s t r u c t u r e o f i s o l a t e d lysosomes  and  c h i c k e n p e c t o r a l i s muscle as s t u d i e d by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y and ning e l e c t r o n microscopy.  scan-  3.  1.2.  LITERATURE REVIEW  1.2.1. E f f e c t o f I o n i z i n g R a d i a t i o n on Meats I r r a d i a t i o n has been n o t i c e d t o e x e r t a t e n d e r i z a t i o n e f f e c t i n b e e f and p o r k ( B a i l e y and Rhodes, 1964) doses o f 4 Mrad., d i a t i o n was  at  Coleby e t a l . (1960) r e p o r t e d t h a t  irra-  effective i n controlling microbial spoilage i n  whole e v i s c e r a t e d c h i c k e n when s t o r e d a t 1°C,  but the  of i r r a d i a t e d c a r c a s s e s tended t o d e t e r i o r a t e d u r i n g  quality the  s t o r a g e , and f l a v o r changes were not masked by r o a s t i n g . These f i n d i n g s d i f f e r from t h o s e o f P r o c t o r e t a l . (1956) who,  w o r k i n g w i t h c h i c k e n meat b l a n c h e d  i r r a d i a t i o n , r e p o r t e d t h a t f l a v o r was a f f e c t e d by 2 Mrep.  by s t e a m i n g  not  before  significantly  I t i s p o s s i b l e t h a t enzymes, which  o t h e r w i s e might cause o f f - f l a v o r s , were i n a c t i v a t e d by blanching  the  treatment.  I r r a d i a t e d f r e s h meats have been r e p o r t e d t o d e v e l o p a b i t t e r t a s t e , thought t o be due t o the a c c u m u l a t i o n f r e e t y r o s i n e ( C a i n e t a l . , 1958;  of  Drake e t a l . , 1957b).  Drake e t a l . (1961) r e p o r t e d a decrease i n consumer t a s t e panel preference  during u n r e f r i g e r a t e d storage of i r r a d i a t e d  raw ground b e e f , which c o r r e l a t e d t o some e x t e n t w i t h  the  a c t i v i t y o f i n t r a c e l l u l a r t i s s u e p r o t e o l y t i c enzymes  —  cathepsins. Doty e t a l . (1958) found an i n c r e a s e i n  non-protein  4. nitrogenous compounds i n ground beef a f t e r i r r a d i a t i o n treatment, and i t was accompanied by an appreciable in the concentration  of soluble proteins.  decrease  Zender et a l .  (1958) also reported a decrease i n glycine-NaOH-soluble protein content of beef muscle a f t e r i r r a d i a t i o n . Bautista et a l , (1961) reported an increased rate of release of amino nitrogen, t o t a l soluble nitrogen, and  TCA-  soluble nitrogen a f t e r i r r a d i a t i o n treatment of beef. El-Badawi et a l . (1964) found a s i g n i f i c a n t in glycine-NaOH-soluble proteins contained f l u i d from i r r a d i a t e d beef.  increase  i n the drip  On the other hand, Anglemier  et a l . (1964) added the drip f l u i d back to the sample p r i o r to extraction, and did not f i n d a s i g n i f i c a n t difference i n the amount of extractable proteins i n i r r a d i a t e d and uni r r a d i a t e d meat.  They also noticed the disappearance of  one band in the electrophoretic pattern of the glycineNaOH-soluble proteins extracted from muscle and concluded that i r r a d i a t i o n - s t e r i l i z a t i o n results i n a fragmentary action on the structure of meat proteins.  These workers  indicated that the number of electrophoretic bands was  ex-  pected to increase rather than decrease or not be affected. They explained that one p o s s i b i l i t y i s that protein fragments are held together by hydrogen and/or the e l e c t r o s t a t i c bonds.  The other p o s s i b i l i t y i s that an i r r a d i a t i o n frag-  mentation e f f e c t i s exerted mainly on the meat proteins not extracted by the glycine-NaOH buffer.  Uzonov et a l . (1972)  observed that i r r a d i a t i o n of beef resulted i n a decrease i n  5. t o t a l content of soluble proteins, and electrophoretic characteristics of the soluble proteins were changed by a decrease i n the cathodic f r a c t i o n .  Klein and Altman (19 7 2a)  reported changes i n electrophoretic mobility of the soluble proteins from chicken meat; at a 1.0 Mrad dose there  was  a considerable decrease i n high molecular weight bands, and at 3.0 Mrad there was  a nearly complete l e v e l l i n g of the  high molecular weight bands. Anglemier et a l . (1964) reported that i r r a d i a t i o n s t e r i l i z a t i o n decreased  the hydration of beef muscle i n pH range  3.5 - 7.0 with depression maximum near the i s o e l e c t r i c region. These results are i n agreement with Lawrie et a l . (1961). Alteration of hydrogen or e l e c t r o s t a t i c bond of a protein should have maximum effect on hydration around the i s o e l e c t r i c region (Hamm, 1960).  Irradiation may  have some tendency to  tighten the meat protein structure by increasing the number of these weak bonds.  This hypothesis i s i n agreement with  information presented by Drake et a l . (19 5 7a) on bovine serum albumin.  Lawrie et a l . (1961) and Batzer et a l . (1959)  reported that i r r a d i a t i o n caused an increase of muscle pH. Bendall and Wismer-Pedersen (1962) observed an increase i n pH of washed f i b r i l s a f t e r heating.  Such s h i f t i n pH  has  been suggested to be due to release of tyrosine hydroxyl groups on denaturation of proteins (Bendall, 1964). Studies have shown that p r o t e o l y t i c enzymes are not f u l l y inactivated at a radiation dosage required for s t e r i l ization (Drake et a l . , 1957b; Doty and Wachter, 1955).  6. Landmann (196 3) found that one of three active proteinase fractions i n beef retained i t s a c t i v i t y a f t e r i r r a d i a t i o n of meat at s t e r i l i z i n g doses.  Rhodes and Meegungwan (1962) r e -  ported a considerable proteinase a c t i v i t y i n l i v e r after i r r a d i a t i o n with 4 Mrad. higher doses proteases  remaining  They also indicated that at  were radiation r e s i s t a n t .  Proctor and  Goldblith (1951) stated that much higher doses might be required to inactivate the enzymes or reduce t h e i r a c t i v i t i e s to a l e v e l acceptable f o r long-term storage o f meat. Accumulation of free tyrosine i n i r r a d i a t e d meat has been noted, and i t was thought by some workers to be liberated by p r o t e o l y t i c enzymes (Cain et a l . , 1958 ; Drake et a l . , 1957b). Losty et a l . (197 3) reported that 2 - 6 Mrad gamma radiation destroyed up t o 75% of the p r o t e o l y t i c a c t i v i t y i n ground beef. Shults et a l . (1975) observed that gamma i r r a d i a t i o n with 2 Mrad at -80°C resulted i n only a 1% reduction of p r o t e o l y t i c enzymes i n beef, an 11% reduction i n chicken and a 2 6%  reduc-  tion i n pork, while an 8 Mrad dose at 21°C resulted i n 79% 82% reduction i n p r o t e o l y t i c enzymes i n the three kinds of meats.  Klein and Altman (1972b) found that p r o t e o l y t i c enzymes  in chicken breast muscle were more radiation resistant than the enzymes i n leg muscle.  There was 50% i n a c t i v a t i o n of proteases  at 2 Mrad and 85% at 5 Mrad i n the breast muscle, while the leg muscle showed almost complete i n h i b i t i o n of the enzymes.  1.2.2. Effect of Ionizing Radiation on Proteins and Amino Acids Because of the high percentage of water i n meats, the  7. reactions which occur when water i s i r r a d i a t e d are of primary importance.  By r a d i o l y s i s of water H, OH,  H2O2  and H2 are the  f i n a l major products, but free radicals are formed along the path of the primary electron and react with each other as d i f f u s i o n occurs.  Also, some of the products formed along  the track escape and can then react with solute molecules (Kuzin, 1964). Adhikari and Tappel (19 75) reported gamma i r r a d i a t i o n of glutamic acid, phenylalanine, serine, arginine, and methionine in aqueous solutions resulted i n the formation of malonaldehyde, which reacted with other amino acid molecules to form Schiff's bases.  Free arginine, tyrosine, and methionine have  been shown to be destroyed more readily than other amino acids by gamma radiation (Fujimaki et a l . , 1961).  In cysteine, how-  ever, the SH group i s so e f f i c i e n t i n trapping free r a d i c a l s , the rest of the molecule i s almost completely protected from attack, and cystine i s the major product formed (Swallow, 1962). Products from the i r r a d i a t i o n of proteins resemble those of amino acids, except that there i s a greater attack on amino acid side chains when amino acids are combined i n peptide chains.  There i s a general decrease i n the amounts of  unchanged amino acids with almost t o t a l loss of methionine and cysteine, and 25% loss of h i s t i d i n e (Drake et a l . , 1957a; Hedin et a l . , 1960).  When protein solutions are i r r a d i a t e d  in the presence of oxygen, the break i n the peptide chain i s not the usual hydrolytic break, but occurs between theN-C  8. bonds to form amides and carbonyls  (Garrison et a l . , 1962).  Ox-  idative damage t o amino acids, peptides.and proteins as a r e s u l t of i r r a d i a t i o n , have been observed by Ambe and Tappel (1961). Tsaien and Johnson (19 59) reported that the soluble proteins of beef, when exposed to a dose of 5.6 Mrads, suffered severe destruction of certain amino acids as measured by the Moore and Stein method a f t e r acid hydrolysis. that about three-quarters one-half of threonine  Their results indicated  of serine and glutamic  acid, about  and aspartic acid and about o n e - f i f t h of  glycine, methionine, l y s i n e , h i s t i d i n e , and arginine could not be recovered acids.  from i r r a d i a t e d protein as ninhydrin reacting amino  The t o t a l of the amino acids destroyed  of the i n i t i a l content.  amounted t o 2 8%  In contrast to t h i s observation Rhodes  (1966), using ion-exchange chromatography, could not detect any s i g n i f i c a n t change i n the proportion of ninhydrin-reacting components (except ammonia) when meat was exposed at 0°C to gamma radiation at dose levels of 5 or 2 0 Mrads. Increased  s o l u b i l i t y of the collagen i n i n t a c t beef muscle  after i r r a d i a t i o n has been reported (Bailey and Rhodes, 1964), but this e f f e c t diminished when collagen was i r r a d i a t e d i n meat juice or i n s a l i n e , which indicates that the e f f e c t i s due to the i n d i r e c t action of radiation and that these changes may not affect the tenderness of meat.  Grant et a l . (1970) reported  an altered r e a c t i v i t y of both native and cross-linked collagen when i r r a d i a t e d i n a dry or wet condition with 100 Mrads of e l ectron i r r a d i a t i o n .  I t was observed that fibres i r r a d i a t e d dry  i  showed greater damage when examined i n the electron microscope. Similar results were obtained using 100 Mrad gamma radiation by Grant et a l . (1973), who concluded that i r r a d i a t i o n of collagen in the dry state resulted i n scission of the polypeptide chains but i n the presence of water,this was accompanied  by the forma-  tion of intermolecular bonds, thus changing the configuration of polypeptide chains.  Jelenska and Dancewicz (1972) noted an  apparent decrease i n the content of free e-amino groups i n i r r a d i a t e d tropocollagen, indicating that there were conformat i o n a l changes due t o formation of new interchain cross-linking bonds.  Coelho (196 9) reported fragmentation as well as r e t i -  culation through intermolecular cross-linking of gamma i r r a diated actomyosin. Alteration i n s o l u b i l i t y characteristics o f i r r a d i a t e d c o l lagen (Grant et a l . , 1970, 1973; Bowes and Moss, 1962) and acto myosin  (Coelho, 1969) have been reported.  Braams (1961, 1963)  found reduced tensile strength of bovine tendon due to i r r a d i a tion treatment.  Friedberg (1969) observed that gamma i r r a d i a -  tion caused chain breaks resulting i n a decrease i n molecular weight of dry collagen.  This e f f e c t decreased when metal ions  were present i n the system (Friedberg et a l . , 1975). Paul and Kumta (197 3) observed increased t r y p t i c hydroly- . sis of irradiated horse heart myoglobin.  McArdle and Desrosier  (1955) reported an increased hydrolysis of i r r a d i a t e d casein, and egg albumin by t r y p s i n , and concluded that ionization of the bonds by radiation caused an opening of the molecules,  10. rendering them more susceptible to t r y p t i c digestion. et a l . (1970, 1973)  observed an altered r e a c t i v i t y of both  native and cross-linked collagen with collagenase —  wet  Grant  and  elastase  (but not dry) i r r a d i a t e d native collagen became  resistant to collagenase;  cross-linked collagen, normally  resistant to enzyme attack, was  found to be more sensitive  after'irradiation. McArdle and Desrosier  (1955) reported a change i n e l e c -  trophoretic m o b i l i t i e s and patterns of i r r a d i a t e d casein and egg albumin.  Both fragmentation (Carrol et a l . , 1962)  and polymerization et a l . , 1973)  (McArdle and Desrossier, 1955;  of proteins have been reported.  Zakrzewski  The predominant  effect of i o n i z i n g radiation on proteins i n aqueous solution consists of aggregation, leading ultimately to p r e c i p i t a t i o n of insoluble material.  Aggregation of human serum albumin  (Hay and Zakrzewski, 1968), myoglobins (Brown and Akoyunoglou, 1964;  Satterlee et a l . , 1971;  clease (Mee  Paul and Kumta, 1973), ribonu-  et a l . , 1972), and egg-white lysozyme (Stevens,  196 7) are a few examples. Studies on hemoproteins have shown s p e c t r a l changes due to i r r a d i a t i o n .  Such changes  have been attributed to  a l t e r a t i o n i n heme group (Brown and Akoyunoglou, Giddings and Markakis, 1972;  1964;  Satterlee et a l . , 1972)  as well  as the globin group of the protein (Clarke and Richards, 1971;  Lycometros and Brown, 1973).  Brown and Akoyunoglou  (1964) noted that gamma i r r a d i a t i o n of metmyoglobin s p l i t small peptides from globin.  Satterlee et al.(1971) suggested  11. that a l t e r a t i o n i n i r r a d i a t e d metmyoglobin might be due  to  loss of amide nitrogen from the protein. Radiation damage to sulfur-containing amino acids often been reported.  has  In proteins i r r a d i a t e d i n the dry state  the number of sulfhydryl groups may  decrease, as i n serum albu-  min  and riboriuclease (Hunt and  (Alexander and Hamilton, 1960)  Williams, 1964), or increase, as i n lysozyme (Stevens et a l . , 1967), accompanied by migration  of disulphide.  In proteins  i r r a d i a t e d i n solution, the number of sulfhydryls decreases rapi d l y and mixed d i s u l f i d e s are formed (Augenstine, 1962).  An-  other important e f f e c t , observed i n proteins i r r a d i a t e d i n s o l ution i n the presence of oxygen , i s the formation of  carbonyl  groups, attributed to oxidation s c i s s i o n of peptide bonds (Jayko and Garrison, 1958).  1.2.3. E f f e c t of Ionizing Radiation on Enzymes and Enzyme Activity The  absorption  of radiation energy by the water phase  of food materials leads to the formation of reactive water radicals which very quickly interact with c e l l u l a r constituents.  Damage caused by water radicals i s usually referred to  as an i n d i r e c t action of i o n i z i n g radiation. caused by energy absorption to as a d i r e c t action.  Radiation changes  i n organic molecules are referred  The observed destruction of i n t r a -  c e l l u l a r enzymes i s due to the combined e f f e c t of direct and indirect action.  The r e l a t i v e contribution of these mech-  anisms to radiation damage may  vary f o r d i f f e r e n t proteins ,  12. depending on t h e i r l o c a l i z a t i o n and microenvironments  (Pihl  and Sanner, 1963). The i n a c t i v a t i o n dose f o r enzymes decreased with increasing temperature (Proctor and Goldblithi 19 51).  This e f f e c t  was also reported by Bellamy and Lawton (1955) f o r electron i r r a d i a t i o n of aqueous pepsin, tyrosinase, and polyphenol oxidase; and neutron i r r a d i a t i o n of dry catalase (Setlow and Doyle, 1953).  It has been suggested that hydrogen bonds i n  the enzyme molecule are weakened by the higher temperatures (Scheraga, 1963), making the molecule subject to i n a c t i v a t i o n by excitation as well as i o n i z a t i o n .  Low s e n s i t i v i t y has  been suggested to result from changes i n the water sheath surrounding an enzyme (Klotz, 1958), modification i n enzyme conformation or changes i n the d i s t r i b u t i o n of radiation products i n preparations i r r a d i a t e d i n the frozen state (Augenstine, 1962). The effect of pH on radiation s e n s i t i v i t y of enzymes has been found to be considerable but unpredictable both i n solution (Pihl and Sanner, 1963; Brustad, 1966; Robins and Butler, 1962) and i n the dry state (Wilson, 1959).  This i s largely  due to the fact that pH influences the change i n dissociable protein groups.  This would influence the radiation s e n s i t i v i t y  by a l t e r i n g protein conformation (Sanner and P i h l , 1968), and d i s t r i b u t i o n of r a d i c a l species produced by i r r a d i a t i o n  (Rob-  ins and Butler, 1962; Okada, 1957)* The i n a c t i v a t i o n dose f o r enzymes increases with concentration as revealed by the studies on carboxypeptidase  (Dale, 1952), trypsin (Sanner and P i h l , 1967), and a-chymotrypsin (Butler et a l . , 1960).  A s i m i l a r e f f e c t has been  reported by Giovannozzi-Sermanni (1969) f o r cathepsin C. These results are supported by the.findings  of Vas  (1969)  for pectin methylesterase and c e l l u l a s e preparations. Regarding effects of oxygen on radiation  resistance  of enzymes, a number of published works indicate an increase in r a d i o s e n s i t i v i t y in the presence of oxygen, while others do not  (Marples and Glew, 1958).  Alexander (1957) reported  a marked s e n s i t i z i n g effect of oxygen on trypsin when i r r a diated i n dry state.  Hunt and Williams (1964) showed a sensi-  t i z i n g effect of oxygen on ribonuclease.  Lynn and  Skinner  (1974) reported equal rates of loss of alkaline phosphatase a c t i v i t y i n aqueous solution'under 0 , N , and N 0; 2  2  2  composition of the radiolysed enzyme proteins  however,  varied depending  on gaseous atmosphere used. < It has been suggested that oxygen concentration w i l l influence the course of r a d i o l y s i s of ter.  When the i r r a d i a t e d solutions  are i n equilibrium  wa-  with  a i r , the e aq w i l l be e f f e c t i v e l y trapped by oxygen to give 0  2  radicals (Sanner and P i h l , 1967).  enzymes (Sanner and P i h l , 1967; 0  2  Experiments on other  Bustard, 1966)  indicated  that  radicals possess low a c t i v i t y and i n general do not par-  t i c i p a t e i n enzyme inactivations. Presence of other compounds i n the medium plays an portant role i n inactivation of enzymes by radiation. centration  and nature of these compounds influences  the interaction of radicals with enzymes.  The  imCon-  greatly  efficiency  14. of a compound to protect an enzyme by a r a d i c a l scavenger mechanism in solution depends on i t s rate of i n t e r a c t i o n with the water radicals responsible f o r the i n a c t i v a t i o n of enzymes.  Since the interaction of water r a d i c a l s with  an organic molecule can lead to the formation of a new r a d i c a l , the r e s u l t w i l l depend on the r e a c t i v i t y of the new  radical.  Protection w i l l be observed when the  organic  r a d i c a l i s less capable of i n t e r a c t i n g with the enzyme molecule than the parent water r a d i c a l (Sanner and 1969).  Radio-protective  Pihl,  effects of g l y c y l - g l y c i n e on trypsin  in solution have been reported by Sanner and P i h l (1967). Jung (196 7) observed s i m i l a r protection of RNase by cystamine in the dry as well as the aqueous state. observed that i n a c t i v a t i o n of RNase was presence of EDTA.  Schuessler  (1973)  reduced i n the  Many other substances, when present i n  enzyme solutions, exert a radio-protective e f f e c t (Dale, Kuzin, 1964).  It has been demonstrated that two  1962;  solutes  could compete f o r the same kind of r a d i c a l and thus "protect" each other (Dale, 1962).  In addition to such  "competitive  protection", some protectors might react with enzyme molecules to a l t e r the c r i t i c a l surface s i t e or make i t unavailable to i n a c t i v a t i n g r a d i c a l s .  Such "reactive protection"  could be provided by mixed d i s u l f i d e formation P i h l , 1955;  Eldjahrn et a l . , 1960)  (Eldjahrn and  or by masking of c r u c i a l  groups with other chemicals (Barron, 1954;  Sutton, 1956).  The reactive protection r e s u l t i n g from an enzymessubstrate complex was  predicted (Augenstine, 1959), but  15. Robinson and P h i l l i p s (1960) could not demonstrate i t with l i v e r alcohol dehydrogenase.  However, other studies  suggested  that enzymes complexed with t h e i r substrates can thus be protected  (Okada, 1957;  Sutton, 1956).  supported by Lynn's studies  Recently t h i s has been  (1972) on t r y p s i n .  demonstrated that enzyme a c t i v i t y was f o l d by complexing i t with s i l i c a .  I t has been  protected at least  fifty-  Further, the r e l a t i v e  ex-  tent and nature of the radiation damage to amino acid residues of the suspended enzyme were d i f f e r e n t from those found with dissolved a l . , 1966)  trypsin.  Trypsin  i n a matrix of agar (Holladay et  and RNase mixed with glycylglycine  196 7) showed s i m i l a r protection  due  (1974) observed a marked protection  (Copeland et a l . ,  to complexing.  Lynn  of t r y p s i n , chymotrypsin,  and chymotrypsinogen when i r r a d i a t e d complexed with DNA. was  It  concluded that active s i t e s were protected either by  geometry of the DNA/protein complex or by nucleic acid  the  acting  to divert the free r a d i c a l attack. Dry enzymes are generally than those in solution. required  more stable to i r r a d i a t i o n  For example, the electron dose  for complete i n a c t i v a t i o n of dry trypsin was  170  times that for trypsin in solution (Bier and Nord, 19 52). Vas  (1969) found that pectin methylesterase and  were highly radiation resistant i n the dry The  cellulase  state.  loss of b i o l o g i c a l a c t i v i t y , when enzymes are  irradiated in the dry state i s associated  with d i f f e r e n t  types of radiation damage, compared with i r r a d i a t i o n in the  16. soluble  state.  Rupture of hydrogen bonds occurs with sub-  sequent unfolding of molecules. may  aggregate, then may  Furthermore, the molecules  disassociate  breakages of peptide bonds may  into subunits , or  cause fragmentation.  In  addition, alteration in the amino acid side chains i s a p o s s i b i l i t y (Sanner and P i h l , 1969).  Radical reactions  play an important role i n the radiation effects observed in the dry state  (Braams, 1963).  by Copeland et a l . (1968), who  This view has  been supported  studied the formation  and  reactions of radiation-induced radicals i n the dry RNase. Relatively l i t t l e i s known about the s p e c i f i c damage responsible for the inactivation of enzymes in the dry The  state.  available data indicate that no general rules exist  concerning the type of damage responsible for the This i s well i l l u s t r a t e d with the two papain.  inactivation.  enzymes RNase and  In the case of RNase, Jung and  Schussler (1966)  have succeeded in separating the active RNase molecules from inactivated  ones, and have shown that the  inactivation  is associated with molecular aggregates, indicating loss of a c t i v i t y was  not due to destruction  cular group within the enzyme.  of any p a r t i -  Copeland (1975) suggested  that i r r a d i a t i o n inactivation of RNase may  be due  to forma-  tion of s u l f u r radicals at ruptured d i s u l f i d e bonds. has  that  Papain  a single essential SH group, and i t has been shown that  s p e c i f i c protection  of t h i s group, e.g.  by mixed d i s u l f i d e  formation, provides s i g n i f i c a n t protection  of the enzyme  against inactivation i n dry state ( P i h l and Sanner, 1963).  Enzymes in solution are more radiation sensitive than in the dry state.  In the dry state, the effects are mostly  mediated by d i r e c t action of r a d i a t i o n , but i n s o l u t i o n , i n d i r e c t action i s more important.  The  i n a c t i v a t i o n of  enzymes i r r a d i a t e d in dilute solutions i s due to the  action  of the radiation products of water, the most important being the OH*  and H' radicals and  I  For certain enzymes, i t appears that the  radiation  i n a c t i v a t i o n i s due predominantly to destruction  of the  active s i t e , while for other enzymes, the i n a c t i v a t i o n seems to be due  to general denaturation.  mechanisms contribute  In many cases both  to the i n a c t i v a t i o n .  Sulphur-containing  and aromatic amino acid residues have both been separately implicated  i n the radiation damage of enzymes.  Thus, i n  lysozyme (Adams et a l . , 1969), trypsin (Lynn, 1971), and chymotrypsin (Lynn, 1972), tryptophan and tyrosine were found to be the residues f i r s t affected by r a d i o l y s i s . (Gaucher et al.,1971; Lynn and  Louis, 1973)  (Schlissler and Jung, 1967), cysteine were modified.  ribonuclease  and cystine residues  P i h l and Sanner (1963) demonstrated that  inactivation of papain in solution was to destruction  and  In papain  of the active SH group.  due  almost exclusively  As destruction  of  SH group p a r a l l e l l e d closely the loss of enzyme a c t i v i t y , s p e c i f i c protection protection  of SH group provided a very strong  against inactivation of enzymes.  This has  also  been found in the case of other sulfhydryl enzymes, such as l a c t i c dehydrogenase (Adelstein, 1965), phosphorylase b  18. (Damjanovich et a l . , 1967) and glyceraldehyde-3-phosphate dehydrogenase  (Lange and P i h l , 1960).  The loss of a c t i v i t y on i r -  radiation could be almost completely accounted f o r by destruction of s p e c i f i c sulfhydryl groups.  On the other hand, with  RNase and chymotrypsin a general protein denaturation has been found to be of major importance f o r enzyme i n a c t i v a t i o n .  Sev-  e r a l studies have shown aggregation of ribonuclease on i r r a d i a tion ( H a s k i l l and Hunt, 1967; Schuessler and Jung, Schuessler, 1973).  1967;  It was demonstrated by Schuessler (1967)  that EDTA had protective effects on ribonuclease, and prevented the formation of aggregates.  From studies with chymotrypsin  (Mounter, 1960) i t was concluded that 50% of the loss of act i v i t y could be due to protein denaturation.  With RNase, i t  has been found that the pattern of amino acid destruction was the same i n active and inactive  enzyme, molecules (Jung, 1967).  Enzymes within c e l l s or tissues r e s i s t i r r a d i a t i o n much more strongly than those i n homogenates or i n pure solution. The ribonuclease and deoxyribonuclease a c t i v i t y of Tetrahymena g e l e i i was  not affected when a c e l l suspension was  given 300,000 to 500,000 r X-rays, while an homogenate of the organism underwent 50% i n a c t i v a t i o n under the same conditions (Eichel and Roth, 1953).  The mean l e t h a l dose f o r  catalase i n crushed potatoes was 5,000,000 rep electrons as compared with only 2 5,000 rep f o r catalase i n pure solution (Bellamy and Lawton, 1955).  With 500,000 rep gamma i r r a d i a -  t i o n , l i t t l e or no reduction of p r o t e o l y t i c a c t i v i t y of beef  19. muscle tissue was noticed, with dosage of 1,BOO, 0 00.rep about 50% of the a c t i v i t y was destroyed (Doty , and-Wachter, 1955). ", The increased radiation resistance of enzymes i n vivo may be attributed to the presence of various compounds i n c e l l u l a r environments which may act as radioprotectors or modifiers, etc. Enzymes complexed with other materials were found to be highly radioresistant (Lynn, 1972, 1971; Holladay et a l . , 1966).  Hutchinson (1957) reported that enzymes  embodied i n lipoproteins were only s l i g h t l y - a f f e c t e d by radiation as compared with free enzymes.  The 50% i n a c t i v a -  tion dose for cathepsin C, i n crude extract, was  significantly  higher as compared with the enzyme i n p u r i f i e d fractions (Giovannozzi-Sermani et al,,, 1969).  Musch (1969) found  that i o n i z i n g radiation of muscle of cod, red f i s h , coal f i s h , and haddock required doses of 10 - 20 Mrad f o r p a r t i a l inactivation of cathepsin D i n tissue, while a f t e r extract i o n , i t became several times more sensitive to radiation. Several studies have demonstrated high radiation resistance of p r o t e o l y t i c enzymes i n tissue (Klein and Altman, 1972b; Shults et a l . , 1975 ; Losty et a l . , 1973). An increase i n ATPase a c t i v i t y has been observed on low dose i r r a d i a t i o n of actomyosin (Coelho, 1969), but higher doses resulted i n progressive i n h i b i t i o n of enzyme.  Myosin A  showed a s i m i l a r pattern of enhanced enzymatic a c t i v i t y up to a maximum followed by i n h i b i t i o n when submitted to i n creasing doses of X-rays (Szabolcs et a l . , 1964).  There  is ample evidence that under the influence of i r r a d i a t i o n ,  20. ATPase a c t i v i t y i n tissues i s considerably increased (Dale, 1952;  Maxwell and Ashwell, 1953). Giovannozzi-Sermani et a l . (1969) found a c t i v a t i o n of  invertase when i r r a d i a t e d i n s i t u .  This view was  supported  by some e a r l i e r work on plant tissues (Kuzin and Kopylov, 1960 ; Berizina, 1962 ; Vas, 1966). catalase a c t i v i t y was  A seven-fold increase i n  observed i n i r r a d i a t e d yeast several  hours after i r r a d i a t i o n (Aronson et a l . , 1956).  Activation  of enzymes has been reported mostly i n tissues and not i n homogenates or solutions, which indicates that there i s some i n d i r e c t mechanism involved.  Desorption of enzymes  from c e l l organelles plays an important  role i n the altered  metabolism of the c e l l (Okada and Peachy, 1957) , but cannot explain the general increase i n a c t i v i t y observed when organelles are completely destroyed.  Kuzin (1964) suggested  the p o s s i b i l i t y of a decrease i n concentration of i n h i b i t o r s of the enzymes, either by disruption of t h e i r synthesis or by a change i n the permeability of the c e l l , enhancing the removal of the i n h i b i t o r s from tissue. was  supported  by Kurnick et al. (1959).  This, hypothesis According to Pauly  and Rajewsky (19 55), an a l t e r a t i o n i n permeability of c e l l membranes i s closely related to an increase i n enzyme a c t i v i t y after i r r a d i a t i o n . Roth et a l . (1962) reported an increased s p e c i f i c act i v i t y of 3-glucuronidase  and acid phosphatase i n rat spleen  after 700 r of X-radiation.  They interpreted t h i s increase  to be due to selective retention of enzymes during loss  21. of spleen nitrogen.  Rahman (1962) also found an increase i n  s p e c i f i c a c t i v i t y of these enzymes i n lysosome-rich fractions from rat thymus, after whole body i r r a d i a t i o n .  He  suggested  that t h i s increase was due to selective nitrogen loss of the lymphoid tissue. the tissue.  He also noted an increase of dense bodies i n  These dense bodies are considered to be  lysosomes  (Novikoff et a l . , 1956), and i t was postulated that the i n crease i n enzyme a c t i v i t y was due to a selective retention of enzyme or possibly de novo formation of the 1.2.4. Lysosomal  lysosomes.  Cathepsins  Cathepsins are a group of i n t r a c e l l u l a r enzymes of animal tissue o r i g i n which hydrolyse proteins under a c i d i c conditions.  Currently f i v e groups of cathepsins are recog-  nized, although revision i s l i k e l y as t h e i r functions are further characterized.  Cathepsins have been reviewed by  Barrett (1969, 1972), Barrett and Dingle (1971), and Mycek (1970).  A summary of the cathepsin classes from the above  reviews i s presented here. 1.2.4.1. Cathepsin A (EC 3.4.2.-). This enzyme s p l i t s N-carbobenzoxy-ct-L-glutamyl-L-tyrosine, a synthetic substrate f o r pepsin.  Its s p e c i f i c i t y i s towards  the carboxy terminal L-amino acid residue of a polypeptide. Cathepsin A probably has l i t t l e action on proteins alone but acts s y n e r g i s t i c a l l y with endopeptidases such as cathepsin D. Cathepsin A has optimum pH of 5.0 - 5.4; i t i s not activated by cysteine and iodoacetamide does not i n h i b i t i t s a c t i v i t y . i i  22. 1.2.4.2. Cathepsin B (3.4.4.-). O r i g i n a l l y defined as the enzyme from bovine spleen s p l i t t i n g benzoyl - arginine  amide (BAA), i t now seems that at least  two enzymes with this a c t i v i t y were present.  The best known of  these i s cathepsin B l , or simply cathepsin B.  I t hydrolyses  benzoyl-arginine p - n i t r o a n i l i d e (BAPA) and the corresponding 2-naphthylamide (BANA) as well as BAA.  A second enzyme, cath-  epsin B2 , hydrolyses BAA but not BAPA or BANA. clear whether i t i s an endopeptidase.  I t i s not yet  Endopeptidase a c t i v i t y  as well as transpeptidation reactions are c h a r a c t e r i s t i c of cathepsin B.  The pH optima f o r synthetic substrates range from  5.0 to 6.5, but with hemoglobin, i t s pH optimum i s near 4.0. Since a cysteine residue i s i n the active s i t e , activation can be achieved with cysteine and other sulfhydryls. mide and p-chloromercuribenzoate  Iodoaceta-  inhibit i t s activity.  1.2.4.3. Cathepsin C (EC 3.4.4.9.). Cathepsin C f i r s t was recognized as the enzyme  deamidating  glycyl-L-phenylalaninamide, and i s now known to act on g l y c y l L-phenylalanine p - n i t r o a n i l i d e and 2-naphthylamide. sin  C i s an exopeptidase  Cathep-  with broad s p e c i f i c i t y f o r s p l i t t i n g  of dipeptide naphthylamides and removal of dipeptides sequent i a l l y from the amino terminus of a polypeptide chain (hence the alternative names dipeptidyl transferase and d i p e p t i d y l amino-peptidase I ) .  Optimum pH i s near 5.0; i t requires a  t h i o l reagent and C l ~ for maximum a c t i v i t y and i s i n h i b i t e d by t h i o l blocking reagents.  23. 1.2.4.4. Cathepsin D (EC 3.4.4.23). This i s the major acid protease of animal tissue.  It  has few low molecular weight substrates and i s inactive towards the synthetic substrates of cathepsins A, B and C. is an endopeptidase,  Cathepsin D  i t s assays are based on release of hydro-  l y t i c products from proteins.  Maximum a c t i v i t y i s toward hem-  oglobin i n the range of pH 3.0 - 4.0.  Cathepsin D a c t i v i t y i s  unaffected by standard t h i o l reagents, t h i o l blocking reagents, or metal activators or i n h i b i t o r s . 1.2.4.5. Cathepsin E (EC 3.4.4.-). An endopeptidase of much more limited tissue d i s t r i b u t i o n than cathepsin D, but closely s i m i l a r s p e c i f i c i t y .  Cathepsin E  is d i f f e r e n t i a t e d by higher a c t i v i t y at pH 2.5 on serum albumin as substrate. 1.2.4.6. Neutral Proteinases.(EC 3.4.4.-). There are many reports of neutral tissue proteinases , but l i t t l e i s known about t h e i r properties.  A requirement  for  t h i o l reagent i s common but not invariable, and some enzymes 2+ are activated by L'DTA, whxle other require Ca Other lysosomal p r o t e o l y t i c enzymes are: caroxypeptidase, dipeptidase, and dipeptidyl aminopeptidase.  Enzymes  acting on collagen represent a group of neutral proteinases in various tissues, but Schaub (1964) and Etherington (1972) described a collagenase from rat organs and muscle that was  active on insoluble collagen at pH 3.3 - 3.5.  The  study of collagenase and neutral proteinases i s made d i f f i cult due to i n s t a b i l i t y of the enzymes and occurance of  24. potent i n h i b i t o r s in many tissues.  1.2.5. Lysosomes and Lysosomal Cathepsins i n Tissue Autolytic degradation of major tissue components has been attributed to the action of a group of enzymes of particulate hydrolases with acid pH optima (de Duve, 1963) as well as neutral protease (Okitani and Fujimaki, 1972). The cytoplasmic p a r t i c l e s which envelop these enzymes are called lysosomes.  The lysosomal complex includes several  cathepsins as well as other hydrolytic enzymes. Cathepsins have been i s o l a t e d from a variety of tissues. Three enzyme fractions were separated from beef muscle, with optima at pH 5, 8-9, and 10, when using soluble protein from muscle of this species as substrate (Sliwinski et a l . , 1961).  Parrish et a l . (1969) presented evidence that cathep-  sin from bovine diaphragm muscle was  lysosomal.  The com-  prehensive work of Stagni et a l . (1968) showed that lysosomes are present i n the r a t and bovine s k e l e t a l muscles.  Landmann  (1963) indicated that p r o t e o l y t i c a c t i v i t y i n beef muscle was due to two enzyme systems: one strongly activated by ferrous ions with optimum a c t i v i t y at pH 5.0, and the other activated by EDTA with optimum a c t i v i t y at pH 9.0. demonstrated the presence of cathepsins B and C.  They Randall  and MacRae (196 7) also reported presence of cathepsins B and C i n water soluble proteins of bovine s k e l e t a l muscle. Bodwell and Pearson  (1964) were unable to attribute  the p r o t e o l y t i c a c t i v i t y i n an extract of bovine s k e l e t a l  25. muscle to either cathepsin B or C.  They concluded  that  the enzyme fraction obtained displayed endopeptidase a c t i v i t y s i m i l a r to that of cathepsin A.  Lutalo-Bosa and MacRae  (196 9) demonstrated the presence of cathepsins B, C, and D in bovine s k e l e t a l muscle. Caldwell and Grosjean  (1971) reported the presence of  cathepsins A, B, C, and D i n chicken breast' muscle, which was confirmed by the observations of Iodice et a l . (1972). Martin and Whitaker (196 8) i s o l a t e d and p u r i f i e d cathepsin D from chicken leg muscle using ammonium sulfate fractionation and chromatographic techniques.  Fukushima et a l . (1971)  were able to obtain highly p u r i f i e d cathepsin D from chicken muscle by column chromatography and gel f i l t r a t i o n .  Barret  (19 70) demonstrated the presence of three isoenzymes of cathepsin D i n chicken  liver.  Various chemical changes take place i n muscle proteins during storage.  Sarcoplasmic  proteins are readily hydro-  lysed by cathepsins as compared with fibrous and e x t r a c e l l u l a r proteins (Sharp, 196 3; Bodwell and Pearson, 1964). et a l . (1969) reported that soluble protein was  Suzuki  most rapidly  hydrolysed, followed by myosin A, actin and myosin B, extracted from rabbit muscle. It has been demonstrated that cathepsin D contributes to the f i r s t step of the degradation (Iodice et a l . , 1966).  of i n t r a c e l l u l a r proteins  Caldwell (1970) suggested that  hydrolysis of proteins by cathepsin D could be augmented by supplementary action of other cathepsins present i n  26. tissue.  This view was supported by the studies of Liao-Haung  et a l . (1971).  Firfarova and Orekhovich (1971) suggested  the existence of an inactive precursor of cathepsin D, which is activated on release from lysosomes. Ever since the early report of Hoagland  (Hoagland et a l . ,  1917) that proteolysis was an important factor contributing to post-mortem changes in muscle, i t has been a t t r a c t i v e to suppose that p r o t e o l y t i c enzymes, possibly cathepsins , were causative agents of many of' the important post-mortem changes observed i n muscle.  It has been observed that certain  areas of the myofibrils such as junction of l i g h t and heavy meromyosin sections of myosin and the tropomyosin-troponin complex are very vulnerable to p r o t e o l y t i c cleavage (Ebashi and Kodama, 1966).  The other s i t e f o r post-mortem proteoly-  s i s of myofibrils i s at or near the Z-line.  Stromer et a l .  (1967a) have shown that trypsin very quickly removes the Z-lines from myofibrils, and they also showed evidence that post-mortem storage causes extensive degradation of the Z-line.  Moreover, both Busch (1969) and Penny (1968) found  that Z-line degradation occurred only during post-mortem storage of i n t a c t muscle, which would contain catheptic enzymes found i n s i t u i n either blood or muscle and not during storage of myofibrils prepared from at-death muscle; such myofibrils would not contain catheptic enzymes.  Henderson  et a l . (1970) also observed degradation of Z-lines i n postmortem muscle.  Reville et al.(1971) reported disruption  of myofibrils accompanied by an increase i n non-sedimentable  cathepsin D a c t i v i t y a f t e r 15 days post-mortem storage. Even though some of the studies have discredited the role of proteolysis i n post-mortem muscle changes (Goll et a l . , 1970; Sharp, 1963; Locker, 1960), the concept of limited proteolysis has been put forward by G o l l et a l . 2+ (1971). They have suggested that uncoupling of the Ca 2+ D u m p by proteolysis may r e s u l t i n loss of Ca accumulating a b i l i t y of sarcoplasmic r e t i c u l a r membranes, causing an 2+ . increase i n free i n t r a c e l l u l a r Ca concentration. An 2+ increased free i n t r a c e l l u l a r Ca concentration causes shortening or isometric tension development i n either l i v i n g or post-mortem muscle. 2+ involving Ca  They also theorized that  causes modification  proteolysis  of actin-myosin i n t e r -  action as well as loss of Z-disk i n t e g r i t y ; these are considered to be possible causes of the resolution of r i g o r mortis (Goll et a l . , 1970). Recently Busch et a l . (1972) i s o l a t e d an endogenous 2+ factor (CASF - Ca -activated sarcoplasmic factor) from 2+ rabbit s k e l e t a l muscle which i n the presence of Ca removal of Z-lines. have been released  They proposed that t h i s factor might from lysosomes and that following removal  of Z-lines, the rest of the myofibrils may be to catheptic enzymes. characterized  causes  susceptible  Recently, Suzuki and G o l l (1974)  CASF as a p r o t e o l y t i c enzyme which, besides  removing Z-lines, s o l u b i l i z e d proteins  from myofibrils.  2 8. 1.2.6.  Lysosomal Concept  In the early 1950s, de Duve and co-workers r e a l i z e d that rat l i v e r acid phosphatase was  associated with a new  class of cytoplasmic granule, the "lysosomes" (Appelmans et a l . . 1955; 1963b).  de Duve et a l . , 1955; de Duve, 1964,  1963a,  The lysosomes have since been shown to contain  over 50 enzymes capable of catabolizing macromolecules of the c e l l .  A p a r t i a l l i s t of the enzymes includes: deoxy-  ribonuclease,  ribonuclease, esterases, lipases , phos^-  phatases, glucoside hydrolases and peptidyl amino acid hydrolases.  Some of these enzymes may  a l l lysosomes.  not be present i n  In general these enzymes are hydrolytic and  have a c i d i c pH optima (Barrett, 1972). Lysosomes are characterized by a general property, the structure linked latency of t h e i r enzymes. was  This latency  considered to be due to the presence of a l i m i t i n g  membrane-like b a r r i e r of l i p i d - p r o t e i n which r e s t r i c t e d the a c c e s s i b i l i t y of the i n t e r n a l hydrolases to any external substrate (Tappel et a l . , 1963).  The examination  of p e l l e t s  of "lysosome-rich" subcellular fractions of rat l i v e r (Novikoff et a l . , 1956)  and of rat kidney (Shibko and Tappel,  1965)  showed they contained a large number of d i s t i n c t p a r t i c l e s , each limited by a single membrane.  Such p a r t i c l e s were  either dense bodies or showed one or more i n t e r n a l c a v i t i e s , sometimes lined with a broad layer of dense material or containing a clump of material.  Lysosomes may  vary i n shape,  but generally they have.a diameter of 0.2 5 to 0.50u™ (Wilson  29. and Morrison, 1966). The major function of lysosomes appears to be the i n t r a c e l l u l a r digestion of p a r t i c l e s ingested into the c e l l by endocytosis (de Duve and Wattiaux, 1966; Strauss, 1964). Localized autolysis associated with lysosomes has also been demonstrated (deDuve and Wattiaux, 1966 ; M i l l e r and Palade, 1964).  S p e c i f i c functions of lysosomes have been documented  in d e t a i l by de Duve and Wattiaux (196 6) and Dingle and F e l l (1969a, 1969b).  1.2.7. Heterogeneity of Lysosomes Lysosomes may d i f f e r quite widely from each other i n a number of properties such as s i z e , structure, enzyme content, density i n various media, and sedimentation coefficient.  Lysosomes from various species were shown to  have lower s p e c i f i c a c t i v i t i e s of 3-galactosidase, a r y l sulfatase, and 3-glucuronidase i n muscle r e l a t i v e to organ tissue (Shibko et a l . , 1963; Shibko and Tappel, 1964).  It  is unlikely that a l l the lysosomal hydrolases are contained within each lysosome since, f o r example, r a t l i v e r lysosomes do not appear to behave as  enzymically homogeneous p a r t i c l e s  (de Duve, 1963). Shibko and Tappel (1964) reported that l i v e r and kidney lysosomes appeared to have the same enzyme complement, but the l i v e r lysosomes sedimented mainly i n the l i g h t mitochondrial f r a c t i o n , while kidney lysosomes sedimented between the nuclear and mitchondrial fractions.  After gradient  30. centrifugation of a crude rat l i v e r lysosomal f r a c t i o n the ratios of three acid hydrolases were found to vary between different fractions of the gradient, i n d i c a t i n g the heterogenity of lysosomal enzyme content (Futai et a l . , 1972). Zonal gradient c e n t r i f ugation of r a t live " lysosomes by -1  Rahman et a l . (19.67) indicated that acid phosphatase and cathepsin C belonged to one group of lysosomes , and acid ribonuclease and cathepsin D to another.  Harikumar et a l .  (1974) found that chicken muscle lysosomes were  relatively  more stable than l i v e r lysosomes under s i m i l a r conditions of incubation and osmotic protection. These observations indicate that lysosomes are heterogenous organelles and that they d i f f e r among and within the  tissues of the same animal.  1.2.8. S t a b i l i t y Stability  of Lysosomes  of isolated lysosomes i s affected considerably  by environmental conditions l i k e osmotic pressure, temperature, pH, ambient ions, and other physical and chemical treatments. Appelmans and de Duve (1955) demonstrated the importance of osmotic protection by a rapid release of acid phosphatase from rat l i v e r lysosomes that were suspended i n d i s t i l l e d o o  water.  Increasing sucrose concentration at 0 -4 C had a  stabilization  effect on lysosomes; maximum s t a b i l i t y  could  be achieved by 0.20 - 0.25M sucrose i n the case of lysosomal suspensions of rat l i v e r and spleen (Appelmans  and de Duve,  31. 19 55;  Gianetto and de Duve, 1955; Rahman, 1963), guinea  pig l i v e r (Turnbull and N e i l , 1969), and muscle (Stagni and de Bernard, 1968). Elevated incubation temperatures decrease the s t a b i l i t y of lysosomes.  Dingle (1961) found that incubation tempera-  tures above 30 C increased the s u s c e p t i b i l i t y of lysosomes 0  to rupture, while t h i s e f f e c t was not prominent when rat l i v e r lysosomes were incubated i n 0.25M sucrose at temperatures over the range of 1°-30°C for 45 minutes; longer incubation times decreased the s t a b i l i t y of lysosomes at temperatures from 5°-30°C (Sawant et a l . , 1964c).  Acid  phosphatase was almost completely released from rat l i v e r lysosomes after two hours at 37°C, but l i t t l e release occurred at 0°C (Rahman, 196 4).  Higher temperature  of 4 5°C caused  comparatively more release of enzyme than at 37°C (Ignarro, 1971).  To study the l a b i l i z a t i o n ,  incubation at 37°C has  been used (Weissmann and Thomas, 1963; Sawant et a l . , 1964a; Balasubramaniam  and Deiss , 1965 ; Bird et a l . , 1968).  Sawant et a l . (1964a) reported an increased  availability  of rat l i v e r lysosomal enzymes i n acid and alkaline pH ranges, with a maximum s t a b i l i t y between pH 6.8 and 7.2. Rat l i v e r lysosomes were found to be l a b i l e at or below pH 5.0 (Appelmans and de Duve, 1955; Dingle, 1961).  Bovine  thyroid lysosomes were most stable between pH 5.1 and 7.3 (Balasubramaniam and Deiss, 1965), rat kidney lysosomes between pH 6.0 and 7.0 (Shibko and Tappel, 1965), and chicken  32 . muscle lysosomes between pH 5.0 and 6.0  (Caldwell and Gros-  jean, 1971). Inorganic s a l t s , depending on i o n i c strength, have been shown to enhance or retard the release of enzymes.  lysosomal  Gianetto and de Duve (1955) reported i s o t o n i c  NaCl at 0 °C was not s u f f i c i e n t to retain the i n t e g r i t y of rat l i v e r lysosomes without the presence of 0.25M sucrose. Ignarro (1971)- reported a decreased in the presence of sodium ion. 2+ was unaffected by ImM by C u  2+  and H g  2+  Ca  lysosomal  stability  Rat l i v e r lysosomal 2+  , s t a b i l i z e d by Zn  (Chvapil et a l . , 1972).  , and  stability labilized  Sawant et a l .  (1964a) reported an increased a v a i l a b i l i t y of a r y l sulfatase 2+ 2+ with addition of 5mM of Ca or Mg , but t h i s e f f e c t was reduced with EDTA. Hayashi et a l . (1973) reported enhanced 2+ proteolytic a c t i v i t y caused by addition of Mg to intact rat l i v e r lysosomes. Increasing concentrations of Na and + 2 + 2 + +  K  had a l a b i l i z i n g effect on lysosomes, but Ca  and  resulted i n a biphasic s o l u b i l i z a t i o n of lysosomal  Mg  hydrolases,  indicating reduced s o l u b i l i t y of enzymes at lower concentration (2: - lOmM) and increased s o l u b i l i z a t i o n at higher concentration (Verity et a l . , 1968). reported a l a b i l i z i n g effect of K  A l l e n and Lee (1972) +  on i s o l a t e d lysosomes.  Release of lysosomal hydrolases by severe mechanical blending i s well known (Gianetto and de Duve, 1955; et a l . , 1971;  Parrish and Bailey, 1967).  Baccino  Freezing and  thawing also released lysosomal enzymes (Gianetto and de Duve,  1955; Sawant et al.,"1964c; Parrish and Bailey, 1967; et a l . , 1971).  Baccino  Nonionic detergents such as Igepal-630 and  Triton X-100 were found to be quite e f f e c t i v e i n l a b i l i z i n g the lysosomal membrane (Rahman, 1963; Stagni and de Bernard, 1968;  Baccino et a l . , 1971). Radiation treatment has been found t o decrease the s t a b i l i t  of lysosomes.  Desai et a l . (1964) reported release of l y -  sosomal hydrolases after exposure to gamma radiation.  Watkins  (1970) reported s o l u b i l i z a t i o n of 3-glucuronidase, acid phosphatase, and N-acetyl-3-glucosaminidase  from rat l i v e r ,  kidney, and spleen lysosomes a f t e r gamma radiation t r e a t ment.  Electron and neutron i r r a d i a t i o n also released lysoso-  mal enzymes from rat spleen lysosomes (Watkins and Deacon, 1973).  Release of lysosomal hydrolases i n various tissues  a f t e r whole body i r r a d i a t i o n has been reported (Warrier et a l . , 1972; Rahman, 1963; Valet and Bauer, 1969;  Kocmierska-  Grodzka and Gerber, 1974 ; Krasnikov et al,. , 1973 ; Roth et a l . , 1962).  However, Sottocasa et a l . (1965) f a i l e d t o find  any release of 8-glucuronidase or 3-galactosidase from the mitochondrial-lysosomal f r a c t i o n of heart a f t e r i r r a d i a tion . treatment. The o r i g i n a l lysosomal concept envisioned simultaneous release of the enzymes when the lysosomes ruptured.  However,  the release of various enzymes i n response to the different l a b i l i z i n g treatments documented above was not always uniform. Weissmann and Thomas (1963) reported a release o f 3-glucuroni-  34. dase to the extent of 7% to 9% , while acid phosphatase increased from 15% to 23% during incubation.  In rat l i v e r ,  a r y l : s u l f a t a s e , acid phosphatase and ribonuclease showed d i f f e r e n t i a l a v a i l a b i l i t y after incubation at different times, pHs, osmotic pressures, and temperatures (Sawant et a l . , 1964a).  Freeze-thawing treatment was e f f e c t i v e i n releasing  a l l the a r y l sulfatase and B-glucuronidase , but acid phosphatase and ribonuclease  remained partly associated with  the membrane (Sawant et a l . , 1964c).  Five enzymes from  beef heart lysosomes were liberated i n different  propor-  tions with various concentrations of sucrose or Triton (Romeo et a l . , 1966). B-glucuronidase,  X-100  A graded release of B-galactosidase,  cathepsin, and ribonuclease from rat and  beef s k e l e t a l muscle lysosomes resulted from increasing Triton X-100  concentrations (Stagni and de Bernard, 1968).  Verity et a l . (1968) found that acid phosphohydrolase, N-acetyl glucosaminidase i n d i v i d u a l l y to mono  and B-glucuronidase  responded  and divalent ion concentrations.  Differences i n s o l u b i l i z a t i o n of the same enzyme from different tissues was  reported by Rahman (1964).  Rat  liver  lysosomes almost completely released acid phosphatase i n two hours at 37°C, but l i t t l e release occurred at 0°C.  Spleen  and thymus lysosomes f a i l e d to release acid phosphatase into solution at either 0°or 37°C.  Watkins (1970) demon-  strated that rate of p o s t - i r r a d i a t i o n release of B-glucuronidase d i f f e r e d among rat kidney, l i v e r , and spleen. D i f f e r e n t i a l release of a r y l sulfatase,B-glucuronidase,  and acid phosphatase from gamma i r r a d i a t e d lysosomes has been reported by Desai et a l . (1964).  A r y l sulfatase and  B-glucuronidase were most readily released followed by acid phosphatase, while ribonuclease showed no release. Watkins (1970) found a two-fold increase i n B-glucosaminidase and B-glucuronidase, but acid phosphatase had an increased soluble a c t i v i t y of only 50% a f t e r a dose of 20 Krads. Watkins and Deacon (1973) found a bi-phasic dose response curve f o r the release of 3-glucosaminidase whereas the curve f o r B-glucuronidase was l i n e a r with the dose; moreover, response to exposure of electron or neutron i r r a d i a t i o n was  different. The release of enzymes from lysosomes r e f l e c t e d both  the membrane character of the organelle and the presence of different enzyme-membrane bonds conferring structurelinked latency upon i n d i v i d u a l lysosomal enzymes (Sawant et a l . , 1964a; Verity et a l . ,  1968).  1.2.9. Ultrastructure of Lysosomes The work of de Duve and his associates (1959, resulted i n the concept of lysosomes as  1963)  membrane-limited  cytoplasmic p a r t i c l e s containing hydrolytic enzymes.  This  concept has been further supported by morphological studies (Essner and Novikoff, 1961; Novikoff, 1963) which have shown that p a r t i c l e s marked by a histochemical acid phosphatase reaction were surrounded by a single outer membrane. Novikoff et a l . (1956) found the a c t i v i t y of acid phosphatase  36 . associated with "dense bodies" 0.2 5  to 0.5um i n diameter  in t h e i r isolated subcellular f r a c t i o n .  Baudhuin et a l .  (1965) observed s i m i l a r "dense bodies" and found good c o r r e l a tion between s p e c i f i c a c t i v i t i e s of acid phosphatase and deoxyribonuclease and frequency of dense bodies i n p a r t i culate fractions from r a t l i v e r .  The lysosomal nature  of these dense bodies i s well established and supported by both biochemical data (Wattiaux et a l . , 1963) and cytochemical staining f o r acid phosphatase at the electron microscopy  l e v e l (Essner and Novikoff, 1961; Trump and  Ericsson , 1964). Lysosomes form an extremely heterogeneous population, at least i n the morphological sense, as a result of t h e i r functional a c t i v i t i e s being responsible f o r the digestion and f o r the storage of material ingested by the c e l l (Daems et a l . , 1972; de Duve, 1963; de Duve and Wattiaux, 1966). However, lysosomes have a single l i m i t i n g membrane which, because of i t s larger dimensions  and i t s asymmetri-  cal structure, often can be distinguished from the l i m i t i n g membranes of other c e l l organelles (Daems et a l . , 1972). Primary lysosomes contain a number of hydrolases not d i r e c t l y observable i n routine preparations, while secondary lysosomes contain, i n addition to t h i s , material previously ingested by the c e l l .  The ingested material may vary widely i n  nature and also can be present i n various stages of digestion (de Duve and Wattiaux, 1966; Daems and Wisse, 1966).  37. A general feature of lysosomes i s electron density of t h e i r matrix, limited by a single membrane (Novikoff et a l . , 1956; Baggiolini et a l . , 1969).  Most of the lysosomes could be  characterized by the presence of an e l e c t r o n - l u c i d rim or h a l o beneath t h e i r l i m i t i n g membrane (Daems, 1966). Electron microscopical methods f o r the demonstration of enzyme a c t i v i t y established the presence of acid phosphatase a c t i v i t y i n s t r u c t u r a l l y characterizable single-membranelimited bodies i n many types of c e l l s (Straus, 1967).  This  group includes c e l l organelles known as multivesicular bodies (Sotelo and Porter, 1959).  Acid phosphatase  activity  has been demonstrated i n such multivesicular bodies i n several types of c e l l s (Novikoff et a l . , 1964; Smith and Farquhar, 1966 ; Bjbrkman and S i b a l i n , 1967 ; Friend and Farquhar, 1967; Holtzman and Dominitz, 1968; Locke and C o l l i n s , 1968), and they are also reported to be involved i n the uptake and digestion of proteins (Becker et a l . , 1967; Smith and Farquhar, 1966).  It was f i r s t suggested by Bennet (1956) that as  a r e s u l t of lysosomal digestion of material ingested by the c e l l , i n d i g e s t i b l e residues remain behind i n the secondary lysosomes.  Since secondary lysosomes can perform  successive acts of digestion (de Duve, 1963; Gordon et a l . , 1965; de Duve and Wattiaux, 1966), they show accumulation of residues.  Secondary lysosomes, having reached;the stage  of being thus f i l l e d with the residues of digestion, develop into what de Duve and Wattiaux (1966) suggested to be c a l l e d  38. telolysosomes.  Residue-loaded bodies have been character-  ized by the abundant presence of myelin figures, electron dense pigments, and droplets having a l i p i d nature and do not show c h a r a c t e r i s t i c a l l y high enzymatic a c t i v i t i e s on the basis of t h e i r lysosomal o r i g i n (Beck et a l . , 1972: de Duve and Wattiaux, 1966; Smith and Farquhar. 1966;  Beaulton,  1967 ; Parker et a l . , 1965 ; Lane and Novikoff, 1965 ;. M i l l e r and Palade, 1964; Frank and Christensen, 1968; Essner and Novikoff, 1961; Fischer et a l . , 1966).  These telolysosomes  s t i l l contain the lysosomal enzymes; these enzymes are not renewed and are destroyed, resulting i n a functionally and enzymatically inactive structure consisting s o l e l y of digestive residue.  For such bodies, the terms post-lysosome  or residual bodies, have been preferred by de Duve and Wattiaux  (1966).  Unfortunately, as yet knowledge of i n t e r n a l structure of lysosomes  i s rather limited and quite i n s u f f i c i e n t to  explain many of the known metabolic features; however, Maunsbach (1969) has l i s t e d some s t r u c t u r a l features and functional equivalents of lysosomes c e l l s of the rat kidney.  in the proximal tubule  Although s p a t i a l d i s t r i b u t i o n  within the organelle must await further information as to internal structure of lysosomes, t h e i r content of hydrolases capable of s p l i t t i n g various types of peptide bonds i s well established (Barret and Dingle, 1971; Tappel, 1969; 1972).  Barrett,  Structure-linked latency of the lysosomal enzymes  is a well-known phenomenon.  Two mechanisms have been proposed  39. to account f o r the latency of these enzymes, the membrane theory and the matrix binding theory.  The membrane theory  has been suggested by de Duve and associates (de Duve et a l . , 195 5; de Duve, 1959, 196 3) and proposes that a lysosome is a simple osmotic sac which i s delimited by a lipoprotein membrane.  The impervious membrane serves as a physical  barrier to r e s t r i c t the physical freedom and substrate a c c e s s i b i l i t y of the enclosed enzymes , which are thought to be present within the intact lysosomes i n a diffusable f u l l y active form (de Duve, 1963).  Koenig (1962, 1964a) ad-  vanced the matrix binding theory as a membrane-limited polyanionic lipoprotein granule, containing hydrolytic enzymes in an inert state bound to lipoprotein matrix.  Cytochemical  observations revealed the acid phosphatase reaction product in electron micrographs  associated with lysosomal matrix,  but no enzyme product was found i n the normally occurring electron-lucent vacuoles of neuronal lysosomes-(Koenig, Kreutzberg and Hager, 1966).  1968a;  6-glucuronidase reaction  product seemed to be absent from electron-lucent vacuoles of l i v e r lysosomes (Hayashi et a l . , 1968). Electron microscopic studies have shown that the f i r s t u l t r a s t r u c t u r a l change in lysosomes during d i l u t i o n of the medium was a swelling and increased electron-lucency of the matrix; subsequently, lysosomes rupture i n increasing numbers concomitantly with release of acid hydrolases (Koenig, 196 7a, 1967b).  Shibko et a l . (1965) observed that i n i t i a l l y r a t  kidney lysosomes were electron dense and had an intact  40. membrane.  After three hours of incubation at 37°C, when  most of the enzyme was s o l u b i l i z e d , the membranes were i n t a c t , although the lysosomes l o s t t h e i r matrix completely. They concluded that release of acid phosphatase had occurred in two stages: f i r s t l y , the enzyme became available to the substrate but remained sedimentable; l a t e r , the quantities of soluble enzymes approximately equalled the t o t a l amount of available enzyme.  Small membranous vesicles inside the  lysosome "ghosts" could then be observed, and Shibko et a l . (1965) concluded that these represented the remains of an internal membrane structure of unknown function.  Electron  micrographs of lysosomes that were repeatedly frozen and thawed revealed that complete disruption of p a r t i c l e s occurred. Twenty per cent of the enzyme, a c t i v i t y remained with the membrane f r a c t i o n and i t was presumed by these workers that lysosomal enzymes were n o n - s p e c i f i c a l l y adsorbed or mechanic a l l y trapped with t h i s f r a c t i o n .  Lucy (1969) suggested  that association of enzyme a c t i v i t y with membrane f r a c t i o n was due to membrane bound enzymes.  Beck and Tappel (196 8)  have studied rat l i v e r 3-glucosidase and found that the enzyme remained firmly bound to the membrane a f t e r p a r t i c l e s were ruptured.  It i s known that many acid hydrolases remain  bound to some extent to the insoluble f r a c t i o n ; part of t h i s binding may be non-specific adsorption, but i t i s d i f f i c u l t to rule out a genuine appertenance of some hydrolase acti v i t i e s to the lysosomal membrane or matrix (Thines-Sempoux, 1973).  Experimental evidence based mostly on biochemical studies has accumulated  to indicate that lysosomes can be  l a b i l i z e d by a great number of substances and (Weissman, 1969 ; Whiting, 1974; of Lysosomes).  i  n  see also section on S t a b i l i t y  most of the studies of a biochemical  nature, measurement of s h i f t s between "free", and "unsedimentable" performed  treatments  "sedimentable",  a c t i v i t i e s of lysosomal enzymes was  on the lysosome-rich f r a c t i o n s .  In investigations  of this type, the leakage of hydrolytic enzymes from lysosomes has been demonstrated, but changes occurring at the u l t r a structural l e v e l have not been elucidated.  Brunk and E r i c s -  son (1972) observed leakage of acid phosphatase from struct u r a l l y intact lysosomes while there was no leakage of thorium dioxide p a r t i c l e s , concluding that the enzyme could diffuse through the lysosomal membrane when there were no large holes or ruptures i n the membrane.  Lodin et a l . (1974)  observed that at a terminal stage of degeneration, lysosomes lost t h e i r simple membranes and dense granular content was d i f f u s e l y spread i n cytoplasm of neurons and g l i a l c e l l s in v i t r o under malnutritional stress.  They also noticed  the disappearance of cytoplasmic p a r t i c l e s and the appearance of empty spaces. High doses of whole body or p a r t i a l body radiation has been reported to produce changes i n neuron lysosomes (Kagan et a l . , 1962; Pick, 1965; Roizin et a l . , 1966)  and  r e d i s t r i b u t i o n of lysosomal acid phosphatase (Greenspan et a l . , 1964).  Changes i n s p e c i f i c a c t i v i t y of lysosomal enzymes  42. and i n the number or i n t r a c e l l u l a r d i s t r i b u t i o n of cytochemical demonstrable lysosomes have been observed i n a number of i r r a d i a t e d tissues, both normal (Horvath and Touster, 196 7; Hugon and Borgers, 1966) and malignant (Carney, 1965; Leener and Evans, 1969).  Rahman (1962) observed an  increase i n s p e c i f i c a c t i v i t i e s of acid phosphatase and B-glucuronidase i n the lysosome-rich f r a c t i o n of rat thymus after whole body X-radiation; moreover, some of the lysosomes showed an i n t e r n a l cavity. Disruption of the lysosome-rich granule f r a c t i o n has been reported (Harris, 1966a, 1966b).  Structural changes  or altered permeability of lysosomal membrane have been suggested as the cause of enzyme leakage from lysosomes a f t e r radiation (Aikman and W i l l s , 1974a, 1974b; Wills and Wilkinson, 1966; Desai et a l . , 1964; Watkins, 1970; H a r r i s , 1970), or due to other treatments (see section on S t a b i l i t y of Lysosomes).  Most of these studies are based on biochemical  and/or cytochemical data, but detailed u l t r a s t r u c t u r a l studies have not been reported.  1.2.10. UItrastrueture of Muscle Transmission electron microscopy has been used to • study the u l t r a s t r u c t u r a l details of s t r i a t e d s k e l e t a l muscle (Bendall, 1969; Slautterback, 1966).  The presence  of i n t e r d i g i t a t i n g filaments of actin and myosin are revealed i n electron micrographs of longitudinal sections. The hexagonal arrangement of actin filaments around the  43.  centrally located myosin can be seen i n cross-sectioned fibres (Huxley, 1965). During post-mortem storage of muscle, u l t r a s t r u c t u r a l changes have been observed.  Storage of muscle at low tem-  peratures has been shown to cause cold shortening of the muscle during the onset of r i g o r (Bate-Smith and Bendall, 1949; Marsh, 1953).  Stromer and G o l l (1967) reported a  decrease i n the length of I bands, but some lengthening of sarcomeres i n beef f i b r i l s was observed between 24 and 312 hours post-mortem at 2°C.  Some studies have indicated  that during aging of beef and chicken muscle, the Z-line disappeared and the actin filaments were weakened at the Z-I junction (Davey and G i l b e r t , 1967; Fukazawa et a l . , 1969).  I t has been observed that treatment of myofibrils  with trypsin results i n a rapid loss of Z-lines (Goll et a l . , 1969; Stromer et a l . , 1967).  In addition to Z-line removal,  structural degradation of Z-lines i s also observed during post-mortem storage of muscle (Davey and G i l b e r t , 196 7, 1969; Fukazawa and Yausi, 1967; Henderson et a l . , 1970; Stromer et a l . , 1967).  Post-mortem degradation of Z-lines has been  most extensively studied by Henderson et a l . (1970), who found that Z-lines i n rabbit and porcine muscle were more susceptible to post-mortem degradation than Z-lines of bovine muscle.  Busch et a l . (1972) i s o l a t e d a protein  fraction from sarcoplasm of rabbit s k e l e t a l muscle which 2+ caused removal of Z-lines i n the presence of Ca  , but had  no u l t r a s t r u c t u r a l l y detectable effect on the m y o f i b r i l .  44. Considerable attention has been given to transmission electron microscopy of sarcoplasmic reticulum, a complex network of vesicles on surfaces of f i b r i l s (Peachy, 1970). The reticulum i s considered to be capable of c o n t r o l l i n g the calcium ion concentration i n the sarcoplasm.  It has  been shown that release of calcium ions from the reticulum causes muscle contraction (Ebashi, 1961; Weber et a l . , 196 3; Ohnishi and Ebashi, 1964:  Schmidt et a l . , 1970).  It i s  known that sarcoplasmic r e t i c u l a r membranes lose t h e i r 2+ a b i l i t y to accumulate Ca during post-mortem storage of muscle (Greaser et a l . , 1967; Uauss and Davies, 1966), thus Busch et a l . (1972) suggested that such a release of bound 2+ Ca  might activate the "sarcoplasmic f a c t o r " , r e s u l t i n g  in Z-line degradation  observed during post-mortem storage  of muscle (Henderson et a l . , 1970).  Greaser et a l . (1969)  pointed out that s t r u c t u r a l features of mitochondria in porcine muscle were changed during a 2 4 hour aging period, but no structural alterations were observed i n sarcoplasmic 2+ reticulum. accumulating  West et a l . (1974) observed a loss of Ca a b i l i t y i n sarcoplasmic reticulum following  degradation by trypsin, crude cathepsin f r a c t i o n , and cathepsin Bl . Scanning electron microscopy has proven to be a v a l uable tool for evaluating topographical details of a variety of b i o l o g i c a l systems.  Schaller and Powrie (1971) studied  the surface ultrastructure of cryofractured s k e l e t a l muscle  from rainbow t r o u t , turkey, and beef at pre-rigor and various post-rigor times.  In pre-rigor samples they found elevated  transverse elements, presumably a part of sarcoplasmic  reti-  culum which collapsed a f t e r storage, and breaks across the f i b r i l occurred at the positions of transverse elements.  They  also observed perforation of the sarcolemma a f t e r post-mortem storage of muscle.  Eino and Stanley (197 3a) studied surface  ultrastructure of post-mortem beef by scanning electron microscopy, and observed a depression of transverse s t r i a t i o n s followed by extensive fibre breakage during two week storage. Cathepsin treatment of muscle fibres has been reported to cause disappearance of transverse s t r i a t i o n s , multiple breakage , and some disruption of fibres (Eino and Stanley, 1973b). These authors also reported that collagenase treatment brought about shredding and d i s s o l u t i o n of collagen fibres , followed by d i s i n t e g r a t i o n of sarcolemma, but had l i t t l e effect on m y o f i b r i l s .  CHAPTER 2 .  EFFECT OF GAMMA RADIATION ON RELEASE OF CATHEPSINS FROM LYSOSOMES  2.1. INTRODUCTION The existence of p r o t e o l y t i c enzymes of lysosomal origin i n various s k e l e t a l muscle tissues i s well established. The i n t r a c e l l u l a r enzymes responsible f o r the a c t i v i t y i n the tissues have been named cathepsins Bamann (1929).  by W i l l s t a t t e r and  There are several studies i n d i c a t i n g that  lysosomal cathepsins  are capable of i n t r a c e l l u l a r protein  degradation (Bohley et a l . , 1971; de Duve, 1963a; R e v i l l e et a l . , 1971).  Isolated lysosomal proteases have been shown  to degrade large proteins to free amino acids or small peptides (Coffey and de Duve, 1968; Huisman et a l . , 1974). The role of catheptic enzymes i n post-mortem proteolys i s and meat tenderization i s not c l e a r , but there are several reports which suggest release of cathepsins lysosomal hydrolases  during aging  and other  (Dutson and Lawrie, 1974;  Eino and Stanley, 1973a; Lutalo-Bosa, 1970; Ono, 1971). P r o l e o l y t i c enzymes have been held responsible f o r agingtenderization of meats (Zender et a l . , 1958; Davey and G i l b e r t , 196 6) and f o r the proteolysis i n post-mortem muscle (Sharp, 1963; Khan and van de Berg, 1964a, 1964b; Suzuki et a l . , 1967; Parrish et al.,1969; Okitani and Fujimaki, 1972; Okitani et a l . , 1973). Protein breakdown by lysosomal extracts and proteolysis i n tissue i s considered  to be due to the involvement  of various cathepsins as well as other lysosomal hydrolases. Caldwell (1970) suggested j o i n t action of several muscle cathepsins on endogenous muscle proteins.  Liao-Haung and  Tappel (1971) observed that degradation of hemoglobin was i n i t i a t e d by cathepsin D, and products were further u t i l i z e d by cathepsin C.  Goettlich-Riemann  et a l . (1971) demonstrated  a synergistic and concerted action of cathepsins A, B, and D. Similar results have been reported f o r cathepsins A and D (Iodice et a l . , 1966).  Lazarus (1973) suggested that probably  collagenase and the lysosomal protease such as cathepsin D (Dingle et a l . , 1971) work s y n e r g i s t i c a l l y i n connective tissue degradation. The lysosome i s a membrane-limited subcellular organelle containing cathepsins and other hydrolytic enzymes capable of i n t r a c e l l u l a r degradation.  While these enzymes remain  within an intact lysosome they are inactive on external substrates (de Duve, 1963a).  For the cathepsins to have  a role i n the post-mortem modification of muscle, they must be released from the lysosome. Cathepsins have been thought to cause the proteolysis observed during extended storage of i r r a d i a t i o n - s t e r i l i z e d meats (Cain et a l . , 1958 ; Pearson et a l . , 1960 ; Coleby et a l . , 1961; Bailey and Rhodes, 19 6*+) .  Studies of Klein and Altman  (1972b) showed an increase i n free c a t h e p t i c . a c t i v i t y i n chicken s k e l e t a l muscle a f t e r i r r a d i a t i o n treatment.  These  observations indicate possible release of cathepsins by radiation-induced f r a g i l i t y of tissue lysosomes.  However,  48. i t i s d i f f i c u l t to determine the extent of lysosomal damage and release of enzymes i n intact tissue.  Isolated  lysosomes  o f f e r a good system f o r such studies, but i t i s d i f f i c u l t to obtain enough intact lysosomes from s k e l e t a l muscle due to few numbers of lysosomes present and the vigorous disruptive treatment needed to extract lysosomes from muscle. For these reasons, most of the studies have been conducted on i s o l a t e d lysosomal fractions obtained from selected organ tissues. The release of various enzymes from i s o l a t e d lysosomes i n response to the conditions l i k e pH, osmotic strength, temperature, and other physiochemical treatments i s not uniform (Sawant et a l . , 1964a; Verity et a l . , 1968; Stagni and de Bernard, 1968).  Gamma radiation has been shown to  result i n d i f f e r e n t i a l release of acid phosphatase, B-glucuronidase and a r y l sulphatase, but had no e f f e c t on ribonuclease from rat l i v e r lysosomes (Desai et a l . , 1964).  Similar  d i f f e r e n t i a l release of B-glucuronidase, B-glucosaminidase, and acid phosphatase due to i o n i z i n g i r r a d i a t i o n treatment has been reported by other workers (Watkins, 1970; and Deacon, 1973).  Watkins  Sottocassa et a l . (1965), however,  f a i l e d to find any effect of X - i r r a d i a t i o n on release of lysosomal B-glucuronidase or B-galactosidase from the mitochondrial-lysosomal fraction of heart t i s s u e .  Large variations  in radiation s e n s i t i v i t y of various lysosomal enzymes from d i f f e r e n t tissues as well as under d i f f e r e n t conditions have been encountered i n the l i t e r a t u r e , which makes i t  d i f f i c u l t to draw general conclusions regarding enzymes, especially cathepsins.  lysosomal  In the present study,  release of cathepsins A, B, C, and D from i s o l a t e d chicken l i v e r lysosomes has been investigated.  50. 2.2. EXPERIMENTAL 2.2.1. Materials Commercial broiler-type chickens (8 - 12 weeks old) were obtained from the University Poultry Farm, and maintained under standard husbandry conditions f o r about a week p r i o r to slaughter.  The birds were s a c r i f i c e d by  exsanguination a f t e r fasting f o r 40 - 48 hours.  The l i v e r s  were rapidly removed and washed free of blood with cold 0.2 5M sucrose solution and c h i l l e d i n crushed i c e . Excessive f a t and connective'tissue were removed.  A l l the  subsequent operations were carried out at 4°C unless otherwise indicated.  2.2.2. General Sample Preparation 2.2.2.1. Chicken l i v e r tissue Chicken l i v e r t i s s u e , cooled i n crushed i c e , was mince into small s l i c e s with scissors p r i o r to i r r a d i a t i o n . portion was kept as a non-irradiated control.  One  Immediately  after i r r a d i a t i o n , the samples were homogenized i n d i s t i l l e d water 1:20 (w/v), using a S o r v a l l Omnimixer.  The homo-  genization was done at high speed f o r four 15 second bursts with 30 second cooling intervals between bursts.  During  homogenization, the mixer container was kept immersed i n a crushed i c e slurry.  The tissue homogenate was centrifuged  at 2 0,2 00 x g at 4°C f o r 3 0 minutes i n a S o r v a l l RC2-B r e frigerated centrifuge.  The supernatant  was used f o r deter-  51. mination of free cathepsin D a c t i v i t y and expressed as per cent of non-irradiated control values. 2.2.2.2. Isolation of lysosomes. Deionized water and a n a l y t i c a l grade sucrose were used for preparation of sucrose solutions.  The solutions con-  taining 0.001M tetrasodium s a l t o f EDTA (Ethylenediaminet e t r a acetic acid) were adjusted t o pH 7.0.  The l i v e r s  were minced and homogenized i n 0.25M sucrose (1:8, W/V). Homogenization was f o r 3 0 seconds i n a Waring blender at top speed.  The pH of the homogenate was adjusted t o 7.2  with 5 N KOH. The homogenate was f i l t e r e d through four layers of cheese cloth and then fractionated by sucrose density gradient and d i f f e r e n t i a l centrifugation technique according to the scheme outlined i n Figure 1, using S o r v a l l RC2-B refrigerated centrifuge (Sawant et a l . , 1964c). The p u r i f i e d lysosomal fraction (F IV) was suspended i n 0.7M sucrose solution and a 1:2 (w/y) d i l u t i o n was made on the basis of l i v e r tissue used f o r i s o l a t i o n of lysosomes. This f i n a l suspension was i r r a d i a t e d as described i n the i r r a d i a t i o n section.  2.2.3. Irradiation The samples were subjected to varying doses of gamma radiation i n a Gamma Cell-220 (Atomic Energy of Canada Ltd.). During i r r a d i a t i o n , the samples were kept i n crushed i c e which was changed p e r i o d i c a l l y when necessary.  The chicken  Figure 1.  Scheme f o r the preparation of lysosomes  Homogenate 1. Centrifuged at 750 x g f o r 10 min. 2. Centrifuged at 3,300 x g f o r 10 min. I  "  P e l l e t (discard)  .  —  1  :  -I  ,  Supernatant Centrifuged at 16,300 x g f o r 20 min.  =  '  •  Pellet Light mitochondrial fraction (F I)  r Supernatant  (discard)  Resuspended c a r e f u l l y i n 0.3M sucrose and centrifuged at 9,500 x g f o r 10 min. '  r-  :  Pellet Light mitochondrial fraction (F II)  -  i  Supernatant  (discard)  Resuspended i n 0.4 5M sucrose and layered over a discontinuous gradient of 0.7M sucrose (bottom layer) and 0.6M sucrose (middle l a y e r ) , centrifuged at 9,500 x g f o r 30 min. r—  1  :  .  P e l l e t (F III)  Supernatant (discard) Resuspended i n 0.7M sucrose, centrifuged at 5,900 x g f o r 30 min.  i  :  Pellet (discard)  V Supernatant c a r e f u l l y decanted, centrifuged at 17,000 x g f o r 20 min. P e l l e t washed again and centrifuged to y i e l d p u r i f i e d lysosomes (F IV) 1  :  53. l i v e r tissue s l i c e s were subjected to radiation doses of 0, 0.05, 0.10, 0.25, 0.50, or 1.00 Mrad.  The lysosomal  suspensions received radiation doses of 0, 0.1, 0.25, 0.50, or 1.00 Mrad.  The dose rate at the time of i r r a d i a t i o n GO  was 0.52 - 0.71 Mrad/nr., using ' Co as radiation source. 2.2.4. Light Scattering Properties of Lysosomes After i r r a d i a t i o n , the lysosomal suspensions were incubated at 37°C i n a constant temperature water bath. The absorbance of the suspensions was read at 540 nm a f t e r 0, 15, 30, 60, 120, and 180 minutes of incubation using a Spectronic 20 spectrophotometer (Bausch and Lomb).  The  instrument was set at zero by using i r r a d i a t e d 0.7M sucrose solutions.  The decrease i n absorbance was used as an index  of release of lysosomal enzymes (Sawant et a l . ,  1964c).  2.2.5. Release of Cathepsins from Lysosomes After i r r a d i a t i o n , the release of catheptic enzymes from lysosomal suspensions was followed at two different temperatures.  One portion of. the suspension was incubated  at 37°C i n a constant temperature water bath and samples were drawn after 0, 15, 30, 60, 120, and 180 minutes.  The  other portion of lysosomal suspension was kept at 4°C and samples were drawn after 0, 24, 48, and 72 hours.  To  determine free catheptic a c t i v i t y as a measure of released enzyme, the samples were centrifuged at 17,000 x g f o r  2 0 minutes and catheptic a c t i v i t y of the supernatant measured.  For determination of t o t a l catheptic a c t i v i t y of l y -  sosomes, samples were given freeze-thaw treatment ten times and then centrifuged at 17,000 x g for 20 minutes and the supernatant used f o r determination of t o t a l catheptic activity.  In some cases Triton X-100  lysosomes  ( f i n a l concentration 0.2%).  was used to disrupt  2.2.6. Enzyme Assays 2.2.6.1. Cathepsin A. Cathepsin A a c t i v i t y was determined according to the procedure of Iodice et a l . (1966) , using N-carbobenzoxyct-L-glutamyl-L-phenylalanine (CBZ-glu-phe ; Sigma Chemical Co.) as substrate.  The reaction mixture (1 ml), containing  0.0152M substrate, 0.04-M sodium acetate buffer pH 5, and enzyme, was incubated at 37°C f o r 2 hours. was stopped by addition of 1 ml of 10% TCA.  The reaction The TCA mix-  tures were heated f o r 10 minutes i n a water bath at 50° - 55°C and centrifuged for 15 minutes at low speed; 0.1 - 0.2  ml  aliquotes were used f o r color development by the ninhydrin procedure of Moore and Stein (1954).  Controls without  substrate were treated i n the same manner. 2.2.6.2. Cathepsins B and C. Cathepsins B and C were measured with benzyol-L-arginine amide and glycyl-L-phenylalanine amide (Sigma Chemical Co.), respectively.  The assays were performed at 37°C f o r  2 hours i n 1 ml reaction mixtures containing 0.01M  substrate,  0.1M  sodium c i t r a t e buffer, pH 5.0, 0.04M cysteine (cys-  teine-HCl) and enzyme (Bowers et a l . , 1967).  The reaction  was stopped by addition of 1.0 ml of 5% TCA.  The hydroly-  t i c products were measured by microdiffusion technique (Seligson and Seligson, 1951).  One ml of saturated potassium  carbonate was added and liberated ammonia was absorbed i n H S0 2  by rotating the v i a l s on a rotator f o r 30 minutes and  4  determined spectrophotometrically by the Nesslerization technique. 2.2.6.3. Cathepsin D. Cathepsin D a c t i v i t y of tissue and of lysosomal preparations was determined according to the method of Anson (1938) with some modifications (Berman, 1967). The reaction mixture contained 1 ml of supernatant and 2 ml of 2% hemoglobin (Bovine, Type II - Sigma"Chemical Co.) i n 0.2M  acetate buffer pH 3.8.  The reaction was  carried  out at 37°C f o r 2 hours i n a constant.temperature water bath with shaking and the reaction was terminated by addition of 2 ml of 10% TCA.  The samples were kept overnight at U°C  and then f i l t e r e d through Whatman No. 4 f i l t e r paper.  The  blanks were also prepared i n a similar manner, but were kept at 4°C instead of incubating at 37°C.  The  absorbance  of the f i l t r a t e was read against respective blanks at 2 80 nm on a Unicam SP 800B.U.V. spectrophotometer Ltd.).  (Pye-Unicam  The increase i n absorbance was expressed as enzyme  activity.  A l l measurements were performed i n duplicate.  2.3. RESULTS AND DISCUSSION 2.3.1. Proteolytic  A c t i v i t y of Tissue as Affected by Gamma  Radiation To evaluate the effect of i r r a d i a t i o n on catheptic enzymes and t h e i r release from lysosomes, chicken l i v e r was selected as the source of lysosomes because s k e l e t a l  muscle  contains a r e l a t i v e l y low lysosomal content as well as low catheptic a c t i v i t y .  In l i v e r tissue, a low dose of i r r a d i a  tion (50 and 100 Krad) significantly  enhanced the catheptic a c t i v i t y  (P<0.05) as shown i n Table 1, but higher  doses (0.25, 0.5, and 1.00 Mrad) resulted i n r e l a t i v e l y smaller increases (Table 2).  Table 1. Effect of low doses o f gamma radiation on free catheptic a c t i v i t y of chicken l i v e r tissue. Dose (Krad)  A c t i v i t y (%)  Control 50 100 Means with different (P<0.05). n =4  l e t t e r s are s i g n i f i c a n t l y  different  57. Table 2. E f f e c t of high doses of gamma radiation on free catheptic a c t i v i t y of chicken l i v e r tissue. Dose (Mrad)  A c t i v i t y (%)  Control  100.0  a  0.25  113.2  b  0.50  112.4  b  1.00  111.9  b  Means with different l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). n =5  Higher doses of i r r a d i a t i o n might have resulted i n p a r t i a l inactivation of enzymes or radiation denaturation of the tissue proteins, thus decreasing extraction of enzymes, resulting i n a s l i g h t drop i n free enzyme a c t i v i t y 1 and 2).  (Tables  These results are. i n agreement with the work o f  Coelho (1969), who noticed an increase i n ATPase a c t i v i t y at lower doses and progressive i n h i b i t i o n at higher doses of gamma radiation.  A s i m i l a r pattern f o r ATPase was reported  by Szabolcs et a l . (1964) upon X-radiation of myosin A. Other studies have indicated extensive protein alterations in i r r a d i a t e d animal tissue (Cain et a l . , 1958; Bautista et a l . , 1961).  Klein and Altman (1972b) observed  an increased  p r o t e o l y t i c a c t i v i t y i n chicken leg and breast muscle at low i r r a d i a t i o n doses up to 0.2 Mrad, but higher doses inhibited enzyme a c t i v i t y ; they also observed  changes i n  58. soluble protein fractions a f t e r incubation using disc e l e c trophoresis.  Some studies have indicated that i r r a d i a t i o n  has fragmentary action on tissue proteins (El-Badawi et a l . , 1964); such changes would render the tissue proteins more susceptible to enzyme attack. tidase, may  Cathepsin D, an  endopep-  have a l i m i t e d contribution to such changes,  but other enzymes l i k e cathepsins  A, B, and C might be  involved i n degradation of fragmented proteins. In studies with f i s h muscle, Musch (1969) reported no increase i n catheptic a c t i v i t y , but noticed that cathepsins were highly radiation resistant and required very high doses of 10 - 2 0 Mrad f o r even p a r t i a l i n a c t i v a t i o n . I t i s l i k e l y that small changes i n enzyme a c t i v i t y were not detectable i n the above studies, as muscle was  used as an  enzyme source, and i t contains a very low l e v e l of catheptic activity.  Secondly, small increases i n enzyme a c t i v i t y  probably would be cancelled out due to p a r t i a l i n a c t i v a t i o n of enzymes a f t e r i r r a d i a t i o n . extent, why  This may  explain, to some  Doty and Wachter (1955) found l i t t l e or no  de-  crease i n p r o t e o l y t i c a c t i v i t y of beef muscle a f t e r 500,000 rep gamma radiation. Various explanations  have been given f o r an i r r a d i a t i o n -  induced increase i n enzyme a c t i v i t y .  Okada and Peachy  (1957) supported the view that desorption of enzymes from c e l l organelles plays an important r o l e , but i t can not explain the general increase i n a c t i v i t y observed when the  59. organelles are completely  destroyed.  that i r r a d i a t i o n treatment may  Kuzin (1964) suggested  decrease the enzyme i n h i b i -  tors e i t h e r by disruption of t h e i r synthesis or by a change in permeability of the c e l l enhancing removal of i n h i b i t o r s from tissue.  According to Pauly and Rajewsky (1956), an  a l t e r a t i o n i n permeability of c e l l membranes i s another p o s s i b i l i t y related to enzyme increase.  Roth et a l . (1962),  as well as Rahman (1962), suggested that an increase i n s p e c i f i c a c t i v i t y of acid phosphatase was  due to s e l e c t i v e  retention of enzymes during loss of tissue nitrogen.  In  the present study, a l t e r a t i o n i n c e l l metabolism cannot be a cause of the increased enzyme a c t i v i t y as post-mortem l i v e r tissue was  used; also, c e l l organelles were disrupted,  and there would be no synthesis of i n h i b i t o r s .  I f changes  in c e l l permeability enhance the removal of i n h i b i t o r s from tissue, they would be extracted easily and would have depressed the catheptic a c t i v i t y of tissue supernatant.  Loss  of tissue nitrogen as a possible reason for the increase in the s p e c i f i c a c t i v i t y of tissue enzymes, as suggested by Rahman (196 2) and Roth et a l . (1962) would not affect the results of the present study as the results are not based on nitrogen content of t i s s u e .  Changes i n c e l l per-  meability and desorption of the enzyme seem to be l o g i c a l reasons for an increase i n the e x t r a c t a b i l i t y of enzymes from tissue and hence an increase of free enzyme a c t i v i t y as observed i n this study.  60. It was obvious from these results that the complexity of the tissue system prevented d e f i n i t i v e conclusions from being e a s i l y drawn.  This complexity necessitated the use  of r e l a t i v e l y simpler and well-defined systems.  For this  purpose, i s o l a t e d chicken l i v e r lysosomes were used i n subsequent studies.  2• •2. Light Scattering Properties of Lysosomal Suspension 3  Light scattering has been used as an index of change in the shape of p a r t i c l e s and the i n t e g r i t y of lysosomes under various pH and temperature conditions (Sawant et a l . , 1964c) or i r r a d i a t i o n treatment (Harris, 1966a, 1966b). Badenock-Jones  and Baum (1974) have recently shown that  l y s i s of rat kidney lysosomes resulted i n a decrease i n the AJ_2Q  of suspensions, and this decrease i n absorbance paral-  l e l l e d quantitatively and temporally the release of soluble acid phosphatase.  The authors concluded that measurement  of changes i n absorbance i s a v a l i d method to measure the changes i n lysosomal i n t e g r i t y . In the present study, i r r a d i a t i o n of lysosomal suspensions caused a s i g n i f i c a n t (P<0.01) change i n absorbance at 540 nm (Figure 2).  There was a sharp decrease i n absor-  bance during i n i t i a l 1 5 - 3 0  minutes incubation at 37°C i n  a l l the i r r a d i a t e d and control samples, although the decrease due to i r r a d i a t i o n treatment was s i g n i f i c a n t l y (P<0.05) greater than the non-irradiated controls up to 6 0 minutes of incubation.  Upon longer incubation, there was a slow  decrease i n absorbance, and doses up t o 0.50 Mrad s t i l l had s i g n i f i c a n t l y (P<0.05) lower absorbance than the control, while 1.0 Mrad treatment did not produce any further decrease i n absorbance a f t e r 60 minures of incubation.  An  aggregation of the p a r t i c l e s was observed i n 1.0 Mrad t r e a t ment a f t e r 12 0 minutes of incubation, which might have been responsible f o r higher absorbance. The correct interpretation of the decrease i n absorbance of lysosomal suspensions a f t e r exposure to gamma radi a t i o n i s not certain.  I t has been observed that a suspen-  sion of sarcosomes (Carney, 1965) and thymocytes  (Myers  and DeWolf-Slade, 1964) showed decreased t u r b i d i t y as they swelled following i r r a d i a t i o n .  Mitochondria have a higher  capacity for swelling and contraction accompanied bance change (Packer et a l . , 1968).  by absor-  I t i s uncertain whether  such an explanation would be applicable to i r r a d i a t e d lysosomes.  Lysosomal suspensions have been reported to exhibit  decreased absorbance after treatment with various agents such as acid (Cohn and Hirsch, 1960), Triton X-100 (BadenockJones and Baum, 1974), neutral steroids (Weissman, 1965; Badenock-Jones  and Baum, 1974), u l t r a v i o l e t radiation (Cohn  and Hirsch, 1960), and gamma radiation (Harris, 1966a). Concerning the decrease i n the absorbance of lysosomes, the present study agrees with the report of Harris (1966a, 1966b), indicating that radiation damages the lysosomes, perhaps causing alterations i n lysosomal membrane, r e s u l t i n g  62.  F i g u r e 2.  E f f e c t o f gamma r a d i a t i o n on l i g h t s c a t t e r i n g p r o p e r t i e s o f lysosomes. (n = 6)  63. in permeability changes, and subsequently causing disruption of lysosomes.  Such radiation effects seem to be amplified  by elevated temperatures  (Harris, 1966a).  A decrease i n the  absorbance of lysosomal suspensions has been accompanied by an increase i n free a c t i v i t y of various lysosomal enzymes, which supports the concept of irradiation-induced damage to lysosomal structure. i r r a d i a t e d lysosomes  Release of lysosomal enzymes from  i s discussed in the following sections  2.3.3. Release of Lysosomal  Cathepsins  It i s well established that lysosomes  are membrane-  limited p a r t i c l e s containing various hydrolytic enzymes. One important group of these i s tissue proteases, commonly termed, as cathepsins.  These enzymes are considered to  be responsible f o r protein breakdown i n tissue.  Cathepsin D  is an endopeptidase which hydrolyses only proteins , while cathepsins A, B, and C are exopeptidases and degrade peptides, but concerted action of cathepsins A, B, and C along with cathepsin D on proteins has been demonstrated  i n several  studies (Iodice et a l . , 1966; Goettlich-Riemann et a l . , 1971; Liao-Huang and Tappel, 1971).  Isolated lysosomal  proteases are capable of degrading large proteins to free amino acids or small peptides (Coffey and de Duve, 196 8; Huisman et a l . , 1974). has been demonstrated  Intralysosomal protein degradation by studies of Neely and Mortimore  (1974), Brostrom and Jeffay (1970), Segal et a l . (1974), and  Davies et a l . (1971).  A l l these studies indicate that  lysosomal cathepsins are involved i n protein degradation. Intralysosomal protein degradation i s of l i m i t e d  importance  in post-mortem muscle, but extralysosomal proteolysis seems to play a s i g n i f i c a n t r o l e ; i n the l a t t e r case, a v a i l a b i l i t y of lysosomal enzymes by way o f leakage and release from l y sosomes i s a l i m i t i n g factor. of  Some studies on the release  cathepsins A, B, C, and D as influenced by gamma radia-  tion are presented. 2.3.3.1. Cathepsin D. A decrease i n absorbance of i r r a d i a t e d lysosomal suspension when incubated at 37°C (as shown i n Figure 2) i n dicated that gamma radiation affects the i n t e g r i t y of lysosomes , and thereby might result i n release of lysosomal enzymes.  Release of cathepsin D from i r r a d i a t e d lysosomes was  followed at two different temperatures.  In lysosomal samples  incubated at 37°C a f t e r gamma i r r a d i a t i o n , there was a sharp increase i n free cathepsin D i n the f i r s t 15 - 30 minutes, a l most reaching maxima  at 60 minutes i n a l l i r r a d i a t e d samples  (Figure 3). In contrast, free enzyme a c t i v i t y o f non-irradiated control increased more slowly, and reached a maximum after 18 0 minutes of incubation.  The mean free enzyme  contents of suspensions i r r a d i a t e d with 0.10, 0.25, or 0.50 Mrad were s i g n i f i c a n t l y higher (P<0.05) compared with the control throughout  the 180 minutes of incubation at 37°C.  Samples receiving a dose o f 1.0 Mrad followed a s i m i l a r  65.  0.20  Incubation time at 37°C (min) Figure 3.  Free cathepsin D a c t i v i t y of lysosomal suspension as influenced by gamma radiation and incubation at 37°C. (n = 5)  66. pattern of increase i n free enzyme content up to 6 0 minutes of incubation and then slowly decreased ; a reverse pattern was also exhibited by the absorbance readings An aggregation  (Figure 2).  of p a r t i c l e s i n 1.0 Mrad samples was noticed  after 120 minutes of incubation at 37°C, and may be responsible f o r higher absorbance values.  The low free enzyme  a c t i v i t y might be due to i n a c t i v a t i o n of the enzyme on prolonged incubation at 37°C and/or adsorption due to aggregation  of the enzyme  of p a r t i c l e s .  Data summerizing the release of cathepsin D a f t e r i r r a d i a t i o n at 4°C are presented  i n Figure 4.  The increase  in free enzyme a c t i v i t y was considerably slower at 4°C than at  37°C (Figure 3 ). There was a rapid increase i n free  enzyme a c t i v i t y i n i r r a d i a t e d samples i n the f i r s t  24 hours.  The controls exhibited a n e g l i g i b l e increase i n free enzyme content during the i n i t i a l 24 hour incubation at 4°C, then increased steadily during subsequent incubation of 48 and 72 hours.  Control samples had s i g n i f i c a n t l y lower (P<0.01)  free enzyme a c t i v i t y compared with i r r a d i a t e d samples throughout the incubation period of 72 hours.  In Figure 5, results  of the release of cathepsin D at 4°C are presented  as per-  centages of the t o t a l a c t i v i t y of the respective treatments. In t h i s case, the i n a c t i v a t i o n e f f e c t of i r r a d i a t i o n i s excluded and the data r e f l e c t a c t i v i t y i n r e l a t i o n to the actual p o t e n t i a l a c t i v i t y - o f the enzyme.  The general pattern  of enzyme release i s s i m i l a r to that shown i n Figure 4.  6 7 .  0.20  o  0.1 5 H  V.  c o  10  c  CD CL CO  "o £ o  0.1 0 h  CO  O CO  >~>  . -J  O CO OJ  0.0 5 h  24  4 8.  Incubation time at 4°C (hours) F i g u r e 4.  Free c a t h e p s i n D a c t i v i t y o f l y s o s o m a l s u s p e n s i o n as i n f l u e n c e d by gamma r a d i a t i o n and i n c u b a t i o n a t k°C. (n = 5)  6 8. Release of cathepsin D i n a l l the i r r a d i a t e d samples sharply increased during the f i r s t 24 hours. of incubation, and then increased slowly during subsequent incubation.  Control samples  showed very slow release i n the f i r s t 24 hours, but rate of release was higher between 48 - 7 2 hours of incubations. The enzyme release was 55.63, 87.22, 80.35, 84.17, and 81.56  percent i n samples i r r a d i a t e d with 0.0, 0.10, 0.25,  0.50,  and 1.00 Mrad respectively a f t e r 72 hours of incubation. The observed increase i n free cathepsin D a c t i v i t y  from lysosomal p a r t i c l e s i s most probably due to the release of bound enzymes and leakage of enzymes due to radiation damage to lysosomes.  Various radiation-induced mechanisms  are possible f o r such changes i n lysosomal structure.  Desai  et a l . (1964) showed that aryl sulfatase, 6-glucuronidase , and acid phosphatase were released after treatment of lysosomal suspension with gamma radiation.  They suggested a free  r a d i c a l mechanism leading to enhanced l i p i d peroxidation , presumably of lysosomal membrane l i p i d consituents , r e s u l t i n g in lysosomal membrane damage.  S i m i l a r l y , release of lysosomal  enzymes has been correlated with induced l i p i d peroxidation (Tappel, 1962).  Wills and Wilkinson (1966) reported that  electron radiation caused release of lysosomal enzymes, suggesting that l i p i d peroxide formation leads to rupture of lysosomal membrane, causing release of hydrolytic enzymes. Harris (1966a, 1966b) observed a decrease i n absorbance of lysosomal suspensions a f t e r gamma i r r a d i a t i o n , and concluded  100,  I  I  I  I  1  0  24  48  72  INCUBATION  Figure 5 .  TIME  AT 4°C  (hours)  Release of cathepsin D from lysosomes as i n fluenced by gamma radiation. (n = 3 )  70. that a release of enzyme had occurred on the basis of an increase in soluble protein a f t e r i r r a d i a t i o n treatment. Watkins (1970) demonstrated that radiation (electron r a d i a tion 5 - 2 0  Krad) induced release of B-glucuronidase, N-  acetyl-3-glucosaminidase, spleen lysosomes.  and acid phosphatase from rat  Watkins and Deacon (1973) observed that  neutron i r r a d i a t i o n of rat spleen lysosomes was  substan-  t i a l l y more e f f e c t i v e than electrons under s i m i l a r experimental conditions i n stimulating release of enzymes. In t h i s study, the observed slow release of cathepsin  D  at 4°C, compared with 37°C i s possibly due to radiation a f t e r - e f f e c t s , such as free r a d i c a l i n t e r a c t i o n s , formation of secondary r a d i c a l s , and the resultant changes i n the system which would proceed at a slower rate at 4°C et a l . , 1968). formation  (Copeland  I t has been suggested that l i p i d peroxide  i s involved i n the release of lysosomal enzymes  after i r r a d i a t i o n (Desai et a l . , ^ e ! ; Wills and Willkinson, 1  1966).  Formation of l i p i d peroxide has been shown to be  enhanced at 37°C (Wills and Willkinson, 1966,  1967a, 1967b;  Myer and Bide, 1966); this might account at least i n part for the rapid release of cathepsin D at 37°C. Sulfhydryl groups are important components of c e l l membranes and potential "radiosensitive" sites i n i n t r a c e l l ular membranes.  The r e l a t i v e l y high s e n s i t i v i t y of SH  groups to i r r a d i a t i o n has been pointed out i n many studies. Wills and Wilkinson  (1967a) found that 5,000 rads of e l e c -  tron i r r a d i a t i o n decreases the DTNB-reactive SH content  71. of l i v e r lysosomes by 2 3%.  Sutherland  and P i h l (196 8)  reported 20% oxidation o f erythrocyte membrane SH at 100 Krad. DTNB and other substances which react with SH groups have been reported to increase the f r a g i l i t y of lysosomes (Van Caneghem, 1972).  Their studies strongly suggest that radia-  tion-induced oxidation of sulfhydryl groups could cause disorganization of l i p o p r o t e i n structure r e s u l t i n g i n increased membrane permeability and disruption of lysosomes. Peroxidation of membrane l i p i d s may also be responsible,, to some extent, for change i n membrane structure, r e s u l t i n g in increased permeability of lysosomal membrane, but i t has been observed that l i p i d peroxidation requires higher doses of radiation than required f o r oxidation of membrane sulfhydryl groups (Sutherland and P i h l , 196 8; W i l l s and Wilkinson, 1967b).  L i p i d peroxidation has been shown to be  accompanied by an increase i n reactive s u l f h y d r y l groups, suggesting that SH oxidation and l i p i d peroxidation, treated as two d i s t i n c t mechanisms, might actually be i n t e r r e l a t e d (Robinson, 1965, 1966). Ionizing r a d i a t i o n would be expected to a l t e r a l l i n t r a c e l l u l a r components more or less indiscriminately,and many reactions could occur i n the lysosomal membrane as well as i n the lysosomal matrix.  During i r r a d i a t i o n of pure  ;  chemicals i n water, one observes not only oxidation of free t h i o l s to d i s u l f i d e , but also disruption of d i s u l f i d e bonds by reduction ...to. t h i o l , by oxidation, by interchange  of various  72. portions of d i s u l f i d e molecules and even by reaction of moieties  of a d i s u l f i d e such as cystine with a p u r i f i e d  protein or with an unsaturated fatty acid i n solution (Markakis  and Tappel, 1960;  1969).  Kollman and Shapiro, 1966 ; Myers et a l . ,  In view of these studies, i t seems l i k e l y that  disruption of d i s u l f i d e bonds could also play a role i n radiation damage to lysosomal membrane by contributing to the unfolding membrane proteins.  Radiation has been  shown to render the proteins of intact erthrocyte membrane more: susceptible to attack by trypsin and other p r o t e o l y t i c enzymes (Myers et a l . , 1967).  Radiation also accelerated  the t r y p t i c hydrolysis of p u r i f i e d proteins such as lease (Solbodian 1967).  et a l . , 1965)  ribonuc-  or albumin (Myers et a l . ,  In t h i s case, the radiation e f f e c t was  attributed  to cleavage of d i s u l f i d e bonds i n the protein, thus exposing hidden arginine and lysine groups to t r y p t i c attack (Meyers et a l . , 1967 ; Solbodian et a l . , 1965).  Meyers (1970) observed  that p r i o r exposure of yeast c e l l s to 100 - 400 Krad X-radiation rendered the c e l l wall susceptible to l y s i s by an enzyme preparation;  moreover, t h i s p a r t i c u l a r radiation  e f f e c t could be simulated  by pretreating the c e l l with  d i t h i o t h r e i t o l , which reduces d i s u l f i d e bonds.  It i s tempting  to assume that the same mechanism might be involved i n radiation damage to lysosomal structure, r e s u l t i n g i n enzyme release. . ... There are also reports suggesting that membrane-bound  cathepsins may be activated by i r r a d i a t i o n , and i t might p a r t i a l l y digest the lysosomal membrane, thus causing the release of lysosomal enzymes (Wills and Wilkinson, 1967b). Activation of l i p o l y t i c enzymes may also be a contributing factor i n such a mechanism (Beaufay and de Duve, 1959). In present studies, involvement  of these enzymes i n changing  membrane permeability does not seem to be a l i k e l y cause as release i s very rapid immediately especially at elevated temperatures  after irradiation, (Figure 3).  According  to this hypothesis, there should be slow release i n i t i a l l y , with a subsequent r i s e i n free enzyme content on prolonged incubation.  Moreover, i t i s hard to explain how cathepsins  can cause t h e i r own release even though a f t e r prolonged incubation, i t i s possible that activation of other lysosomal hydrolases might play some role. Firfarova and Orekhovich  (1971) suggested that cathep-  sin D existed i n an inactive precursor form i n chicken l i v e r ; i t i s possible that this i s true also f o r other lysosomal hydrolases.  Lysosomal enzymes might be activated by r a d i a t i o n -  induced dissociation of a precursor complex, thus t r i g g e r i n g further release of enzymes by coordinated enzyme action. It has been demonstrated that the carbohydrate chains afford the lysosomal glycoproteins limited protection against proteolytic attack by cathepsins, and s p l i t t i n g of the carbohydrate moiety expedites the autolysis of the protein component of lysosomal glycoproteins (Goldstone and Koenig,  74. 1974).  Radiation-induced  damage to glycoproteins might  make them susceptible to enzyme attack.  In other cases  of radiation activation of lysosomal hydrolases, neuraminidase and glucosidases might s p l i t the moiety, thus i n i t i a t i n g the a u t o l y s i s .  liver carbohydrate  Such autolysis  would affect the s t r u c t u r a l i n t e g r i t y of lysosomes and might cause, to some extent, enhanced release of lysosomal enzymes. Radiation-induced (Copeland et a l . , 1967,  alterations i n lysosomal proteins 1968), carbohydrates  1972), and l i p i d s (Nawar, 1972;  (Phillips,  Scott et a l . , 1964)  may  cause permeability changes i n the lysosomal membrane and/or dissociate bound lysosomal enzymes from intralysosomal structure. 2.3.3.2. Cathepsins A, B, and C. Lysosomes are the source of p r o t e o l y t i c enzymes involved in i n t r a c e l l u l a r protein breakdown (Coffey and de Duve, 1968). Caldwell (1970) suggested the joint action of several muscle cathepsins on endogenous muscle proteins.  Liao-Haung and  Tappel (1971) observed i n the chromatographic analysis of hydrolytic products of hemoglobin that oligopeptides produced by p r i o r action of cathepsin D were degraded by cathepsin C to smaller oligopeptides, dipeptides, and free amino acids. Goettlich-Riemann  et a l . (1971) reported a s y n e r g i s t i c and  concerted action of cathepsins A, B, and D i n protein hydrolysis.  These workers pointed out that cathepsin D i n i t i a t e d  protein hydrolysis and cathepsins A and B u t i l i z e d the hydrolysis products f o r further degradation.  Iodice et a l .  75. (1966) demonstrated that cathepsin A had l i t t l e or no a c t i v i t y on hemoglobin unless cathepsin D was present, and concluded that cathepsin A was r e s t r i c t e d i n i t s a c t i v i t y to break down products of proteins r e s u l t i n g from the p r i o r action of cathepsin D.  I t i s evident from these studies that  to the extent that cathepsins are involved i n protein breakdown i n post-mortem muscle, the a v a i l a b i l i t y of cathepsins A, B, and C l i k e l y would be important.  In the previous  section release of cathepsin D was discussed; this section deals with cathepsins A, B, and C as influenced by gamma radiation. Free enzyme a c t i v i t y of cathepsin A i s shown i n Figure 6. Immediately following i r r a d i a t i o n , a s l i g h t decrease i n free cathepsin A a c t i v i t y was noticed, which might be due to p a r t i a l inactivation of the enzyme already present i n free state or released during i r r a d i a t i o n .  Free a c t i v i t y  of cathepsin A i n a l l the i r r a d i a t e d samples increased rapidly during incubation at 4°C, and the increases were d i r e c t l y dose-related.  The increase i n free enzyme a c t i v i t y  of cathepsin A was s i g n i f i c a n t l y affected by r a d i a t i o n t r e a t ment as well as incubation time (P<0.01).  After 72 hours  incubation at 4°C, release of cathepsin A was 64.44, 75.00, and 80.19 percent for 0.25, 0.50, and 1.00 Mrad r e s p e c t i v e l y , while i n control samples, the release was very slow, reaching 36.7 8 percent a f t e r 72 hours of incubation. Release of cathepsin B (Figure 7) was enhanced by  76.  100  A Control  Figure 6.  Release of cathepsin A from lysosomes as i n fluenced by gamma radiation. (n = 5)  77.  100i  Figure 7.  Release of cathepsin B from lysosomes as influenced by gamma radiation. (n = 5 )  78. radiation dose and incubation time at 4°C (P<0.01). An increase i n cathepsin B was observed immediately a f t e r i r radiation (zero  incubation time) at 0.5 and 1.0 Mrad, i n  contrast to cathepsin A (Figure 6) and cathepsin C (Figure 8) which showed s l i g h t declines immediately a f t e r i r r a d i a t i o n . Free a c t i v i t y increased s t e a d i l y over the entire incubation period, with a s l i g h t l e v e l l i n g o f f between 48 hours and 72 hours, e s p e c i a l l y i n 0.25 Mrad treatment.  At the end  of 72 hours incubation, free enzyme content i n 0.50 and 1.00 Mrad reached a s i m i l a r l e v e l , but the 0.25 Mrad treated samples had a d i s t i n c t l y lower mean l e v e l of free cathepsin B After 72 hours incubation at 4°C, the free a c t i v i t y of cathep sin B was 26.13, 54.70, 70.14, and 70.48 percent of t o t a l a c t i v i t y f o r radiation treatments of 0.0, 0.25, 0.50, and 1.00 Mrad respectively.  Cathepsin  B had a s l i g h t l y slower  release and a generally lower l e v e l of free a c t i v i t y compared with cathepsin A. Release of cathepsin C following radiation treatment (Figure 8) exhibited a s i m i l a r pattern to that f o r cathepsins A and B, but rate of release and l e v e l of free a c t i v i t y were both higher.  Cathepsin  C was released to the extent  of 43.86, 72.68, 82.00, and 85.47 percent of t o t a l enzyme a c t i v i t y a f t e r 0.0, 0.25, 0.50, and 1.00 Mrad of i r r a d i a t i o n respectively when incubated at 4°C.  Free enzyme a c t i v i t y  of i r r a d i a t e d sample increased rapidly during 4 8 hours of incubation, and then l e v e l l e d o f f on further incubation.  100,  Figure 8 . Release of cathepsin C from lysosomes as i n fluenced by gamma radiation. Cn = 5 )  80. In non-irradiated samples, release of the enzyme increased slowly during the f i r s t 48 hours of incubation, and more rapidly during subsequent incubation at 4°C.  Radiation  doses had s i g n i f i c a n t e f f e c t (P<0.01) on the release of lysosomal cathepsin  C.  Figure 9 compares the e f f e c t of a 1.00  Mrad dose on  the release of cathepsins A, B, C, and D during incubation at 4°C.  The highest rate of increase and l e v e l of free  enzyme a c t i v i t y were observed f o r cathepsin D, followed by cathepsins C, A, and B.  It i s i n t e r e s t i n g to note that  free a c t i v i t y of cathepsins C and D was  11% - 13%  higher  than cathepsins A and B immediately a f t e r i r r a d i a t i o n . the cases of a l l four enzymes, the rate of release quite rapid up to 4 8 hours of incubation.  In  was  The rate of  release of cathepsins C and D declined a f t e r 48 hours of incubation, but cathepsins A and B increased much more steadily u n t i l the end of 72 hours incubation, i n d i c a t i n g that cathepsins C and D are r e l a t i v e l y e a s i l y released from lysosomes and reach a maximum l e v e l i n less time than cathepsins A and B.  A s i m i l a r s i t u a t i o n i s observed when the ef-  fects of radiation dose on release of cathepsins  are compared.  Control samples consistently had a higher free a c t i v i t y of cathepsins C and D than A and B a f t e r 48 hours incubation t h i s difference increased with increasing  radiation dose  (Figure 10 and Table 3). The present studies on cathepsins demonstrate that  and  81.  Figure 9.  Comparative e f f e c t of gamma radiation (1.0 Mrad) on the release of lysosomal cathepsins A, B, C, and D. (n = 5)  82.  IOOI  o CATHEPSIN C • CATHEPSIN D  0.25  0.50  RADIATION  F i g u r e 10.  DOSE  1.00 (Mrad)  Comparative e f f e c t o f v a r i o u s r a d i a t i o n doses on r e l e a s e o f c a t h e p s i n s from lysosomes a f t e r 48 hours i n c u b a t i o n a t 4°C. (n = 5)  83. t h e i r release i s s i g n i f i c a n t l y (P<0.01) enhanced by gamma radiation, thus increasing the free enzyme content i n the system.  This i s i n agreement with other workers' obser-  vations (Desai et a l . , 1964; Wills and Wilkinson, 1966) that lysosomal enzymes l i k e acid phosphatase and 8-glucuronidase are released from gamma i r r a d i a t e d  lysosomes.  Watkins and Deacon (1973) demonstrated s i m i l a r release of 3-glucuronidase, 3-glucosaminidase, and acid phosphatase after treating rat spleen lysosomes with 5 - 2 0 Krad of neutron or electron i r r a d i a t i o n . Release of lysosomal enzymes has been attributed to membrane damage caused by free r a d i c a l s , formation of l i p i d peroxide (Desai et a l . , 1964-; Wills and Wilkinson, 1966), as well as oxidation of SH groups of the i n t r a c e l l u l a r membranes (Wills and Wilkinson, 1967a, 1967b).  The possible  mechanisms of lysosomal enzyme release has been discussed in d e t a i l in the previous section, regarding cathepsin D. Present studies also demonstrated that lysosomal cathepsins are d i f f e r e n t i a l l y released a f t e r i r r a d i a t i o n treatment -- cathepsins C and D are more readily released than cathepsins A and B. This phenomenon of d i f f e r e n t i a l release has been reported by Desai et a l . (1964).  They  demonstrated that aryl sulfatase and 3-glucuronidase were most readily released, while acid phosphatase showed a slow release, and ribonuclease was not affected.  Watkins  and Deacon (1973) found that following a dose of 20 Krad,  84. electrons affected the release of 22.9, of B-glucuronidase,  13.0,  and 9.9  percent  B-glucosaminidase, and acid phosphatase  a c t i v i t i e s respectively. D i f f e r e n t i a l s o l u b i l i z a t i o n of lysosomal  B-glucuronidase  and acid phosphatase was  reported by Watkins (1970).  also  Lysosomal enzymes have been  reported to show d i f f e r e n t i a l release under various conditions, l i k e incubation (Weissmann and Thomas, 1963), d i f f e r e n t  pH,  osmotic strength, or temperature (Sawant et a l . , 1964b), freeze-thaw treatment (Sawant et a l . , 1964a), T r i t o n concentration (Romeo et a l . , 1956; or change of mono et a l . , 1968).  X-100  Stagni and de Bernard, 1968),  and divalent ion concentrations (Verity  ,  Most of the studies c i t e d above have reported^  such a behaviour of lysosomal enzymes other than cathepsins. No previous reports on cathepsins regarding the influence of radiation treatment have appeared i n the l i t e r a t u r e . This d i f f e r e n t i a l release of cathepsins as well as other lysosomal enzymes i s possibly due to differences i n structure, binding s i t e s , or l o c a l i z a t i o n of the enzymes i n the lysosomes.  The data suggest that enzymes are bound d i f f e r e n -  t i a l l y within lysosomes, or that they might be in different p a r t i c l e s (Dianzani, 1963) s e n s i t i v i t y to external damage.  localized  possessing varying  Enzymes l i k e cathepsin C  might be l o c a l i z e d i n r a d i a t i o n - s e n s i t i v e p a r t i c l e s or i n radiation-sensitive areas within the p a r t i c l e , or cathepsins A and B might be r e l a t i v e l y t i g h t l y bound as compared with cathepsin C.  Unfortunately, information oh intralysosomal  85.  Table 3.  Release of cathepsins A, B, C, and D from lysosomes at 4°C after i r r a d i a t i o n .  Free enzyme a c t i v i t y (% of t o t a l ) Radiation dose (Mrad)  Incubation time (hrs)  0.0  0..25  0.,50  Cathepsin A  0 24 48 72  20. 9 22. 2 27. 8 36. 8  12,,9 32..0 51.. 8 64.,7  13., 2 36.,5 57., 8 75.,6  14.,0 43.,0 67.. 9 80.,2  Cathepsin B  0 24 48 72  11. 9 14. 0 21. 6 26. 2  11..9 25..1 46..9 54..7  14..7 32.,6 53.,4 70.,1  16.,7 39 . .5 61., 5 70..5  Cathepsin C  0 24 48 72  31. 0 28. 7 33. 5 43. 9  28..3 42..4 61..9 72..7  28.,1 50,. 9 72.,4 82 . .0  27., 7 63.,1 83..9 85.,5  Cathepsin D  0 24 48 72  21. 0 29. 0 3 7.0 55 .6  26..1 64., 4 72,. 8 80.,4  27.,4 63.,4 79., 9 84..2  26.,6 77.. 8 88,.4 81..6  Enzyme  n = 5 f o r cathepsins A, B, and C n = 3 f o r cathepsin D  1., 00  86.  l o c a l i z a t i o n of enzymes i s obscure.  Lysosomes are generally  considered as membrane-limited p a r t i c l e s containing  lysosomal  hydrolases, which would be l a b i l i z e d by various treatments, causing an injury to p a r t i c l e membrane (Beaufay and de Duve, 1959). D i f f e r e n t i a l release of enzymes raises an i n t e r e s t i n g question concerning  the enzymes of lysosomes —  whether  they are free i n the i n t e r i o r of the p a r t i c l e s or bound to the enclosing membrane or lysosomal matrix.  Sawant et a l .  (1964b) obtained d i f f e r e n t i a l a v a i l a b i l i t y of lysosomal enzymes at acid, a l k a l i n e , and neutral pH, which they indicated that lysosomal  considered  enzymes are bound to d i f f e r i n g  charged s i t e s of the membrane.  These workers also observed  an increased a v a i l a b i l i t y of acid phosphatase on addition of 0.2M NaCl, i n d i c a t i n g the p o s s i b i l i t y of s a l t between the enzymes and the membranes.  linkages  These observations  are based on a v a i l a b i l i t y of enzymes rather than release of enzymes and increased a v a i l a b i l i t y could be due to e i t h e r release o f enzymes i n soluble form, or to the greater accessib i l i t y of substrate to the bound enzyme.  Beck and Tappel  (196 8) concluded from t h e i r studies that lysosomal enzymes might be bound to membrane. 2+ that both inorganic Hg chloromercuribenzoate,  Verity and Reith (196 7) reported  ions and organic mercurials (pphenylmercuric acetate) induced an  i r r e v e r s i b l e loss of the structure-linked latency of lysosomal enzymes i n suspensions of lysosomes.  A s i m i l a r effect of  87. p-chloromercuribenzene  sulfonate (pCMBS) has been reported  on neutrophil granules (Hariss, 1968).  This indicates the  importance of the t h i o l groups of proteins i n maintaining both the i n t e g r i t y of lysosomal membranes and the latent properties of lysosomal enzymes.  I t i s also possible that  proteins as well as l i p i d s and lysosomal membranes may be involved i n binding of some of the enzymes to the l i m i t i n g membranes. There i s evidence that lysosomal enzymes might be bound to the lysosomal matrix.  Shibko et a l . (1965) observed  loss of lysosomal matrix and release of acid phosphatase and aryl sulfatase during incubation of lysosomal suspension at 37°C.  Koenig (1962) suggested that h y d r o l y t i c enzymes are  contained within the lysosomes by e l e c t r o s t a t i c binding to the a c i d i c groups of the lipoprotein matrix.  T h i o l groups  in some hydrolases may play an a n c i l l a r y role i n the struct u r a l latency of some hydrolases, possibly through formation of d i s u l f i d e bonds with t h i o l groups i n lysosomal lipoprot e i n , or through some interaction with polyunsaturated fatty acids i n the l a t t e r (Robinson, 1966). Cytochemical observation of hydrolase a c t i v i t y at the fine u l t r a s t r u c t u r a l l e v e l demonstrates  association of reac-  tion products with lysosomal membrane as well as the matrix (Daems et a l . , 1972).  I t i s also well-established that a  number of isoenzymes of lysosomal cathepsins e x i s t , especially of cathepsins D and B (Barret, 1971; Keilova, 1973) which  88. might be d i f f e r e n t i a l l y bound or l o c a l i z e d within lysosomes. In view of the above studies, i t i s obvious that various lysosomal enzymes are l o c a l i z e d i n a unique fashion with a d i f f e r e n t i a l a f f i n i t y or binding for the intralysosomal structure.  These differences i n l o c a l i z a t i o n as well as  binding might be responsible for d i f f e r e n t i a l radiation sensit i v i t y and the resultant pattern of release of lysosomal cathepsins. These studies demonstrate that radiation causes release of lysosomal cathepsins, thus making them available f o r hydrolysis of various components of animal t i s s u e .  I t has  been demonstrated that lysosomal cathepsins are capable of degrading proteins (Coffey and de Duve, 1968).  Ethrington  (1972) demonstrated that r a t l i v e r extracts degraded insoluble collagen and suggested the presence of c o l l a g e n o l y t i c cathepsin which was d i f f e r e n t than cathepsin B or cathepsin D, the two known endopeptidases.  Cathepsin B l has been considered a  major contributor to the enzymic degradation  of collagen by  rat l i v e r lysosomes (Anderson, 1969; Burleigh, 1973).  Several  studies on hemoglobin digestion by p a r t i a l l y p u r i f i e d cathepsin preparations have suggested a s y n e r g i s t i c action of cathepsins A, B, and C, and cathepsin D (Iodice et a l . , 1966 ; Liao-Huang and Tappel, 1971; Goettlich-Riemann  et a l . , 1971).  Tappel  (196 9) postulated that during the degradation of denatured proteins by lysosomal extracts, the action was i n i t i a t e d by cathepsin' D, and large peptide fragments, were broken down by  89.  other proteases and dipeptidases. have suggested an important  Coffey and de Duve (196 8)  role for cathepsin D i n hemoglobin  breakdown, and Dingle (1971) has shown i t s function i n cartilage degradation.  Recently Huisman et a l . (1974) suggested that  either cathepsin Bl or some t h i o l enzymes other than cathepsins B l , C, or D are involved i n lysosomal protein degradation. Glycoproteins have been shown to be degraded by rat kidney lysosomes; moreover, i t was observed that s p l i t t i n g of carbohydrate chains of glycoprotein by neuraminidase and glycosidases made the protein moiety more susceptible to proteinase attack (Aronson and de Duve, 19.6 8; Goldstone and Koenig, 1974).  The great majority of glucosidases are located i n l y -  sosomes (Barrett, 1969), and some of them have been shown to be released from lysosomes after i r r a d i a t i o n treatment (Desai et a l . , 1964;  Watkins, 1970; Watkins and Deacon, 1974).  Besides i n v i t r o studies using p u r i f i e d substrates, there i s evidence that lysosomal cathepsins are active on endogenous proteins.  Eino and Stanley (1973a) reported catheptic a c t i v i t y  on salt-soluble and insoluble proteins of beef muscle, moreover, incubating muscle fibres i n a cathepsin solution at pll 5.5  and 0° — 5°C reduced the t e n s i l e strength of muscle  fibres (Eino and Stanley, 1973b).  Their crude enzyme prepara-  tion might have contained cathepsins A, B, and C besides cathepsin D, and there i s a strong p o s s i b i l i t y that the e f f e c t was  due to involvement of a l l the cathepsins, especially at  pK 5.5, as cathepsins A, B, and C are able to degrade the  90. oligopeptides produced by cathepsin D attack ( G o e t t l i c h Reimann et a l . ,. 19 71; Liao-Huang and Tappel, 1971).  Autolysis  of chicken skeletal' muscle extract has been attributed to a combined action of cathepsins  (Caldwell, 1970).  There i s  ample evidence of proteolysis i n post-mortem muscle (Sharp, 196 3; Khan and van den Berg, 1964a, 1964b; Suzuki at a l . , 1967; Parrish et a l . , 1969;  Okitani and Fujimaki, 1972;  Okitani et a l .  1973) , but i t s extent and importance has been disputed. U l t r a s t r u c t u r a l observations  show a disorganization or  absence of Z-line i n aged muscle, and a tendencey f o r a myof i b r i l to be broken at the former location of the Z-line (Davey and G i l b e r t , 1967).  Trypsin has been shown to quickly  remove the Z-line from myofibres (Goll et a l . , 1970). dence that cathepsins was  may  Evi-  cause the degradation of Z-lines  presented by Penny (1968) and Fukazawa et a l . (1969).  They found that f i b r i l s prepared post-mortem without the presence of cathepsins  and other soluble c e l l u l a r material  did not show a loss of Z-line.  Busch et a l . (1972) demon2+  strated that Z-line degradation was  caused by CASF (Ca  -  activated sarcoplasmic factor) i s o l a t e d from muscle; i t has been suggested that CASF might be l o c a l i z e d i n lysosomes, and the leakage might have occurred during t h e i r i s o l a t i o n procedure.  Recently, CASF has been characterized as proteo2+  l y t i c enzyme, having optimum pH and optimum Ca tion of 7.0  and 1.0 mM  concentra-  respectively, and i t removes soluble  material from myofibrils (Suzuki and G o l l , 1974).  91. West et a l . (1974) observed that incubation of i s o l a t e d sarcoplasmic reticulum with cathepsin f r a c t i o n at pH resulted i n loss of calcium accumulating plasmic reticulum. Bl treatment  Such an effect was  7.0  a b i l i t y of sarco-  lower with cathepsin  as compared with treatment with a f r a c t i o n con-  taining cathepsins A, B l , B2 , and C.  However, no studies  have been reported with cathepsin D or other lysosomal proteases, but b r i e f t r y p t i c digestion produced s i m i l a r results (West et a l . , 1974).  Loss of calcium accumulating  ability  of sarcoplasmic r e t i c u l a r membranes causes the onset of r i g o r mortis (Greaser et al,, 1969 ; Schmidt et a l . , 1970), which in turn i s associated with the post-mortem changes i n muscle (Herring et a l . , 1965; Marsh and Leet, 1966).  Degradation  of sarcoplasmic reticulum i n muscle would result i n release of calcium, thus activating CASF, which i s capable of removing Z-lines (Busch et a l . , 1972) (Suzuki and G o l l , 1971).  and s o l u b i l i z i n g myofibrils  However, i t remains to be demon-  strated whether muscle cathepsins can cause such changes i n sarcoplasmic reticulum .in post-mortem muscle. Evidence of proteolysis affecting the texture was  noted  in studies with i r r a d i a t i o n - s t e r i l i z e d meat (Cain et a l . , Pearson et a l . , 1958 , 1960 ; Coleby et a l . , 1961; Rhodes, 1964).  1958  Bailey and  Radiation did not inactivate the p r o t e o l y t i c  enzymes, and with storage, extensive degradation of muscle occurred.  Klein and Altman (1972b) found an increased pro-  t e o l y t i c a c t i v i t y i n chicken breast and leg muscle after  92. 0.2 Mrad of i o n i z i n g radiation.  Extensive degradation and  increased proteolysis i n i r r a d i a t e d meat i s possibly due to lysosomal f r a g i l i z a t i o n and release of lysosomal cathepsins. Results of this investigation suggest that i r r a d i a t i o n induced damage to lysosomal structure markedly enhanced the release of lysosomal cathepsins.  Release of other lysosomal  enzymes such as collagenase and neutral protease i s a strong possibility.  Similar damage to lysosomes and increases i n  free enzyme contents might occur i n i r r a d i a t e d meats, r e s u l t i n g in an extensive breakdown of tissue proteins.  Such an e f f e c t  would considerably influence the ultimate quality of i r r a d i a tion-preserved foods of animal o r i g i n .  i  93.  2.4.  SUMMARY AND CONCLUSIONS The effect of gamma radiation on chicken l i v e r tissue  and on i s o l a t e d lysosomes was studied.  The release  of chicken l i v e r cathepsin D by gamma radiation treatment was observed.  The enzyme release was greater at low doses  (50 and 10 0 Krad)  compared with high doses (0.25, 0.50,  1.0 Mrad); possibly at high doses p a r t i a l i n a c t i v a t i o n occurred, or radiation denaturation Of tissue proteins might have decreased the extraction of the enzyme. Lysosomes were i s o l a t e d from chicken l i v e r by sucrose gradient and d i f f e r e n t i a l centrifugation techniques.  Light  scattering studies of i s o l a t e d lysosomes showed a rapid drop i n absorbance at 5 40 nm a f t e r i r r a d i a t i o n treatment and p o s t - i r r a d i a t i o n incubation at 37°C, i n d i c a t i n g a strong p o s s i b i l i t y of leakage of lysosomal enzymes. The results of studies on release of lysosomal enzymes indicate that free a c t i v i t y of cathepsins A, B, C, and D was substantially increased by i r r a d i a t i o n treatments of isolated lysosomes.  Release of cathepsin D was very rapid  at 37°C compared with 4°C incubation.  Release of cathepsin D  from i r r a d i a t e d lysosomes almost reached maximum l e v e l i n one hour at 37°C,-while the non-irradiated control showed a slow release over 3 hours of incubation. Release of cathepsins A, B, C, and D from i r r a d i a t e d lysosomes was maximal during the f i r s t 2 4 - 4 8 hours of incubation at 4°C, but non-irradiated controls showed slow  94.  i n i t i a l release (during f i r s t 24 - 48 hours) and a higher rate of release a f t e r 48 hours; the l e v e l of free enzyme content remained considerably lower than the i r r a d i a t e d lysosomes. Lysosomal cathepsins A, B, C, and D exhibited a d i f f e r e n t i a l release pattern under the influence of i r r a d i a t i o n .  Cathep-  sins C and D were more r e a d i l y released than cathepsins A and B. Radiation-enhanced release of lysosomal enzymes i s i n dicative of damage to lysosomal  structure from such treatment.  It i s l i k e l y that radiation damage to membrane structure causes permeability changes, and possibly the creation of weak points results i n disruption of some lysosomes during incubation.  Another possible reason may be disassociation  of lysosomal  cathepsins  from intralysosomal structure by  radiation-induced damage to binding s i t e s .  Radiation-  induced oxidation of SH groups, disruption of d i s u l f i d e bonds, or l i p i d peroxidation also may be responsible f o r damaging lysosomal  structure.  Denaturation  or fragmentation  of l y -  sosomal proteins could also contribute to radiation-induced alterations i n lysosomes. Radiation-induced  release of cathepsins A, B, C, and D  makes them freely available f o r degradation  of tissue proteins.  Synergistic action of these enzymes may cause extensive protein degradation. sin  The observation that release of cathep-  D was very rapid at the i n i t i a l stage of incubation makes  i t available for i n i t i a t i o n of protein breakdown as suggested  95. by various workers.  It i s also important to point out that  other lysosomal enzymes l i k e neutral proteases, a p r o t e o l y t i c 2 +  enzyme c a l l e d Ca  -activated sarcoplasmic factor (CASF),  or some other cathepsins yet uncharacterized, might also be released and have s i m i l a r degradative action on tissue proteins.  96,  CHAPTER 3.  RADIATION SENSITIVITY OF LYSOSOMAL CATHEPSINS AND  HEMOGLOBIN SUBSTRATE  3.1. INTRODUCTION The application of i o n i z i n g radiation f o r the preservation of foods i s of great interest to r a d i a t i o n research workers, as micro-organisms responsible f o r food spoilage can be e f f e c t i v e l y eliminated by s t e r i l i z i n g doses of r a d i a t i o n . Results of various investigations show that a number of enzymes are radiation resistant (Coelho, 1969.; Klein and Altman, 1972b; Losty et a l . , 1973).  The r e s i d u a l proteoly-  t i c a c t i v i t y has been held responsible f o r a u t o l y t i c degradation of i r r a d i a t e d foods of animal o r i g i n (Drake et a l . , 1961; Coleby et a l . , 1960).  Several studies have established the  presence of lysosomal cathepsins i n s k e l e t a l muscle tissue (Caldwell and Grosjean, 1971;  Parrish et a l . , 1969;  et a l . , 1967); moreover, j o i n t action of d i f f e r e n t  Randall cathepsins  in degradation of proteins have been suggested (Caldwell, 1970 ; Iodice et a l . , 1966).  It i s quite l i k e l y that r e s i d u a l •  a c t i v i t y i n i r r a d i a t e d meats i s not only due to cathepsin but also cathepsins A, B, and C.  D,  Inactivation of tissue  p r o t e o l y t i c enzymes has been interpreted as i n a c t i v a t i o n of cathepsin D, but information on radiation s e s i t i v i t y of cathepsins A, B, and C i s needed.  Due to low enzyme a c t i v i t y  of s k e l e t a l muscle, i s o l a t e d lysosomal f r a c t i o n from chicken l i v e r was  used as a source of enzymes.  Tissue proteins undergo various changes due to i r r a d i a -  tion treatment (Klein and Altman, 1972a; Uzunov et a l . , 1972), which might affect the course of proteolysis during of i r r a d i a t e d meats.  storage  I t has been shown that i r r a d i a t e d a l -  bumin and casein become susceptible to t r y p t i c digestion (McArdle and Desrosier, 1955), but studies on collagen i n dicate that protein becomes resistant to collagenase possibly  attack,  due to intermolecular c r o s s - l i n k i n g . Studies on  degradation  of i r r a d i a t e d proteins by cathepsins  seems highly  appropriate to better understand proteolysis occurring i n i r r a d i a t e d meats.  In t i s s u e , i t i s ' d i f f i c u l t to d i f f e r e n -  t i a t e between the effects of i r r a d i a t i o n on various protein components, and to distinguish the separate e f f e c t s of i n d i v i d u a l enzymes towards autolysis.  In the present  study,  an attempt has been made to evaluate the e f f e c t of i r r a d i a tion on lysosomal  cathepsins and model substrate, and i t s  digestion by lysosomal  cathepsins.  98.  3.2. EXPERIMENTAL 2.2.1. Treatment  of Samples f o r Enzyme Study  Chicken l i v e r s were removed from 3 or 4 birds  immediately  a f t e r k i l l i n g , washed free o f blood with 0.25M sucrose solution, and c h i l l e d i n crushed i c e . Excessive f a t and connective tissue were removed; tissue was minced with s c i s sors and homogenized i n 0.25M sucrose (1:8, w/v) using a Waring blender at top speed f o r 30 seconds 'at 4 C. The lysosomes were i s o l a t e d by sucrose density gradient and d i f f e r e n t i a l centrifugation techniques (Sawant et a l . ,  1964c).  The lysosomal p e l l e t was suspended i n 0.7M sucrose solution and a 1:2 (w/v) d i l u t i o n was made on the basis of l i v e r tissue.  Samples of this f i n a l suspension received varying  doses of gamma radiation i n a Gamma Cell-220.  Details o f  these procedures are given i n Chapter 2.  3.2.2. Enzyme Assay Conditions f o r determination of cathepsins A, B, C, and D are outlined i n Table 4.  Detailed procedures are given  in Chapter 2 under "Enzyme Assays" section.  3.2.3. Enzyme A c t i v i t y i n Lysosomal  Suspensions  3.2.3.1. Residual a c t i v i t y . After i r r a d i a t i o n , lysosomal suspensions received freezethaw treatment ten times, or were treated with Triton X-100, 0.2%  f i n a l concentration, and kept at room temperature f o r  at least 30 minutes to disrupt the lysosomal p a r t i c l e s . This suspension was centrifuged at 17,000 x g f o r 20 minutes, and the supernatant used for determination of enzyme a c t i v i t y . Catheptic a c t i v i t y remaining after i r r a d i a t i o n treatment was expressed as percentage of control value and designated "residual  activity".  3.2.3.2. Enzyme a v a i l a b i l i t y after i r r a d i a t i o n of lysosomes in the intact and ruptured state. The lysosomal p e l l e t was suspended solution  i n 0. 7M sucrose  at pH 7.2. One portion was given freeze-thaw t r e a t -  ment ten times to disrupt lysosomal p a r t i c l e s p r i o r to i r r a d i a t i o n , while the other portion was subjected to freeze-thaw treatment a f t e r i r r a d i a t i o n .  Doses of 0.25,  0.50, and 1.00 Mrad were given to the above samples, and non-irradiated samples were kept as controls.  Available  enzyme a c t i v i t y of cathepsins A, B, and C was determined using the disrupted p a r t i c l e suspensions without c e n t r i fugation. 3.2.3.3. pH during i r r a d i a t i o n . The lysosomal p e l l e t was suspended i n 0.7M sucrose solution.  Lysosomes were disrupted by Triton X-100 t r e a t -  ment and centrifuged at 17,000 x g f o r 20 minutes.  pH of  the supernatant was adjusted to 4.0, 5.5, 7.0, or 8.5 p r i o r to i r r a d i a t i o n . or 1.00: Mrad  Irradiation  doses of 0.10, 0.25, 0.50,  were given to these samples.  samples were used as controls.  Non-irradiated  Cathepsins A, B, C, and D  Table 4. Substrates and incubation conditions for cathepsin assays  Enzyme  Substrate*  Buffer  Cathepsin A  0.0152M N-carbobenzoxy-a-Lglutamyl-L-tyrosine  0.04M acetate  5.0  Cathepsin B  0.01M  benzoyl-L-arginine amide  0.1M  citrate  5.0  0.04M cysteine HC1  Cathepsin C  0.01M  glycyl-L-tyrosine amide  0.1M  citrate  5.0  0.04M cysteine HC1  Cathepsin D  2.0%  Hemoglobin  0.2M  acetate  3.8  Reaction time: 2 hours at 37 C. * A l l substrates were purchased from Sigma Chemical Company.  pH  Additions -  101. a c t i v i t i e s were determined.  Enzyme a c t i v i t y of i r r a d i a t e d  samples was expressed as a percentage of the respective controls. 3.2.3.4. Sucrose concentration during i r r a d i a t i o n . The lysosomal p e l l e t was suspended i n 0.10, 0.25, 0.50, or 7.0M sucrose solution pH 7.2, and given a dose of 0.50 Mrad, while non-irradiated samples served as controls. After i r r a d i a t i o n , lysosomes were disrupted with Triton X-10 0 treatment and cathepsin D a c t i v i t y was determined. 3.2.3.5. Temperature  during i r r a d i a t i o n .  Lysosomes were suspended i n 0.7M sucrose solution pH 7.0 and administered a dose of 0.5 0 Mrad of gamma r a d i a tion either at room temperature or crushed i c e temperature and then stored at 4°C.  Samples were drawn at 0 hours and  48 hours after i r r a d i a t i o n , and centrifuged at 17,000 x g for 20 minutes; cathepsin D a c t i v i t y of the supernatant was designated as "free a c t i v i t y " .  Total cathepsin D ac-  t i v i t y was determined after Triton X-100 treatment. 3.2.3.6. Irradiation - heat combination treatment. Lysosomal suspension was treated with Triton X-100 and centrifuged at 17,000 x g f o r 20 minutes.  The super-  natant samples were subjected to 0.0, 0.25, 0.5, or 1.00 Mrad gamma radiation.  Samples were drawn from each dose  and heated at 50 , 60 , 70 , 80 , or 90°C f o r 10 minutes in a constant temperature water bath, and then cooled to room temperature immediately.  Cathepsin D a c t i v i t y was  determined according to the procedure described i n Chapter 2.  102. 3.2.4. Hydrolysis of Irradiated Hemoglobin 3.2.4.1. In solution. A 10% aqueous solution of hemoglobin was i r r a d i a t e d for 0.0, 0.25, 0.5, or 1.00 Mrad at ambient chamber temperature.  Hydrolysis of 2.5% hemoglobin by cathepsin D was  followed at pH 3.8 or pH 5.0 i n 0.2M acetate buffer or pH 7.0 i n 0.2M phosphate buffer. 3.2.4.2. In dry state. Dry powdered hemoglobin was i r r a d i a t e d at room temperature f o r 0.0, 0.25, 0.5, 1.00, or 5.0 Mrad.  The i r r a d i a t e d  hemoglobin was used as substrate f o r cathepsin D hydrolysis at pH 3.8.  The procedure f o r determination of cathepsin D  has been outlined i n the "Enzyme assay" section of Chapter 2.  3.2.5. Absorption Spectrum of Irradiated Hemoglobin Absorption  spectrum of hemoglobin substrate i r r a d i a t e d  for 0.0, 0.2 5, 0.5, and 1.0 0 Mrad i n solution were taken after appropriate d i l u t i o n .  Samples were scanned using  Pye-Unicam SP-800B spectrophotometer (Pye-Unicam Ltd.).  3.2.6. Agarose Gel Electrophoresis of Hemoglobin a f t e r Hydrolysis by Cathepsin D Irradiated as well as non-irradiated hemoglobin was hydrolysed by cathepsin D at pH 3.8 i n 0.2M acetate buffer using 5% substrate and 5 hours reaction time at 37°C. Control samples were kept at 4°C.  A f t e r h y d r o l y s i s , 10pi  103. of sample was applied to agarose g e l . 3.2.6.1. Electrophoresis procedure. F i f t y grams of urea were dissolved i n 200 ml of Barb i t a l buffer pH 8.6, 0.05M with 0.035% EDTA. film (Agarose Universal Electrophoresis  Agarose gel  Film - ACI) was  soaked i n the above buffer f o r 15 minutes; 0.6 ml of 2mercaptoethanol was added and soaking continued f o r another 15 minutes.  The gel was dried and lOul of hydrolysed hemo-  globin was applied into the dried sample s l o t s .  The cas-  sette c e l l compartments were f i l l e d with B a r b i t a l buffer pH 8.6 and the agarose gel f i l m slipped into a cassette c e l l cover.  The samples were run at 4°C f o r one hour.  After electrophoresis, staining was done i n 0.2% amido black i n 5% acetic acid f o r 15 minutes, followed by 30 seconds washing i n 5% acetic acid.  The f i l m was dried f o r  20 minutes i n an a i r oven, cleared by soaking i n 5% acetic acid f o r one minute, and then dried at 70° - 85°C f o r 2 0 minutes.  104. 3.3.  RESULTS AND  DISCUSSION  3.3.1. Radiation Inactivation of Cathepsins A, B, C, and  D  3.3.1.1. Lysosomal suspension. Radiation doses of 0.25,  0.5,  and 1.0 0 Mrad s i g n i f i c a n t  (P<0.01) inactivated cathepsins A, B, and C (Figure A dose of 0.25  ID.  Mrad caused a marked decrease i n residual  enzyme a c t i v i t y and the curves l e v e l l e d o f f as the dose was  increased  to 0.5  and 1.00  Mrads.  Cathepsin B was  most  sensitive to r a d i a t i o n , followed by cathepsins C, D, and A (Figure 11).  After 1.00  Mrad r a d i a t i o n , the residual enzyme  a c t i v i t y dropped to 78.7,  6 7.0,  and  70.2  percent of control  in the case of cathepsins A, B, and C respectively.  Higher  radiation s e n s i t i v i t y of cathepsins B and C might be due  to  the presence of sulfhydryl groups i n t h e i r active s i t e . This i s i n agreement with the report that in sulfhydryl enzymes l i k e papain, destruction  of SH groups leads to  rapid inactivation of enzymes ( P i h l and Sanner, Lynn and  1963;  Louis, 1973).  Radiation doses of 0.25,  0.50,  and 1.00  Mrad caused  appreciable inactivation of cathepsin D (Figure 11).  En-  zyme a c t i v i t y of i r r a d i a t e d samples was s i g n i f i c a n t l y (P<0.01) lower than that of control, and the 1.00  Mrad  treatment had s i g n i f i c a n t l y lower a c t i v i t y compared with other i r r a d i a t i o n treatments. 1.00  Doses of 0.25,  0.50,  and  Mrad caused a decrease in the enzyme a c t i v i t y of  15.42, 18.46, and  27.42 percent respectively.  The  results  105.  100  £80  > i—  u <  • A o A  ^401 >M  z  CATHEPSIN A CATHEPSIN B CATHEPSIN C CATHEPSIN D  LU  < 20}  Q to LU  0.25  ±  1.00  0.50  RADIATION  DOSE  (Mrad)  Figure 11. Radiation i n a c t i v a t i o n of cathepsins A, B, C, and D i n intact lysosomes. (n = 5)  106. of this report  are i n general agreement with other workers'  observations of radiation i n a c t i v a t i o n of enzymes.  It has  been observed that tissue proteases are r e l a t i v e l y radiationresistant (Doty and Wachter, 1955; Landman, 1963).  Drake et a l . , 1957b;  Klein and Altman (1972b) reported 50% i n -  activation of p r o t e o l y t i c enzyme i n chicken breast and muscle a f t e r 1.0 h i b i t i o n by 5.0  Mrad gamma i r r a d i a t i o n , and Mrad.  leg  85% - 100%  in-  Losty et a l . (1973) observed up to  reduction of p r o t e o l y t i c a c t i v i t y i n ground beef a f t e r  75% 2-6  Mrad i r r a d i a t i o n . Giovannozzi-Sermanni (1969) reported that cathepsin C was  highly radiation-resistant  with p u r i f i e d fractions.  in ox spleen t i s s u e , compared  In t i s s u e s , the low  catheptic  a c t i v i t y makes i t d i f f i c u l t to conduct such studies ; moreover, vigorous extraction procedures are required lysosomes.  to extract  P u r i f i e d cathepsins might exhibit d i f f e r e n t  radiation s e n s i t i v i t y due to change of environment, and p a r t i a l l y due  to p u r i f i c a t i o n procedure.  Isolated lysosomes  from soft organ tissue such as l i v e r provide the optimum system to study radiation s e n s i t i v i t y of cathepsins close to t h e i r natural environments.  Information on  radiation  inactivation of cathepsins, especially i n lysosomes, i s lacking in l i t e r a t u r e .  However, Desai et a l . (1964) re-  ported that lysosomal B-glucuronidase was  readily i n a c t i -  vated compared with a r y l sulfatase and acid phophatase, while ribonuclease did not show i n a c t i v a t i o n a f t e r 10 - 50  107. Krads of gamma radiation administered to i s o l a t e d rat l i v e r lysosomes.  In the present study, lysosomal cathepsins  A, B, C, and D showed s l i g h t differences i n t h e i r r a d i a tion i n a c t i v a t i o n .  The difference observed by Desai et a l .  (1964) i n the case of non-catheptic lysosomal hydrolases was quite large as compared with the present study. Inactivation of enzymes could be due to general denaturation, destruction, or modification of active centres (Sanner and P i h l , 196 9) and formation of i n h i b i t o r y substances by r a d i o l y s i s .  Variations i n enzyme l o c a l i z a t i o n ,  active centre, and general structure would a f f e c t the extent of i n a c t i v a t i o n of each enzyme.  In view of these  studies, i t seems that net i n a c t i v a t i o n would be complex, and the mode of i n a c t i v a t i o n might be different f o r each of the cathepsins. The results indicate that lysosomal cathepsins are f a i r l y r a d i a t i o n - r e s i s t a n t , and even higher resistance has been reported when i r r a d i a t e d i n tissue (Rhodes and Meegungwan, 1962; Siebert and Musch, 1969), thus residual p r o t e o l y t i c a c t i v i t y may  cause extensive protein breakdown  during post-mortem storage. 3.3.1*2. Soluble enzyme f r a c t i o n at various pHs. The disrupted lysosomes were centrifuged and the super natant fraction i r r a d i a t e d at pH of 4.0, The results of the experiment  5.5, 7.0, or 8.5.  are presented i n Table 5.  Comparative effects of pH 5.5 and 7.0 on radiation i n a c t i v a  108. tion of cathepsins A, B, C, and D are shown i n Figures 12 and 13 respectively.  pH during i r r a d i a t i o n s i g n i f i -  cantly (P<0.01) affected the r a d i o - s e n s i t i v i t y of cathepsins A, B, C, and D.  Table 5.  Although the extent of i n a c t i v a t i o n  E f f e c t of pH on radiation s e n s i t i v i t y of soluble lysosomal cathepsins A, B, C, and D. Residual enzyme a c t i v i t y (% of control) Dose (Mrad)  pH 4. 0  pH 5.5  pH 7.0  pH 8.5  0.10 0.25 0.50 1.00  59. 28 41. 07 17. 14 7. 7 8  55. 77 37. 62 25. 08 22.,77  63. 57 50. 00 31. 78 15. 35  68. 00 51. 11 40. 89 23. 11  0. 10 0.25 0. 50 1.00  91. 80 72. 13 68. 85 65. 57  81., 82 71.,20 53.,63 48.,20  98.,44 90.,62 85.,93 68., 75  83., 78., 70., 47.,  Cathepsin C  0.10 0.25 0. 50 1.00  61. 60 51. 10 32. 54 23. 48  94.,42 47.. 37 36..53 28,.48  73., 78 68..92 50,.95 35,.52  91..58 87..50 65,.49 47,.28  Cathepsin D  0.10 0.25 0.50 1.00  94. 30 76. 25 46. 56 2 8.50  77,.29 54,.46 38,.07 15,. 59  69,.45 53,.17 50,.28 26,. 86  75,.79 63,. 57 46,.04 30,.20  Enzyme Cathepsin A  Cathepsin B  n =5  75 75 00 50  Figure  12,  R a d i a t i o n i n a c t i v a t i o n of s o l u b l e l y s o s o m a l c a t h e p s i n s at pH 5,5. (n = 5)  Figure 13.  Radiation inactivation of soluble lysosomal cathepsins at pH 7.0. (n = 5 )  111. varied with the change of pH, cathepsin B showed highest radiation-resistance over the entire range of pH, followed by cathepsin C.  Cathepsin A was found to be most sensi-  tive to i r r a d i a t i o n under these conditions. E f f e c t of pH on i r r a d i a t i o n i n a c t i v a t i o n of lysosomal cathepsins was different f o r cathepsins A, B, G, and D (Table 5).  Cathepsins A and C were most sensitive at pH  4.0 and least sensitive at pH 8.5.  Cathepsins B and D  had the highest i n a c t i v a t i o n at pH 5.5 and the lowest at pH 7.0 and 8.5 respectively.  A l l the enzymes studied  showed r e l a t i v e l y high radiation s e n s i t i v i t y at a c i d i c pH, the optimum pH range f o r t h e i r reactions.  Results  of the present study f o r cathepsin C are contradictory to studies of Giovannozzi-Sermanni  et a l . (1969) on p u r i f i e d  cathepsin C from ox spleen; they found an increased radiation s e n s i t i v i t y with increasing pH.  These differences  might be due to o r i g i n of cathepsin C as well as state of p u r i t y , as presence of other components w i l l greatly affect the r a d i o s e n s i t i v i t y of enzymes (Sanner and P i h l , 1969).  Results of t h i s study agree with the report of  Robins and Butler (1962), who found an increased radiation resistance of trypsin with increasing pH of s o l u t i o n , and the study of Delincee and Radola (1974), who observed higher i r r a d i a t i o n i n a c t i v a t i o n of horseradish peroxidase at pH 4.0 as compared with pH 7.2 and 10.0.  The effect  of pH i s largely due to i t s influence on dissociation  112.  of  p r o t e i n s , which i n turn may influence the radiation  s e n s i t i v i t y by a l t e r i n g protein conformation (Sanner and P i h l , 1969), and d i s t r i b u t i o n of r a d i c a l species produced by r a d i o l y s i s i n aqueous system (Robins and Butler, 1962). The difference i n i n a c t i v a t i o n pattern between cathepsins i r r a d i a t e d i n lysosomal suspension and i n soluble form indicates that different mechanisms of i n a c t i v a t i o n are  probably involved i n d i f f e r e n t environments.  In l y -  sosomal suspension, the direct e f f e c t of radiation might be r e l a t i v e l y greater than i n free enzyme systems where the  enzyme i s i n soluble form, and more l i k e l y subject to  i n d i r e c t effects . The i n a c t i v a t i o n pattern of soluble cathepsins (Figure 13) is the reverse of that obtained with i r r a d i a t i o n of intact lysosomal suspensions (Figure 11). Such a change may be due to dissociation of enzymes from lysosomal structure, where enzymes would be protected to a varying degree by binding or association with other lysosomal components; but  after disruption of lysosomes and fractionation of  soluble enzyme as used i n t h i s experiment, no such protective e f f e c t i s involved, hence change i n environment might be responsible f o r this marked change i n response to i r r a diation of cathepsins.  Nature and extent of radiation  damage to the cathepsins might be d i f f e r e n t under changed environments as Lynn (1972) observed that t r y p s i n , when complexed with s i l i c a before i r r a d i a t i o n , showed a change  113. i n i t s r e l a t i v e a c t i v i t y to synthetic substrates.  Radiation  damage to amino acid residues of the complexed enzymes has been shown t o be different as compared with dissolved enzyme (Holladay et a l . , 1966; Copeland et a l . , 1967). Comparison of Figures 11 and 13 shows that r a d i a t i o n inactivation was s u b s t a n t i a l l y increased when soluble enzymes were i r r a d i a t e d .  This i s i n general agreement  with reports that enzymes present i n tissue (GiovannozziSermanni, 1969) or complexed with other materials (Lynn, 1972, 1974) show higher r a d i a t i o n resistance, due to protective effects of the other compounds present i n the system, thus decreasing the damage due to i n d i r e c t e f f e c t s of i r r a d i a t i o n , especially by free r a d i c a l s . Variation i n radiation s e n s i t i v i t y of cathepsins A, B, C, and D may be p a r t i a l l y due to the difference i n l o c a l i z a t i o n as well as binding mechanisms within lysosomes (Koenig, 1969).  3.3.2.  A v a i l a b i l i t y of Cathepsins A, B, and C a f t e r I r radiation of Intact and Disrupted Lysosomes  Lysosomes were i r r a d i a t e d with various doses of gamma r a d i a t i o n , before or after disruption, to study the effect of radiation on available enzymes.  Determinations  of  a c t i v i t y of cathepsins A, B, and C were made using d i s rupted lysosomes without c e n t r i f u g a t i o n , so that a c t i v i t y of p a r t i a l l y bound or adsorbed enzyme could be included.  114. The results f o r cathepsin A are presented i n figure  14.  Cathepsin A a c t i v i t y was found to decrease s i g n i f i c a n t l y (P<0.01) after i r r a d i a t i o n of disrupted or intact  lysosomes.  Available enzyme content i n disrupted lysosomes was found to be s i g n i f i c a n t l y (P<0.05) higher than intact after 0.25  lysosomes  and 0.50 Mrad doses, but at a higher dose of  1.0 Mrad no s i g n i f i c a n t difference was noted. Cathepsin B a c t i v i t y of intact or disrupted  lysosomes  decreased s i g n i f i c a n t l y (P<0.01) after i r r a d i a t i o n ment, but the difference i n available  treat-  enzyme content of  intact and disrupted lysosomes was not s i g n i f i c a n t at any dose (Figure 15). Cathepsin C also followed the f a m i l i a r pattern of radiation  inactivation  (Figure 16), showing  significant  (P<0.01) decrease i n a c t i v i t y i n both intact and disrupted lysosomes.  Enzyme a c t i v i t y i n disrupted lysosomes  was  higher than i n intact lysosomes f o r the 0.5 Mrad dose. These observations suggest that the lysosomal membrane does not have much protective e f f e c t .  However,  enzymes i n the disrupted system might have been adsorbed or bound to lysosomal fragments and have thus been protected from i r r a d i a t i o n inactivation. the p r e - i r r a d i a t i o n  disruptive  I t i s also possible that treatment increased the  a v a i l a b i l i t y of enzymes , and thereby compensated f o r the higher subsequent i n a c t i v a t i o n .  Presence of lysosomal  fragments i n the suspension medium would provide some  Figure 14.  A v a i l a b i l i t y of cathepsin A after i r r a d i a t i o n of disrupted and intact lysosomes. (n = 4)  F i g u r e 15.  A v a i l a b i l i t y of cathepsin B a f t e r i r r a d i a t i o n of d i s r u p t e d and i n t a c t lysosomes, (n = 4 )  Figure  16.  A v a i l a b i l i t y of cathepsin C after i r r a d i a t i o n of disrupted and intact lysosomes. (n = 4)  118. protection to enzymes against i n d i r e c t effects of i r r a diation by trapping water radicals and preventing formation of secondary r a d i c a l s .  3.3.3. Radiation Inactivation of Cathepsin D under Various Conditions 3.3.3.1. Sucrose concentration. Presence of other compounds i n the suspension medium considerably affects the radiation s e n s i t i v i t y of enzymes when i r r a d i a t e d i n solution (Sanner and P i h l , 1969).  In  the present study, sucrose concentration was varied from 0.1M to 0.7M at pH 7.0 p r i o r to i r r a d i a t i o n at 0.50 Mrad. The results are shown i n Figure 17,  At 0.1M sucrose con-  centration, residual a c t i v i t y of cathepsin D f e l l to 52.75% of the control value, but an increase of sucrose  conentra-  tion i n suspension medium to 0.2 5M s i g n i f i c a n t l y (P<0.01) protected the enzyme from radiation i n a c t i v a t i o n . ing  Increas-  sucrose concentration to 0.5M did not provide any  further increase i n radiation protection.  The maximum  protection was observed at 0.7M sucrose l e v e l , when r e s i dual a c t i v i t y was found to be 82.4 8% of unirradiated cont r o l value.  These results are consistent with other studies  (Jung, 1967; Schuessler, 1973; Dale, 1942; Sanner and P i h l , 1967), showing protective effects of various compounds present i n the suspension medium.  Such a protective effect  i s due mainly to interaction of thesei compounds with free  119.  F i g u r e 17.  E f f e c t o f s u c r o s e c o n c e n t r a t i o n on radiation s e n s i t i v i t y of cathepsin D a f t e r 0 . 5 Mrad dose. (n = 3 )  120. radicals which would otherwise interact with enzyme molecules and cause i n a c t i v a t i o n . 3.3.3.2. Temperature. I r r a d i a t i o n of lysosomal suspensions was carried out at ambient chamber temperature or 0°C to determine the influence  of temperature on i n a c t i v a t i o n of lysosomal  cathepsin D.  After an i r r a d i a t i o n dose of 0.50 Mrad at  ambient temperature and 0°C, the residual a c t i v i t y of cathepsin D was 69.59 and 83.38 percent of the control value • respectively.  This increased radiation-resistance  of  cathepsin D at 0°C i s i n agreement with the findings of Schults et a l . (1975), who observed a marked decrease i n radiation s e n s i t i v i t y of p r o t e o l y t i c enzymes at low temperature i n raw beef, pork, and chicken muscle.  A similar  effect was found on p r o t e o l y t i c a c t i v i t y of ground beef after i r r a d i a t i o n at low temperature (Losty et a l . , 1973). It has been suggested that low i r r a d i a t i o n temperatures should be used to avoid irradiation-induced.flavour in radiation processed meats. radiation-resistance  changes  However, the increased .  of p r o t e o l y t i c enzymes at low temp-  eratures suggests a hazard from undesirable residual prot e o l y t i c action.  3.3.4. Radiation - Heat Combination Treatment f o r Inactivation of Lysosomal Cathepsin D Proteolytic enzymes are more radiation-resistant i n  121. tissue than i n lysosomal suspension and t h e i r t o t a l i n a c t i vation would require very high doses of radiation.  How-  ever, the enzymes are heat l a b i l e and a combination of gamma radiation and heat might be expected to produce a synergistic effect based on similar finding by other workers (Glew, 1962; Farkas and Goldblith, 1962).  The  results of a radiation - heat combination treatment are shown i n Table 6 and demonstrated that although both i r r a diation and heat alone could reduce the residual a c t i v i t y of soluble  cathepsin D s i g n i f i c a n t l y (P<0.01); both f a i l e d  to inactivate the enzyme completely at the l e v e l s used.  Table 6.  Effect of i r r a d i a t i o n - heat combination t r e a t ment on inactivation of cathepsin D. • Residual enzyme a c t i v i t y (% of control)  Irradiation dose (Mrad) 0.00 0.50 1. 00 2.00  Room temp. 100.00 31.90 15.23 5.73  Heat treatment 50°C  60°C  70°C  80°C  90°C  89.60  57.70  27.24  19.35  12.19  22.40  6.90 9. 32 0.00  5.91 5. 01  5.91  11.47  6.63 7.45 0.00  0.00  2.11  n =5 * 10 minutes heating at given temperature i n constant temperature water bath  3.51 0.00  A combination treatment of 2.0 Mrad gamma radiation followed by 10 minutes heating at 6 0°C resulted i n complete vation of cathepsin D.  inacti-  I r r a d i a t i o n of 1.0 Mrad alone  reduced the enzyme a c t i v i t y to 15.2 3%, while the r a d i a tion of 0.5 Mrad followed by 10 minutes heating was much more e f f e c t i v e i n reducing residual enzyme a c t i v i t y to 6.9%; thus by radiation - heat combination treatment, the i r r a d i a t i o n requirement can be reduced markedly.  Glew  (1962) has shown that heating at 50°C a f t e r radiation treatment had a synergistic e f f e c t on milk phosphatase inactivation and Farkas and Goldblith (1962) have found similar effects on lipoxidase.  To reduce the r e s i d u a l  a c t i v i t y of p r o t e o l y t i c enzymes i n t i s s u e s , some studies have been conducted using various combination treatments. Cain and Anglemier (1969) reported that heating of beef to 140°F before i r r a d i a t i o n s i g n i f i c a n t l y reduced the soluble nitrogenous constituents during storage.  Losty  et a l . (1973) found that 2 - 6 Mrad gamma i r r a d i a t i o n alone destroyed p r o t e o l y t i c a c t i v i t y of ground beef up t o 75%, but a combination of 4.5 - 5.2 Mrad plus blanching at 6 5° or 75°C reduced residual a c t i v i t y to 5%. Results of present experiment are i n agreement with the abovementioned reports, i n that residual a c t i v i t y can be considerably reduced by combination treatment.  Due t o higher  radiation-resistance of p r o t e o l y t i c enzymes i n t i s s u e , higher radiation dose as well as elevated temperature would be required.  123. 3.3.5. Radiation-induced Changes i n Substrate. Proteolysis  has been shown to be a cause of degra-  dation of i r r a d i a t e d tissue (Cain et a l . , 1958;  Drake et  a l . , 1961), and much attention has been given to the zymes responsible for i t .  en-  Irradiation of tissue would also  affect the endogenous proteins which are l a t e r attacked by tissue proteolytic enzymes.  In tissue autolysis i t  is d i f f i c u l t to d i f f e r e n t i a t e between the r e l a t i v e effects of i r r a d i a t i o n on enzymes and endogenous proteins.  In  the previous section, studies on i r r a d i a t i o n of lysosomal cathepsins have been presented.  This section deals with  the effects of i r r a d i a t i o n on hemoglobin used as substrate for cathepsin D. 3.3.5.1. Hydrolysis of i r r a d i a t e d hemoglobin at different £Hs. Hemoglobin was in 10%  i r r a d i a t e d at ambient temperature  aqueous solutions.  Hydrolysis of 2.0%  irradiated  hemoglobin substrate by lysosomal cathepsins was at pH 3.8, in Table 7.  5.0,  and  7.0.  followed  Results of the study are presented  Irradiation doses of 0.25,  0.5,  and  1.0  each s i g n i f i c a n t l y (P<0.01) reduced the hydrolysis hemogolbin at the three pH conditions used.  Mrad of  124. Table 7.  Hydrolysis of hemoglobin by lysosomal cathepsin D at various pHs a f t e r gamma r a d i a t i o n i n soluble state. Percent hydrolysis  Irradiation dose (Mrad)  pH 3.8  pH 5.0  pH 7.0  0.25  100.00 84.39  100.00 95.17  100.00 97.50  0.5.0 1.00  67.87 44.57  93.79 84.14  96.26 84.11  0.0  n =3 The maximum i n h i b i t o r y e f f e c t was observed at pH 3.8, where cathepsin D has maximum a c t i v i t y ; extent of hydrolysis of hemoglobin at pH 5.0 and 7.0 was s i m i l a r , but s i g n i f i cantly (P<0.05) lower than that observed at pH 3.8. Hemoglobin hydrolysis was maximum at pH 3.8 due t o cathepsin D attack; irradiation-induced denaturation, and changes i n conformation  of protein molecules might have  rendered i t unavailable f o r cathepsin D attack.  Consider-  able a c t i v i t y was observed at pH 5.0 and 7.0, where cathepsin D would s p l i t hemoglobin to a l e s s e r extent, but other enzymes including cathepsins A, B, and C might attack protein or oligopeptides produced by cathepsin D, thus the i n h i b i t o r y e f f e c t of i r r a d i a t i o n i s reduced at higher pH.  Concerted  action of various cathepsins has been r e -  ported by various workers (Liao-Huang et a l . , 1971; Goett-  12 5. lich-Riemann et a l . , 1971;  Iodice et a l . , 1966).  protein breakdown by cathepsin B at pH 5.0 Huisman et a l . , 1974)  (Otto,  1971;  and by neutral protease at pH  (Okitani and Fujimaki, 1972; also been reported.  Moreover,  Okitani et a l . , 1973)  7.0 has  A radiation-induced decrease i n high  molecular weight component and an increase i n low molecular weight components of chicken muscle soluble protein fractions have been observed (Klein and Altman, 1972a). This finding i s consisent with the present results of enzyme hydrolysis of i r r a d i a t e d hemoglobin.  Low  molecular  weight components are not.attacked by cathepsin D at pH thus resulting i n decreased hydrolysis.  Besides  3.8,  the  e f f e c t of reduction i n molecular weight denaturation might also have contributed to reduced susceptability to protein hydrolysis by various cathepsins present i n lysosomal extract used. 3.3.5.2. Hydrolysis of hemoglobin after i r r a d i a t i o n i n soluble or dry state. Irradiation of hemoglobin was  carried out i n soluble  (10%) form or i n the dry state at room temperature.  After  i r r a d i a t i o n , digestion of 2.0% hemoglobin substrate by lysosomal cathepsins was for 2 hours.  determined by incubation at 37°C  Results of this study demonstrated that the  irradiation-induced i n h i b i t o r y e f f e c t was  significantly  (P<0.01) higher on hemoglobin i r r a d i a t e d i n soluble form than i n the dry state (Table 8).  Doses ranging from 0.2 5  126^  to 5.00 Mrads progressively rendered the protein more resistant to attack by cathepsin D.  A high dose of 5.0  Mrads given to dry hemoglobin resulted i n 66.38% h y d r o l y s i s , while an equivalent e f f e c t (67.87%) was observed with only 0.5 Mrad dose administered t o protein i n the soluble state.  Table 8.  E f f e c t of i r r a d i a t i o n of hemoglobin i n the dry and soluble state on i t s hydrolysis by lysosomal cathepsin D at pH 3.8.  Irradiation dose (Mrad) 0.0 0.25 0.50 1.00 5.00  Percent hydrolysis Dry state 100.00 86.58 82.68 77.92 66.38  Soluble  state  100.00 84. 39 67.87 44.57  n = 3  Radiation-induced changes i n substrate might decrease i t s a f f i n i t y f o r enzyme binding.  In dry hemoglobin, radiation  damage would be caused by "direct e f f e c t s " but i n soluble state the protein would undergo additional changes due to " i n d i r e c t e f f e c t s " of i r r a d i a t i o n .  Moreover, i r r a d i a t i o n  damage to hemoglobin i n dry and soluble state might be of a different nature.  This variation i n extent and nature  of i r r a d i a t i o n damage to hemoglobin i s r e f l e c t e d i n i t s  127. hydrolysis by cathepsin D (Table 8). 3.3.5.3. Electrophoretic pattern of i r r a d i a t e d hemoglobin a f t e r hydrolysis by cathepsin D. Irradiated hemoglobin solution was d i l u t e d to 5% and used as substrate  for hydrolysis by cathepsin D at pH 3.8.  After incubation of 5 hours at 3 7°C, samples were drawn and lOul  was employed for electrophoresis.  are presented i n Figure 18.  The r e s u l t s  The electrophoretogram of  control samples showed changes i n various bands (Figure 18, slots 1, 3, and 5) a f t e r i r r a d i a t i o n .  Intensity of band A  decreased with an increase i n band C and D i n 0.5 and 1.0 Mrad i r r a d i a t e d hemoglobin (slots 3 and 5 respectively) compared with unirradiated sample ( s l o t 1).  Hydrolysis of  n o n - i r r a d i a t e d and i r r a d i a t e d hemoglobin showed that bands A and C primarily were decreased a f t e r digestion by cathepsins.  A s l i g h t decrease i n band D was also observed  (slots 2, 4, and 6).  After digestion of i r r a d i a t e d hemo-  globin (slots 4 and 6), band A showed a s l i g h t l y higher intensity and some smearing between bands A and B occurred, as compared with digestion of non-irradiated sample ( s l o t 2). It i s i n t e r e s t i n g to note that mainly bands A and C are affected by i r r a d i a t i o n treatment (slot 1 compared with slots 3 and 5), and the same bands are reduced due to catheptic digestion (slots 2, 4, and 6).  I t i s l i k e l y that  changes i n hemoglobin molecules as observed i n the change of electrophoretic pattern after i r r a d i a t i o n treatment are  128.  1  3  2  Control Figure 18.  4  0.50 Mrad  5  6  1.00 Mrad  Electrophoretic pattern of i r r a d i a t e d hemoglobin before (1, 3, and 5) and a f t e r (2, 4, and 6) catheptic digestion at pH 3.8.  129. responsible f o r i t s reduced digestion by lysosomal sin D at pH 3.8  (Table 8).  cathep-  Delincee and Radola (1974)  observed extensive modification of charge properties i n horseradish peroxidase  protein a f t e r gamma i r r a d i a t i o n  as studied by t h i n - l a y e r i s o e l e c t r i c focusing. noted aggregation  of protein.  They also  A l t e r a t i o n i n electrophoretic  mobility of i r r a d i a t e d horse heart myoglobin (Paul and Kumta, 1973), casein and egg albumin (McArdle and  Desrosier,  1955), myoglobin (Satterlee et a l . , 1972), and soluble muscle proteins (Klein and Altmann, 1972a; Uzunov et a l . , 1972)  also have been observed.  It has been suggested that  protein structure i s altered by radiation-induced breakage of s u l f u r linkages or hydrogen bonds (McArdle and Desrosier, 1955) , covalent bonds of the polypeptide  chains  or hydrogen and i o n i c bonds between the side chains (Uzunov et a l . , 1972). 3.3.5.4. Spectral changes i n i r r a d i a t e d hemoglobin. After i r r a d i a t i o n of a 10% solution of hemoglobin, measurements of the v i s i b l e spectrum were made on diluted samples for evaluation of radiation-induced changes i n the protein used as a substrate f o r cathepsin D. of various doses i s shown i n Figure 19.  The  effect  Increase i n ab-  sorbance i n the 520 - 540 and 560 - 580 nm regions, and decreases in the 480 - 500 and 600 - 630 nm regions were observed.  Moreover, the Soret band peak around 400  decreased as a result of i r r a d i a t i o n .  nm  These observations  130.  A— BCD-  400  Figure 19.  450  CONTROL 0.25 M r a d 0.50 M r a d 1.00 M r a d  500 550 W A V E L E NGTH (nm)  600  650  Radiation-induced spectral changes i n hemoglobin substrate.  131;. are i n agreement with the studies carried out on myoglobins (Clarke and Richards, 1971;  Satterlee et a l . , 1971).  Spectral changes have been attributed to a l t e r a t i o n s in the protein moiety (Lycometros and Brown, 197 3.-, ' Borwn and Akoyunoglou, 1964) nucleus  as well as to rupture of the hemin  (Clarke and Richards, 1971).  Radiation-induced  polymerization of myoglobin has been reported by Lycometros and Brown (1973).  Satterlee et a l . (1972) observed that  aggregation of metmyoglobin increased with radiation dosage They also reported that i r r a d i a t e d metmyoglobin had a lower a - h e l i c a l content, i n d i c a t i n g that some of the i r r a d i a t e d molecules might exist in a p a r t i a l l y unfolded, state.  It  seems quite l i k e l y that such radiation-induced a l t e r a t i o n s in heme-proteins may  be p a r t i a l l y responsible f o r decreased  hydrolysis of hemoglobin by.lysosomal cathepsins, observed in the present  study.  132. 3.4.  SUMMARY AND  CONCLUSIONS  Radiation i n a c t i v a t i o n of lysosomal cathepsins A, B, C, and D has  been studied i n t h i s chapter.  It was  that a l l of the cathepsins showed a high resistance  found to  radiation i n a c t i v a t i o n when i r r a d i a t e d i n lysosomal suspension.  It was  noticed  that cathepsin B was  slightly  more sensitive to radiation compared with cathepsins A, and D, and the Jrighest resistance Residual a c t i v i t y after 1.0 78.7,  67.0,  70.2,  was  C,  shown by cathepsin A.  Mrad dose was  found to be  72.58% for cathepsins A, B, C, and  D  respectively. A soluble enzyme f r a c t i o n i s o l a t e d from lysosomes was  disrupted  found to be considerably more sensitive than  the enzyme i r r a d i a t e d in the lysosomal environment. radiation of disrupted  Ir-  and i n t a c t lysosomes showed s i m i l a r  effects on a v a i l a b i l i t y of the enzymes, suggesting that association  of enzymes with lysosomal fragments imparted a  radioprotective  effect.  However, disruption of lysosomes  p r i o r to i r r a d i a t i o n may  increase the a v a i l a b i l i t y of the  enzyme, and thus compensate for the i n a c t i v a t i o n e f f e c t . Soluble enzymes exhibited acidic pH pH  (7.0  (4.0  and  and  8.5),  5.5),  r a d i a t i o n - s e n s i t i v i t y at  which decreased with  increasing  thus demonstrating that these enzymes  w i l l be more radiation-resistant at physiological pH  and  drop i n pH of post-mortem muscle might have some sensitization effect.  Studies on cathepsin P revealed that sucrose concent r a t i o n i n the suspension medium and temperature of i r r a d i a tion influenced radio s e n s i t i v i t y ; 0.5M sucrose Concentration s i g n i f i c a n t l y (P<0,01) increased radio-protection and 0°C provided a greater degree of radio-protection than 2 3°C.  Heating of i r r a d i a t e d cathepsin D solution decreased  the r e s i d u a l a c t i v i t y , demonstrating that r a d i a t i o n - heat combination process could be used to reduce the i r r a d i a t i o n dose l e v e l , which would be b e n e f i c i a l i n avoiding the undesirable effects of high doses of i r r a d i a t i o n required to reduce r e s i d u a l p r o t e o l y t i c a c t i v i t y i n meats. The mechanism involved i n r a d i a t i o n - i n a c t i v a t i o n of cathepsins is not clear.  Its elucidation i s made more d i f f i c u l t by  i n s u f f i c i e n t characterization of lysosomal cathepsins and information regarding t h e i r l o c a l i z a t i o n within lysosomes. Most of the other studies have concentrated  their  e f f o r t s i n residual p r o t e o l y t i c a c t i v i t y , and less attention has been paid to tissue proteins and t h e i r hydrolysis by proteases.  In the present investigation the observations  made on i r r a d i a t e d hemoglobin used as substrate f o r cathepsin D indicated that hemoglobin i r r a d i a t e d i n soluble form became resistant to enzyme degradation.  Such r e s i s -  tance was higher at pH 3.8 as compared with pH 5.0 or 7.0, thus indicating that i r r a d i a t e d hemoglobin would be markedly resistant to cathepsin D attack but not to other p r o t e o l y t i c enzymes such as cathepsins A, B, or C, which  are active at pH 5.0, or neutral proteases active at pH.7.0. Hemoglobin i r r a d i a t e d i n the dry state exhibited resistance  similar  to degradation, but the l e v e l of resistance  was  lower, indicating that such changes are caused by d i r e c t as well as i n d i r e c t effects of i r r a d i a t i o n treatment. Electrophoretic  pattern and spectral c h a r a c t e r i s t i c s sup-  port the conclusion that i r r a d i a t i o n caused considerable changes i n hemoglobin, which might result i n decreased hydrolysis  by p r o t e o l y t i c enzymes.  tissue proteins to catheptic treatment remains to be  S u s c e p t i b i l i t y of  attack a f t e r i r r a d i a t i o n  established.  This study demonstrated that cathepsins are r e l a t i v e l y radiation-resistant  but the extent of resistance  among lysosomal cathepsins.  Presence of other compounds  and pH of the medium influence of these enzymes.  varies  the radiation  inactivation  Radiation-induced changes i n protein  substrate would also influence  the course of proteolysis.  These results indicate that i r r a d i a t i o n i n a c t i v a t i o n of tissue enzymes i s a complex phenomenon and residual prot e o l y t i c a c t i v i t y of tissue would be influenced factors.  by various  Irradiation - heat combination treatment seems  to be a promising choice to reduce the residual enzyme activity.  135, CHAPTER 4.  EFFECT OF GAMMA RADIATION ON ULTRASTRUCTURE OF LYSOSOMES AND  CHICKEN SKELETAL MUSCLE  4.1. INTRODUCTION 4.1.1. Lysosomes The role of lysosomal cathepsins i n post-mortem meat tenderization i s not well•elucidated, but several studies have demonstrated a release of lysosomal cathepsins and other hydrolases during the aging period (Lutalo-Bosa, 1970; Ono,  1971; Eino and Stanley, 1973a; Dutson and Lawrie, 1974).  Release of lysosomal enzymes from i s o l a t e d p a r t i c l e s under various conditions have been reported (Sawant et a l . , 1964a; Hayashi et a l . , 1973; Verity et a l . , 1968).  Ionizing  radiation has also been shown to decrease the s t a b i l i t y of lysosomes and enhance the release of enzymes (Desai et a l . , 1964; Watkins, 1970; Watkins and Deacon, 1973).  Release  of these enzymes i s probably a result of alterations i n lysosomal structure, especially lysosomal membranes. Shibko et a l . (1965) reported that lysosomes l o s t electrondense material as a r e s u l t of incubation at 37°C f o r 3 hours, but retained intact outer membranes as observed i n electron micrographs.  Brunk and Ericsson (1972) demonstrated cyto-  chemically that acid phosphatase could-leak through s t r u c t u r a l l y intact lysosomal membranes.  ultra-  In the present  study, an attempt has been made to observe the influence of ionizing i r r a d i a t i o n on ultrastructure of  lysosomes,  an  d i t s possible effects on leakage of lysosomal contents.  4.1.2. Muscle During post-mortem storage of muscle, u l t r a s t r u c t u r a l changes have been observed.  Studies have indicated that  during aging of beef and chicken Z-I junction i n myofibrils is weakened and breaks across the f i b r i l s occur near Z-line (Davey and G i l b e r t , 1967; Fukazawa et a l . , 1969).  Similar  breakage of myofibrils occurs i n aged bovine and chicken muscle when subjected  t o mechanical stress (Davey and  Dickson, 1970; Sayre, 1970).  Schaller and Powrie (1971)  demonstrated that besides breaks occurring at weak points l i k e Z-I junction i n aged muscles, disruption of sarcolemma and sarcoplasmic reticulum i s also possible. radiation deposits  Ionizing  energy at random, and i t i s d i f f i c u l t  to predict i t s effects on ultrastructure of muscle, but the above-mentioned weak points might be vulnerable tion damage.  to radia-  In the present study, e f f o r t has been made  to assess the alterations i n ultrastructure of i r r a d i a t e d chicken s k e l e t a l muscle with the help of transmission electron microscopy (TEM) and scanning electron microscopy (SEM).  137. 4.2. EXPERIMENTAL 4.2.1. General Sample Preparation 4.2.1.1. Lysosomes. Irradiated and non-irradiated samples of f r a c t i o n were incubated at 4°C or 37°C.  lysosomal  Samples were  drawn at 0, 48, and 72 hours a f t e r incubation at 4°C after 1 hour at 37°C.  and  A l l the lysosomal suspensions were  centrifuged at 17,0 00 x g for 2 0 minutes at 4°C, and the p e l l e t was used f o r f i x a t i o n and further processing for EM studies. 4.2.1.2. Tissue. Breast muscle (Pectoralis major) samples were rapidly removed and subjected to gamma radiation i n the Gamma C e l l 220 unit.  The cryofracture technique  Powrie (1971) was  of Schaller and  used to prepare samples for f i x a t i o n .  Cubes of t i s s u e , about 1 cm along each edge, were immersed in l i q u i d nitrogen, a frozen sample was placed between two sheets of plexiglass and struck with a hammer head to fracture the f r i a b l e material.  Frozen fragments approx-  imately 3 mm i n diameter were taken f o r f i x a t i o n and  fur-  ther preparation f o r scanning electron microscopy. Tissue samples from chicken breast muscle, about 2 mm i n diameter, were removed immediately a f t e r i r r a d i a tion and further processed for transmission electron microscopy.  138. 4.2.2. Sample Processing f o r Electron Microscopy Lysosomal p e l l e t s and tissue samples were fixed by immersion i n 2% glutaraldehyde i n 0.1M cacodylate-HCl buffer pH 7.2 containing 0.1M sucrose at 4°C f o r 1 hour (Brunk and Ericsson, 1972).  The specimens were washed  3 times with the buffer and placed i n 1% OsO^-O.lM sucrose in 0.1M cacodylate-HCl buffer pH 7.2 at 4°C.  The specimens  were dehydrated s e r i a l l y i n water-ethanol solutions of 30 , 50 , 70 , 80,, 90 , and 100% ethanol. 4.2.2.1. Scanning electron  microscopy.  After dehydration, the specimens were placed i n amyl acetate-ethanol solutions of 25 ,50  , 75 , and  100% amyl acetate to replace ethanol and then dried i n a c r i t i c a l point d r i e r (Parr Instrument with C0  ?  Co;..) by flushing  f o r 20 minutes under pressure, followed by  evaporation f o r 2 0 minutes.  The dried fragments of  tissue and lysosomal p e l l e t were mounted to an aluminum holder with s i l v e r cement and were coated with 60% gold, 40% palladium i n a vacuum evaporator (Hummer I-Technics Inc.) using D. C. Sputtering system f o r coating.  Samples  were examined with an ETEC Autoscan - scanning electron microscope operated.at 2 0 KV.  Images were recorded on  Polaroid type 55P/N f i l m . 4.2.2.2. Transmission electron  microscopy.  After dehydration, the specimens were i n f i l t r a t e d with araldite-epon mixture.  The embedded samples were  139. cured f o r 2 4 hours at 7 0°C.  Sections were cut from the  hardened resin blocks on a Carl Reichert Om U3 Ultramicrotome. Thin sections (approximately  60oR)  were mounted on  uncoated 300 mesh copper grids and stained with uranyl acetate followed by lead c i t r a t e .  The sections were exam-  ined with an AEI Corinth 275 transmission electron microscope operated at 6 0 KV.  The sample chamber was  cool with l i q u i d nitrogen during observations.  kept  A photo-  graphic record of the images was made using Agfa Gevatex T51P  70 mm  film.  140. 4.3. RESULTS AND DISCUSSION 4.3.1. Radiation-induced Changes i n Ultrastructure  of  Lysosomes 4.3.1.1. Internal ultrastructure  of  lysosomes.  The present communication w i l l consider the e f f e c t of radiation on u l t r a s t r u c t u r a l changes i n lysosomes under different incubation conditions, non-irradiated p a r t i c l e s .  and t h e i r comparison with  Transmission electron  micro-  scopy was used to observe the i n t e r n a l structure,  while  scanning electron microscopy was employed to observe the surface ultrastructure  of the lysosomal p a r t i c l e s .  Figures 2.0 and 21 are TEM micrographs control lysosomal p a r t i c l e s .  of non-irradiated  Different shape and size of  p a r t i c l e s i s apparent: most of them have a spherical appearance.  Some p a r t i c l e s have a dense matrix (Figures 2 0 -21,  L I ) , while others have a much l i g h t e r matrix (Figure 20 21, L2).  In most of the p a r t i c l e s , a single layer mem-  brane i s distinguishable. lysosomes (de Duve, 1963a). possibly  This feature i s c h a r a c t e r i s t i c of Some amorphous  material,  c e l l debris or remains of damaged p a r t i c l e s , can  be found among the intact p a r t i c l e s .  Almost a l l of the  lysosomal p a r t i c l e s observed were turgid with a dense granular matrix contained within a l i m i t i n g membrane. After incubation of lysosomal p a r t i c l e s at 4°C, the lysosomal matrix appears less dense (Figure 22).  The l y -  sosomal membrane of some of the p a r t i c l e s i s diffused  and  141.  Figure 20.  Transmission electron micrographs of non i r r a d i a t e d lysosomes without incubation. LI = lysosomes with dense matrix; L2 = lysosomes with l i g h t matrix; MV = multi vesicular body 75,000X.  142,  Figure 21.  Transmission electron micrographs of nonirradiated lysosomes without incubation. LI = lysosomes with dense matrix; L2 = lysosomes with l i g h t matrix; A, 75000X; B, 80.000X; C, 80.000X; D, 120,000X.  143.  Figure 22. Transmission electron micrographs of noni r r a d i a t e d lysosomes after 4 8 hour incubation at 4 C; arrow = leakage of lysosomal content; dm = diluted matrix; mv = multivesicular body. A, 50,000X; B and C, 75,000X.  144. i r r e g u l a r , suggesting  that loss of intralysosomal material  could have occurred as a r e s u l t of weakening of the membrane or a change i n i t s permeability.  Apparent leakage  of i n t e r n a l material can be seen i n Figure 22-C (arrow), through weakened but intact lysosomal membrane. D i l u t i o n of matrix appears to occur i n some p a r t i c l e s (Figure 2 2 - B , dm).  Upon prolonged incubation, most of the p a r t i c l e s  were disrupted (Figure 23); p a r t i c l e s with a low density matrix appeared to disintegrate r e a d i l y .  Lysosomes with  a dense matrix were also subject to disruption (Figure 23-A  and C; arrows), but the dense matrix appeared less  readily dispersible and tended to clump together.  Similar  resistance to dispersion was exhibited by T r i t o n X-100 treated lysosomes (Figure 24).  A l l of the p a r t i c l e s are  disrupted, but few clumps of dense material are s t i l l present. Leakage of lysosomal matrix material appeared to be enhanced by gamma r a d i a t i o n treatment of lysosomal particles.  After 48 hours of incubation at 4°C, most of  the p a r t i c l e s appeared as hollow rings of lysosomal membrane with very l i t t l e dense material contained within (Figure 2 5).  A f t e r 72 hours, very few p a r t i c l e s showed  intact lysosomal membrane; even the dense type p a r t i c l e s were disrupted (Figure 26). amount of scattered lysosomal  lysosomal  A considerable  debris was observed i n the  TEM micrographs, with some dense matrix clumps (Figure 26,  145.  Figure 23.  Transmission electron micrographs of noni r r a d i a t e d lysosomes a f t e r 72 hours incubation at 4°C; arrow = disrupted dense lysosomes; dmv = disrupted multi vesicular body; rm = residual material. A, 30,000X; B, 50.000X; C, 80,000X.  146.  Figure 24. Transmission electron micrographs of lysosomes disrupted by Triton X-100 treatment; arrows = clumps of lysosomal dense matrix. A, 50,000X; B, 80,000X; C, 3O,0O0X  Figure 25.  Transmission electron micrographs of irradiated (1.0 Mrad) lysosomes after 48 hours incubation at 4°C. A, 75.000X; B, 50.000X; C, 150,000X.  148.  Figure 26.  Transmission electron micrographs of irradiated lysosomes (1.0 Mrad) after 72 hours incubation at 4°C; Lr = lysosome after release of i t s contents; rm = released material from lysosome; arrows = clumps of dense lysosomal matrix. A, 45,000X; B, 120.000X; C and D, 50,000X.  149. arrows), showing extensive disruption of the p a r t i c l e s . Incubation of lysosomal p a r t i c l e s at 37°C seemed to enhance the leakage of lysosomal matrix material and disruption of lysosomes (Figures 27, 28).  In non-irradiated  samples, a few of the p a r t i c l e s showed almost complete leakage of intralysosomal material a f t e r 1 hour of incubation (Figure 2 7-A), but most of the p a r t i c l e s part of t h e i r dense material i n t h e i r matrix  contained  (Figure 27,  b and c).  However, some disruption of p a r t i c l e s was also  observed.  I r r a d i a t i o n of p a r t i c l e s considerably enhanced  the leakage of material (Figures 29, 30, 31). The majo r i t y of p a r t i c l e s appear as hollow rings containing very l i t t l e matrix material.  Integrity of the membrane seemed  to be affected by i r r a d i a t i o n treatment, as the membrane looked disorganised.  In Figures 2 9-B and 30-C (arrows),  two i r r a d i a t e d p a r t i c l e s show leakage o f material through weak points i n the lysosomal membrane.  The dense p a r t i c l e s ,  which were found to be r e l a t i v e l y resistant to disruption and leakage of the i n t e r n a l material on incubation (Figures 27, 28), seem to have been affected greatly by i r r a d i a t i o n treatment, r e s u l t i n g i n either disruption (Figure 31, dL) , or extensive leakage of lysosomal  content  (Figure 30, Lc)  through a weakened and disorganised lysosomal membrane. In Figure 31-C (dm), a p a r t i c l e with a disrupted membrane and leaking dense material i s shown.  Another segment of  the membrane appears to be s t i l l organised, with only a few  150.  Figure 27. Transmission electron micrographs of noni r r a d i a t e d lysosomes after 1 hour incubation o at 37 C; a = complete leakage of lysosomal content; b and c = p a r t i a l leakage; cv = cavity. A, B, and D, 50,000X; C, 30,000X.  151.  Figure 28. Transmission electron micrographs of noni r r a d i a t e d lysosomes after 1 hour incubation at 37°C showing various stages of leakage of lysosomal contents (a, b, and c ) . A, 30,000X; B and C, 80,000X; D, 200,000X.  152.  Figure 29. Transmission electron micrographs of irradiated lysosomes (1.0 Mrad) after 1 hour incubation at 3 7°C; arrow weak point i n lysosomal membrane showing leakage. A, B, and C. 80,000X; D, 120,000X. =  153.  F i g u r e 30. T r a n s m i s s i o n e l e c t r o n micrographs o f i r r a d i a t e d lysosomes (1.0 Mrad) a f t e r 1 hour i n c u b a t i o n at 37°C; Lc = l e a k e d l y s o s o m a l c o n t e n t s ; arrow = weak p o i n t i n l y s o s o m a l membrane showing leakage. A, 80,000X; B and C, 50,000X; D, 60,000X.  154.  Figure 31. Transmission electron micrographs of i r r a d i a t e d lysosomes (1.0 Mrad) after 1 hour incubation at 37°C; dL = disrupted lysosomes; mv = multivesicular body; dm = damaged membrane; arrows = weak points i n the membrane. A, 30,000X; B, 80,000X; C, 120,000X; D, 200,OOOX.  weak points (arrows).  Another p a r t i c l e i n Figure 30-D  shows a general weakening and disorganisation of the lysosomal membrane, indicating that membrane structure could be extensively damaged by i r r a d i a t i o n . The present observations agree with the results of Shibko et a l . (1965), who observed the loss of electronopaque contents but retention of i n t a c t outer membranes in r a t kidney lysosomes a f t e r 3 hours incubation at 37°C. They also observed a membranous structure and numerous small vesicles present within the l i m i t i n g membrane a f t e r incubation.  In the present study, no such vesicles or  other material indicating intralysosomal membranes, was observed.  The present study also indicated that lysosomal  membranes might be disrupted from weak points and lysosomal contents might leak from such points; t h i s cannot be observed i n TEM micrographs unless the section i s cut through such a plane (Figures 29-B and 30-C).  Moreover,  irradiation  might cause severe damage at such weak points, thus making the mechanism of leakage more apparent when examined under EM.  Multi-vesicular bodies or residual bodies which contain  undigested material might show presence of small vesicles after leakage of soluble contents (Figure 31-B).  Ericsson  and Brunk (1972) demonstrated that, following severe damage to i n v i t r o cultivated c e l l s induced by photosensitization i n j u r y , leakage of Thorotrast p a r t i c l e s from lysosomes occurred through apparent ruptures or holes i n the lysosomal  membranes; but under osmotic shock, release of acid phosphatase from lysosomes occurred by d i f f u s i o n (Brunk and Ericsson, 1972), as detected by cytochemical study.  As  discussed i n Chapter 2, release of cathepsins i s greatly enhanced a f t e r i r r a d i a t i o n treatment; i t i s possible that i r r a d i a t i o n treatment causes damage r e s u l t i n g i n rupture or hole formation at the s i t e of already e x i s t i n g "pores" in lysosomal membranes.  Radiation treatment might cause  dissociation or s o l u b i l i z a t i o n of lysosomal matrix, thus speeding up the release o f lysosomal contents. 4.3.1.2. Surface ultrastructure of lysosomes. In SEM micrographs  (Figures 32, 33) surface u l t r a -  structure of non-irradiated lysosomal fractions i s presented. After incubation at 4°C f o r 48 hours, some p a r t i c l e s show apparent leakage of lysosomal content, but the majority of the p a r t i c l e s seem to be i n t a c t and no extensive damage i s observed.  At a few places, amorphous material presum-  ably a r i s i n g from the p a r t i c l e s , can be seen.  Many of the  p a r t i c l e s are spherical but some show elongation.  Irradiated  p a r t i c l e s appear extensively damaged and leakage of lysosomal contents i s evident (Figure 34, 35). A considerable amount of amorphous material can be seen i n the v i c i n i t y of d i s rupted p a r t i c l e s .  Some of the p a r t i c l e s show very c l e a r l y  the leakage of the material from holes occurring i n the lysosomal membrane (Figure 35-A). Comparison of the surface ultrastructure of i r r a d i a t e d  Scanning e l e c t r o n micrographs o f n o n - i r r a d i a t e d l y s o s o m a l p a r t i c l e s a f t e r 48 hours i n c u b a t i o n at 4° C; Lc = leakage o f c o n t e n t s ; arrows = damaged p a r t i c l e s ; r s = roughs u r f a c e d p a r t i c l e . A and B, 24,000X; C, 60,000X; D, 50,000X.  158.  F i g u r e 33. Scanning e l e c t r o n micrographs o f n o n - i r r a d i a t e d l y s o s o m a l p a r t i c l e s a f t e r 48 hours i n c u b a t i o n at 4°C; am = amorphous m a t e r i a l ; Lc = leakage o f c o n t e n t s ; dp = damaged p a r t i c l e s ; arrows = s i t e o f l e a k a g e . A and B, 24,000X; C, 90,000X; D, 360,000X.  Scanning electron micrographs of i r r a d i a t e d (1.0 Mrad) lysosomal p a r t i c l e s after 48 hour incubation at 4°C; am = amorphous material; dp = damaged p a r t i c l e s ; Lc = leakage of contents. A, 60,000X; B, 90,000X; C and D, 50,000X  160.  Figure 35. Scanning electron micrographs of irradiated (1.0 Mrad) lysosomal p a r t i c l e s a f t e r 48 hour incubation at 4 C; am = amorphous material; dp = damaged p a r t i c l e s ; Lc = leakage of contents; arrow = point of leakage; de = depression. A, 90,000X; B, 240,000X of p a r t i c l e encircled i n A.  161. and n o n - i r r a d i a t e d p a r t i c l e s i n d i c a t e s t h a t r a d i a t i o n damages the l y s o s o m a l s t r u c t u r e most p r o b a b l y by weakening the l y s o s o m a l membranes, and r e s u l t i n g i n the o f " h o l e s " a t the weak p o i n t s .  These o b s e r v a t i o n s  and support the r e s u l t s o b t a i n e d w i t h TEM the p r e c e e d i n g  section.  formation  as d i s c u s s e d i n  In g e n e r a l , these  o b s e r v a t i o n s p r o v i d e f u r t h e r evidence  confirm  ultrastructural  t h a t r a d i a t i o n damage  t o l y s o s o m a l s t r u c t u r e i s r e s p o n s i b l e f o r t h e marked i n c r e a s e i n the f r e e a c t i v i t y o f l y s o s o m a l c a t h e p s i n s as d i s c u s s e d e a r l i e r i n Chapter 2.  4.3.2. E f f e c t o f I r r a d i a t i o n oh U l t r a s t r u c t u r e o f S k e l e t a l Muscle 4.3.2.1. T r a n s m i s s i o n Transmission  electron  microscopy.  e l e c t r o n micrographs o f untreated c o n t r o l  muscle 3 hours post-mortem are p r e s e n t e d 37.  i n F i g u r e s 36  and  V a r i o u s bands can be e a s i l y d i s t i n g u i s h e d w i t h normal  i n t e r - f i b r i l spaces.  I n c o n t r a s t t o c o n t r o l m u s c l e , the  i r r a d i a t e d m y o f i b r i l s show severe d i s o r g a n i s a t i o n ( F i g u r e s 38, 39, 40).  Transverse  can be observed  breaks i n i r r a d i a t e d m y o f i b r i l s  i n F i g u r e s 38-A,  B, and C.  The most v u l -  n e r a b l e s i t e f o r breaks seems t o be I band, but  general  d i s o r g a n i s a t i o n o f the m y o f i b r i l has o c c u r r e d .  Twisting  of unattached  ends o f m y o f i b r i l s o c c u r r e d ( F i g u r e 38-D).  In F i g u r e 39, a t r a n s v e r s e break at the I band through number o f m y o f i b r i l s i s shown.  The  I band, and i n some  a  F i g u r e 36. T r a n s m i s s i o n e l e c t r o n micrographs o f noni r r a d i a t e d chicken p e c t o r a l i s muscle, 3 hours post-mortem; A = A band; 1 = 1 band; Z = Z l i n e . A, 30,000X; B, 20,000X; C, 80,000X; D, 20,000X.  163.  F i g u r e 37. T r a n s m i s s i o n e l e c t r o n micrographs o f noni r r a d i a t e d c h i c k e n p e c t o r a l i s muscle, 3 hours post-mortem; A = A band; 1 = 1 band; Z = Z l i n e . A, 18,000X; B,  75,000X.  164.  Figure 38. Transmission electron micrographs of irradiated (1.0 Mrad) chicken pectoralis muscle, 3 hours post-mortem; br = breaks; t f = twisted free ends of f i b r i l s . A and B, 20,000X; C, 45,000X; D, 30,000X.  F i g u r e 39. T r a n s m i s s i o n e l e c t r o n micrographs of i r r a d i a t e d (l.OMrad) c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; Z = Z l i n e ; dz = d i s i n t e g r a t e d Z line. A, 30,000X; B, 120,000X; ( a r e a from A ) .  166.  F i g u r e 40. T r a n s m i s s i o n e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) c h i c k e n p e c t o r a l i s muscle, 3 hours p o s t mortem; s f = s e p a r a t e d f i b r i l s . 25,000X.  F i g u r e 41. T r a n s m i s s i o n e l e c t r o n micrographs o f c y t o p l a s mic p a r t i c l e s i n i r r a d i a t e d (1.0 Mrad) c h i c k e n p e c t o r a l i s muscle 3 hours post-mortem; dp = damaged p a r t i c l e s . 75,000X.  167. cases, Z-line material, i s found to be disintegrated,  and  p r e c i p i t a t i o n of t h i s material seems to have occurred i n the i r r a d i a t e d samples.  Some workers have indicated that  during aging of beef and chicken muscle, the Z-line d i s appeared (Davey and Gilbert,1967; Fukazawa et a l . , 1969), and breaks across f i b r i l s occurred near the Z-line (Davey and G i l b e r t , 1969;  Sayre, 1970).  In the present study,  i r r a d i a t i o n resulted i n breaks occurring i n the  fibrils  near the Z-line, but intact Z-lines could be observed in most cases (Figures 38-C,  39).  In some cases, the  Z-line  seems to have disintegrated, but removal or dissolution of Z-line as found i n aged muscle was i r r a d i a t e d muscle.  not observed i n  In some areas, i n t e r f i b r i l  distance  has increased, i n d i c a t i n g that myofibrils have been pulled apart under the influence of i r r a d i a t i o n (Figures 38-A, and 40).  C,  Transmission electron micrographs of lysosome-  l i k e cytoplasmic p a r t i c l e s from i r r a d i a t e d and muscle are shown i n Figures  control  41 and 42 respectively.  The  p a r t i c l e s found i n control muscle exhibit c l e a r l y an i n t a c t l i m i t i n g membrane, but p a r t i c l e s found i n i r r a d i a t e d muscle appear p a r t i a l l y disrupted and t h e i r ultrastructure d i s organised.  This e f f e c t i s s i m i l a r to the observations  already discussed  regarding  i s o l a t e d lysosomes, i . e . ,  dissolution of lysosomal membranes and disruption of the p a r t i c l e s occurs a f t e r i r r a d i a t i o n treatment.  168.  5 F i g u r e 42. T r a n s m i s s i o n e l e c t r o n micrographs o f c y t o p l a s mic p a r t i c l e s i n n o n - i r r a d i a t e d c h i c k e n p e c t o r a l i s muscle, 3 hours post-mortem. A, 30 ,0 0OX; B, 120,OOOX; C, 150,000X; D, 120,000X.  169. 4.3.2.2.- S u r f a c e u l t r a s t r u c t u r e o f muscle. S u r f a c e u l t r a s t r u c t u r e o f c r y p f r a c t u r e d c o n t r o l and i r r a d i a t e d muscle was examined by SEM.  Non-irradiated  muscle samples had a smooth f i b r e s u r f a c e and t h e t r a n s v e r s e break a c r o s s t h e f i b r e s was c l e a n ( F i g u r e 4 3 ) .  A closer  view o f t h e t r a n s v e r s e break i n F i g u r e 44-A r e v e a l s a smooth s u r f a c e w i t h some amorphous m a t e r i a l and some b e a d - l i k e s t r u c t u r e , p o s s i b l y formed by t h e d i s i n t e g r a t e d m a t e r i a l a r i s i n g from t h e breaks.  F i g u r e 44-B shows p a r t o f s a r -  colemma o f n o n - i r r a d i a t e d muscle. sarcolemma  The s u r f a c e o f t h e  appears r o u g h , b u t no breaks i n t h e s t r u c t u r e  can be observed.  I r r a d i a t i o n o f muscle w i t h 1.0 Mrad caused  marked u l t r a s t r u c t u r a l changes as shown i n F i g u r e s 45 - 47. The muscle f i b r e s appear c o n s i d e r a b l y c o n t r a c t e d , and a l t e r n a t e l y e l e v a t e d and depressed a r e a s appeared the l e n g t h o f "the f i b r e s ( F i g u r e 4 5 ) .  along  F i s s u r e s between  f i b r e s were a l s o observed. ( F i g u r e s 45-B, 46-A).  This i s  p o s s i b l y due t o i r r a d i a t i o n - i n d u c e d weakening o f c o n n e c t i v e t i s s u e , which has s u b s e q u e n t l y broken d u r i n g t h e c r y o f r a c t u r e treatment.  Higher m a g n i f i c a t i o n o f the f i b r e  ( F i g u r e 46-B) i n d i c a t e d t h a t t h e sarcolemma samples was e x t e n s i v e l y d i s r u p t e d .  surface  of irradiated  Moreover, t h e f i b r i l s  t h u s exposed showed e x t e n s i v e l o n g i t u d i n a l s e p a r a t i o n between them ( F i g u r e 46-B). The t r a n s v e r s e breaks o c c u r r i n g i n i r r a d i a t e d were d i f f e r e n t t h a n those i n n o n - i r r a d i a t e d f i b r e s .  fibres Trans-  170.  F i g u r e 43. Scanning e l e c t r o n micrographs o f n o n - i r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s muscle. A, 480X; B, 1,600X.  171.  F i g u r e 44.  Scanning e l e c t r o n micrographs o f n o n - i r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s muscle, 3 hours post-mortem; A, t r a n s v e r s e break, 2,0OOX; B, sarcolemma, 20,000X.  F i g u r e 45. Scanning e l e c t r o n micrographs o f i r r a d i a t e d and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; c t = c o n n e c t i v e t i s s u e ; s f = space between f i b r e s . A, 160X; B, 320X; C, 800X.  F i g u r e 46. Scanning e l e c t r o n micrographs o f i r r a d i a t e d (1.0 Mrad) and c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s muscle, 3 hours post-mortem; A, arrow = s e p a r a t i o n o f f i b r e s , 120X; B, p e r f o r a t e d s a r c o lemma, s f = s e p a r a t e d f i b r i l s , 16,000X.  174.  verse breaks i n c o n t r o l f i b r e s were s t r a i g h t w i t h a smooth s u r f a c e ( F i g u r e 44-A), whereas those i n i r r a d i a t e d  fibres  showed an uneven s u r f a c e a t the b r e a k s , and a l s o a " s p i k e y " s t r u c t u r e coming out o f the p l a n e ( F i g u r e 47-A).  Such  an e f f e c t might be due t o i r r a d i a t i o n - i n d u c e d s e p a r a t i o n o f the f i b r i l s , o r breakdown o f c o n n e c t i v e t i s s u e , which c o u l d r e s u l t i n breakage o f f i b r i l s at d i f f e r e n t p o i n t s . An e n l a r g e d a r e a o f the t r a n s v e r s e break o f i r r a d i a t e d f i b r e s i s shown i n F i g u r e 47-B;  f i s s u r e s between i n d i v i d u a l  f i b r i l s and remains o f s a r c o p l a s m i c r e t i c u l u m can be A f t e r a g i n g o f bovine l o n g i s s i m u s d o r s i ,  seen.  breaks  a c r o s s f i b r i l s and c o l l a p s e o f s a r c o l p l a s m i c r e t i c u l u m have been r e p o r t e d by S c h a l l e r and Powrie G i l b e r t ( 1 9 6 9 ) , Davey and Dickson  (1971).  Davey and  ( 1 9 7 0 ) , and Sayre  (1970)  showed t h a t i n aged bovine and c h i c k e n m u s c l e , breakage of f i b r i l s occurred near the Z-disc during stress.  mechanical  I r r a d i a t i o n - i n d u c e d u l t r a s t r u c t u r a l changes as  p r e s e n t e d i n t h i s r e p o r t seem t o be s i m i l a r t o those  resulting  from a g i n g , but breaks a c r o s s f i b r i l s as observed  in  TEM  r e v e a l e d weakening  and  micrographs  are more s e v e r e .  SEM  d i s r u p t i o n o f t h e sarcolemma as w e l l as s a r c o p l a s m i c culum.  I t i s d i f f i c u l t t o determine  reti-  whether t h e mechanism  i n v o l v e d i n i r r a d i a t i o n - i n d u c e d changes and t h a t r e s p o n s i b l e for  changes observed  i n aged muscle are s i m i l a r , but  the  r a p i d and e x t e n s i v e damage t o muscle u l t r a s t r u c t u r e caused by i r r a d i a t i o n i s i n d i c a t i v e o f a d i f f e r e n c e i n mechanisms.  175.  F i g u r e 47-A.  Scanning e l e c t r o n m i c r o g r a p h o f i r r a d i a t e d (1.0 Mrad) c r y o f r a c t u r e d c h i c k e n p e c t o r a l i s m u s c l e , 3 hours post-mortem; f = f i s s u r e s . 2,000X.  176.  F i g u r e 47-B. A r e a o f r e c t a n g l e i n f i g u r e 47-A a t h i g h e r m a g n i f i c a t i o n (20,000X); f = f i s s u r e s ; arrows = p a r t o f s a r c o p l a s m i c r e t i c u l u m .  177.  Damage t o s a r c o p l a s m i c  r e t i c u l u m by i r r a d i a t i o n  could  r e s u l t i n r e l e a s e o f c a l c i u m i o n s and cause c o n t r a c t i o n o f muscle ( E b a s h i , 1 9 6 1 ;  Schmidt e t a l . , 1 9 7 0 ) .  178. .4.4. SUMMARY AND I t was  CONCLUSIONS  found t h a t u l t r a s t r u c t u r e o f lysosomes can  s i g n i f i c a n t l y a l t e r e d by i r r a d i a t i o n t r e a t m e n t .  be  Observa-  t i o n of lysosomal u l t r a s t r u c t u r e i n d i c a t e d that lysosomal contents leaked r e a d i l y a f t e r i r r a d i a t i o n treatment.  Dilu-  t i o n o f dense l y s o s o m a l m a t r i x o c c u r r e d i n n o n - i r r a d i a t e d p a r t i c l e s d u r i n g i n c u b a t i o n , but r e l e a s e o f l y s o s o m a l c o n t e n t s was  r e l a t i v e l y slow.  On p r o l o n g e d s t o r a g e (72 hours at  4°C),  n o n - i r r a d i a t e d lysosomes showed some d i s r u p t i o n , but dense p a r t i c l e s r e t a i n e d t h e i r shape a l t h o u g h t h e i r membranes were n o t c l e a r l y d i s t i n g u i s h a b l e .  I r r a d i a t e d lysosomes  showed e x t e n s i v e r e l e a s e o f dense m a t e r i a l from t h e p a r t i c l e s a f t e r 48 hours o f i n c u b a t i o n ; f u r t h e r i n c u b a t i o n caused d i s r u p t i o n o f most o f t h e p a r t i c l e s .  Clumps o f dense  l y s o s o m a l m a t r i x were found i n t h e medium, i n d i c a t i n g  dis-  r u p t i o n o f dense lysosomes, which i n c o n t r o l samples showed resistance to disruption.  T r i t o n X-100  treatment  dis-  r u p t e d the lysosomes e x t e n s i v e l y . I n c u b a t i o n o f l y s o s o m a l p a r t i c l e s a t e l e v a t e d temperatures  enhanced the leakage  l y s o s o m a l c o n t e n t s , e s p e c i a l l y from i r r a d i a t e d  of  lysosomes,  and e x t e n s i v e damage t o i r r a d i a t e d p a r t i c l e s was  observed.  Leakage o f l y s o s o m a l c o n t e n t s i s p r o b a b l y due t o i n c r e a s e d p e r m e a b i l i t y o f l y s o s o m a l membranes, as most o f t h e p a r t i c l e s had d i f f u s e d o r d i s o r g a n i z e d membranes. i n the membranes o f a few i r r a d i a t e d p a r t i c l e s were  irradiated Holes observed.  I r r a d i a t i o n might c r e a t e weak p o i n t s i n the membranes and  179. such effect would be exaggerated due to incubation and might result i n formation of holes i n the membranes, and also cause disruption of p a r t i c l e s .  SEM studies showed  additional evidence that lysosomal contents leaked from weak areas; deposits of amorphous material s t i l l attached to these areas could be observed. Transmission electron microscopy of i r r a d i a t e d muscle revealed considerable damage occurring to m y o f i b r i l s . Most of the damage was observed at the I band, where breaks across the f i b r i l s were noticed.  Disintegration of Z-lines  was observed at a few breaks, but i n other cases Z-lines remained i n t a c t .  In certain areas, an increase i n i n t e r -  f i b r i l spaces was observed.  Some cytoplasmic p a r t i c l e s  in tissue resembling lysosomes were found to be damaged by i r r a d i a t i o n treatment. Surface ultrastructure of cryofractured muscle was studied under scanning electron microscope.  Radiation  was found to cause extensive contraction of muscle  fibres,  while non-irradiated muscle did not show such an e f f e c t . Transverse breaks i n non-irradiated muscle had a smooth surface, but i n i r r a d i a t e d muscle, f i b r e s showed an uneven surface at these breaks with a spike-like appearance. Fissures between f i b r i l s were caused by i r r a d i a t i o n t r e a t ment, which supports the e a r l i e r finding of TEM study that i n t e r f i b r i l  spaces tend to increase due to i r r a d i a t i o n .  There were indications that the sarcoplasmic reticulum was  180.  a l s o damaged i n i r r a d i a t e d samples.  I t was o b s e r v e d t h a t  the sarcolemma was e x t e n s i v e l y p e r f o r a t e d due t o i r r a d i a t i o n treatment. R a d i a t i o n - i n d u c e d s t r u c t u r a l a l t e r a t i o n s i n lysosomes and subsequent r e l e a s e o f l y s o s o m a l c o n t e n t s , w h i c h c o n t a i n p o w e r f u l h y d r o l y t i c enzymes l i k e c a t h e p s i n s , might cause e x t e n s i v e p r o t e o l y s i s i n t i s s u e d u r i n g post-mortem  storage.  Moreover, r a d i a t i o n - i n d u c e d damage t o m y o f i b r i l s t r u c t u r e might i n c r e a s e t h e a v a i l a b i l i t y and s u s c e p t i b i l i t y o f m y o f i b r i l l a r protein t o cathepsin attack.  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T e c h n o l . 7: 25. -  PUBLICATION Ali,  (Continued)  M. and R i c h a r d s , J . F . 19lM• E f f e c t o f gamma r a d i a t i o n on l y s o s o m e s and l y s o s o m a l c a t h e p s i n s . P a p e r No. D-4, p r e s e n t e d a t 17th A n n u a l C o n f e r e n c e o f C a n a d i a n I n s t i t u t e of Food S c i e n c e and T e c h n o l o g y , . M o n t r e a l , P.Q., June 9-12,  1974.  Ali,  M. and R i c h a r d s , J . F . 1975E f f e c t o f gamma r a d i a t i o n on c h i c k e n l i v e r c a t h e p t i c a c t i v i t y and r e l e a s e o f l y s o s o m a l c a t h e p s i n D. J . Food Sci.. 40: 47.  Ali,  M. and R i c h a r d s , J . F . 1975U l t r a s t r u c t u r a l changes i n c h i c k e n l i v e r l y s o s o m e s and c h i c k e n b r e a s t m u s c l e i n d u c e d by gamma r a d i a t i o n . P a p e r No. B-3. p r e s e n t e d a t l8th A n n u a l C o n f e r e n c e o f C a n a d i a n I n s t i t u t e o f Food S c i e n c e and T e c h n o l o g y , H a l i f a x , Nova S c o t i a , June 2-4, 1975-  

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