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UBC Theses and Dissertations

Phosphodiesterase II in rat intestinal mucosa Flanagan, Peter Rutledge 1970

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PHOSPHODIESTERASE I I IN RAT INTESTINAL MUCOSA by PETER RUTLEDGE FLANAGAN B . S c , U n i v e r s i t y C o l l e g e D u b l i n , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f B i o c h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1970 In presenting th i s thes i s in pa r t i a l f u l f i lment o f the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s f o r f i nanc i a l gain shal1 not be allowed without my wr i t ten permission. Department of ^jJe^iffy The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date i ABSTRACT The d i s t r i b u t i o n and some of the p r o p e r t i e s of phospho-d i e s t e r a s e I I were s t u d i e d i n homogenates of r a t i n t e s t i n a l mucosa i n an attempt t o e l u c i d a t e i t s r o l e i n the n u c l e i c a c i d metabolism of t h i s t i s s u e . In most of the experiments the p-n i t r o p h e n y l e s t e r o f thymidine 3'-phosphate was used as a s u b s t r a t e f o r p h o s p h o d i e s t e r a s e . D u r i n g the work, evidence was accumulated which i n d i c a t e d t h a t p h o s p h o d i e s t e r a s e I I of i n t e s t i n e was lysosomal i n o r i g i n . For i n s t a n c e , when the t i s s u e was suspended (or homogenized) i n media of d i f f e r i n g t o n i c i t y , the ph o s p h o d i e s t e r a s e I I a c t i v i t y i n the h y p o t o n i c p r e p a r a t i o n s i n c r e a s e d markedly over a p e r i o d of 96 hours. Other i n v e s t i g a t o r s have shown t h a t t h i s "osmotic a c t i v a t i o n " i s a c h a r a c t e r i s t i c o f lysosomal enzymes. Subsequently, homogenates of mucosal t i s s u e were f r a c t i o n -a ted by d i f f e r e n t i a l c e n t r i f u g a t i o n and the s u b c e l l u l a r f r a c t i o n s o b t a i n e d were i d e n t i f i e d by known enzyme markers. The d i s t r i b u -t i o n o f p h o s p h o d i e s t e r a s e I I i n the f r a c t i o n s was most s i m i l a r t o t h a t o f the marker f o r lysosomes - a c i d phosphatase. However a l a r g e p r o p o r t i o n of the ph o s p h o d i e s t e r a s e I I a c t i v i t y , g r e a t e r than t h a t of a c i d phosphatase, was found i n the supernatant s o l u t i o n remaining a f t e r the f i n a l high-speed c e n t r i f u g a t i o n s t e p . The h i g h e s t s p e c i f i c a c t i v i t y f o r ph o s p h o d i e s t e r a s e I I was found i n the " l i g h t m i t o c h o n d r i a l " and " f i n a l s upernatant" f r a c t i o n s . S i m i l a r r e s u l t s were o b t a i n e d when homogenates or n u c l e i - f r e e homogenates were f r a c t i o n a t e d by s u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . The d i s t r i b u t i o n p a t t e r n s o f p h o s p h o d i e s t e r a s e I I and a c i d phosphatase were a g a i n s i m i l a r and t h e p a r t i c l e s t o whi c h p h o s p h o d i e s t e r a s e I I were bound e x h i b i t e d t h e h i g h e s t a c i d phospha-t a s e a c t i v i t y . An at t e m p t was made t o c o n f i r m t h e s e r e s u l t s by " p u r i f y i n g " lysosomes from i n t e s t i n a l mucosa u s i n g a combined d i f f e r e n t i a l c e n t r i f u g a t i o n and d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n t e c h n i q u e . D u r i n g t h e p u r i f i c a t i o n , t h e s p e c i f i c a c t i v i t i e s o f p h o s p h o d i e s t e r a s e I I and a c i d phosphatase i n c r e a s e d p a r a l l e l w i t h each o t h e r and t h e " p u r i f i e d l y s o s o m a l " f r a c t i o n s e x h i b i t e d t h e h i g h e s t s p e c i f i c a c t i v i t i e s f o r t h e s e enzymes. However t h e t o t a l a c t i v i t i e s o f t h e two enzymes r e c o v e r e d i n t h e p u r i f i e d f r a c t i o n s were q u i t e s m a l l , i n d i c a t i n g c o n s i d e r a b l e l o s s i n t h e d i s c a r d e d s o l u b l e f r a c t i o n s . O t h e r w o r k e r s have shown t h a t h o m o g e n i z a t i o n r u p t u r e s lysosomes i n c e r t a i n f r a g i l e t i s s u e s , r e s u l t i n g i n h i g h s o l u b l e a c t i v i t i e s o f t h e enzymes c o n t a i n e d i n t h e s e p a r t i c l e s . I t would seem p o s s i b l e t h e r e f o r e t h a t i n i n t e s t i n a l mucosa p h o s p h o d i e s t e r a s e I I i s l o c a t e d i n lysosomes i n v i v o , s i n c e most o f i t s a c t i v i t y was found t o be d i s t r i b u t e d between t h e l y s o s o m a l and s o l u b l e f r a c t i o n s o f homogenates o f t h i s t i s s u e . The p h o s p h o d i e s t e r a s e I I o f i n t e s t i n e was most a c t i v e a t pH v a l u e s around n e u t r a l i t y . A second s u b s t r a t e , 2 , 4 - d i n i t r o -p h e n y l t h y m i d i n e 3 *-phosphate, w h i c h was used i n o n l y " a few e x p e r i m e n t s because o f i t s l i m i t e d a v a i l a b i l i t y , was h y d r o l y z e d a t a r a t e f a s t e r t h a n t h a t o f p - n i t r o p h e n y l t h y m i d i n e 3 1 - p h o s p h a t e . L i t t l e o r no change i n t h e a c t i v i t y o f t h e enzyme was o b s e r v e d i n the p r e s e n c e o f M g + + , C a + + o r EDTA, b u t Z n + + , C u + + and H g + + i n h i b i t e d m a r k e d l y . The enzyme was most a c t i v e a t a t e m p e r a t u r e i i i o f 58° and o n l y 27% of the a c t i v i t y was l o s t on h e a t i n g the p r e p a r a t i o n f o r 1 hour a t 55°. The M i c h a e l i s c o n s t a n t f o r the enzyme w i t h p - n i t r o p h e n y l thymidine 3'-phosphate was 4.5 x 1 0 - 4 M at 37°, and t h e a c t i v a t i o n energy f o r the pho s p h o d i e s t e r a s e I I c a t a l y z e d h y d r o l y s i s o f the same compound was 14.63 k i l o c a l o r i e s / mole. i v TABLE OF CONTENTS Page INTRODUCTION .' 1 ' H i s t o r i c a l 1 N u c l e i c A c i d Metabolism i n I n t e s t i n a l Mucosa 2 C o n t r o l o f Nuclease A c t i v i t y i n v i v o 3 F u n c t i o n of Nucleases i n v i v o 4 N u c l e o l y t i c Enzymes i n I n t e s t i n e 6 Phosphodiesterase I I 7 The P r e s e n t I n v e s t i g a t i o n 10 MATERIALS AND METHODS .......... 15 S y n t h e s i s of S u b s t r a t e s 16 Enzyme Assays 23 U n i t s and C a l c u l a t i o n s 26 P r e p a r a t i o n of I n t e s t i n a l Mucosal Homogenates .... 27 F r a c t i o n a t i o n of Homogenates by D i f f e r e n t i a l C e n t r i f u g a t i o n 28 Sucrose D e n s i t y - G r a d i e n t C e n t r i f u g a t i o n 32 P r e p a r a t i o n o f " P u r i f i e d " Lysosomes from I n t e s t i n a l Mucosa ... 32 RESULTS 35 I n t r o d u c t o r y 35 I n t e s t i n a l O r i g i n of the Phosphodiesterase I I A c t i v i t y 35 V Page L o c a l i z a t i o n o f P h o s p h o d i e s t e r a s e I I A c t i v i t y i n I n t e s t i n a l T i s s u e 41 A. A c t i v a t i o n E x p e r i m e n t s 41 B. D i f f e r e n t i a l C e n t r i f u g a t i o n o f M u c o s a l -C e l l Homogenates 45 C. S u c r o s e D e n s i t y - G r a d i e n t C e n t r i f u g a t i o n o f M u c o s a l - C e l l Homogenates 57 D. P r e p a r a t i o n o f P u r i f i e d Lysosomes from I n t e s t i n a l Mucosa 62 Some P r o p e r t i e s o f P h o s p h o d i e s t e r a s e I I A c t i v i t y i n Rat I n t e s t i n e '.- 64 A. pH and I o n i c Requirements .64 B. E f f e c t s o f Temperature 72 C. D e t e r m i n a t i o n o f K M, V m a x and A c t i v a t i o n Energy. 75 DISCUSSION ......... 80 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 o f t h e P h o s p h o d i e s t e r a s e I I A c t i v i t y i n Rat I n t e s t i n a l Mucosa 80 P r o p e r t i e s o f t h e P h o s p h o d i e s t e r a s e I I A c t i v i t y .. 87 SUMMARY °. 9 0 BIBLIOGRAPHY 94 v i LIST OF TABLES Page I . D i s t r i b u t i o n o f some enzyme a c t i v i t i e s i n a s u s p e n s i o n of r a t i n t e s t i n a l mucosa 38 I I . The PDase I I and a c i d phosphatase a c t i v i t i e s and the p r o t e i n content of the s u c c e s s i v e washings and homogenate of a sample of r a t i n t e s t i n a l mucosa 40 I I I . E f f e c t o f medium t o n i c i t y on the PDase I I a c t i v i t y o f suspensions and homogenates of r a t i n t e s t i n a l mucosa 42 IV. R e d i s t r i b u t i o n o f PDase I I a c t i v i t y between the s o l u b l e and p a r t i c u l a t e components of homogenates and suspensions prepared i n media o f d i f f e r i n g t o n i c i t y 43 V. E f f e c t of a c t i v a t i n g c o n d i t i o n s on the. a c t i v i t y o f PDase I I and some lysosomal enzymes i n an homogenate 46 VI. D i s t r i b u t i o n of the a c t i v i t i e s o f PDase I I , a c i d phosphatase, cytochrome o x i d a s e and glucose-6-phosphatase i n s u b c e l l u l a r f r a c t i o n s o f an homogenate of r a t i n t e s t i n a l mucosa . 47 V I I . Recovery of p r o t e i n and the a c t i v i t i e s of PDase I I and a c i d phosphatase from a d e x t r a n -phosphate homogenate d u r i n g the p r e p a r a t i o n of e p i t h e l i a l - c e l l "ghosts" from r a t i n t e s t i n a l mucosa 50 V I I I . D i s t r i b u t i o n o f the a c t i v i t i e s of PDase I I , a c i d phosphatase, cytochrome oxidase and glucose-6-phosphatase i n s u b c e l l u l a r f r a c t i o n s of an homogenate of e p i t h e l i a l -c e l l "ghosts" 52 IX. D i s t r i b u t i o n o f the a c t i v i t i e s of PDase I I and a c i d phosphatase i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d by p r o t r a c t e d d i f f e r e n t i a l c e n t r i f u g a t i o n o f an homogenate of e p i t h e l i a l - c e l l "ghosts" 55 X. The PDase I I and a c i d phosphatase a c t i v i t i e s of a number of f r a c t i o n s o b t a i n e d d u r i n g an attempt t o p u r i f y lysosomes from r a t i n t e s t i n a l mucosa 63 v i i Page XI. Comparison of the optimum pH and a c t i v i t y of PDase II i n a v a r i e t y of buffers 66 XII. E f f e c t of a number of divalent cations and of EDTA on the PDase II a c t i v i t y of r a t i n t e s t i n a l mucosa . 71 XIII. E f f e c t of temperature on the K M and V m a x of PDase II 77 v i i i LIST OF FIGURES Page 1. " P u r i f i c a t i o n " of lysosomes from r a t i n t e s t i n a l mucosa • • • • 34 2. E f f e c t of time on the h y d r o l y s i s of Tp-NP and Tp-DNP by the PDase I I a c t i v i t y of r a t i n t e s t i n a l mucosa 36 3. The d i s t r i b u t i o n o f PDase I I , a c i d phosphatase, cytochrome oxidase and glucose-6-phosphatase i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d from an homogenate of e p i t h e l i a l - c e l l "ghosts" 53 4. The d i s t r i b u t i o n o f PDase I I and a c i d phosphatase i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d by p r o t r a c t e d f r a c t i o n a t i o n of an homogenate of e p i t h e l i a l -. c e l l "ghosts" . 56 5. Sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n o f s u b c e l l u l a r p a r t i c l e s of r a t i n t e s t i n a l mucosa .... 59 6. Sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n of s u b c e l l u l a r p a r t i c l e s o f r a t i n t e s t i n a l mucosa .... 61 7. PDase I I a c t i v i t y o f a supernatant f r a c t i o n and a "lysosomal" f r a c t i o n as a f u n c t i o n of pH .... 65 8. PDase I I a c t i v i t y i n phosphate b u f f e r s o f d i f f e r e n t c o n c e n t r a t i o n and pH ., 68 9. E f f e c t of c o n c e n t r a t i o n of s u c c i n a t e b u f f e r on the a c t i v i t y o f PDase I I 69 10. E f f e c t o f temperature on the a c t i v i t y of PDase I I . 73 11. Rate o f heat i n a c t i v a t i o n of PDase I I ... 74 12. E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the a c t i v i t y of PDase I I 76 13. A r r h e n i u s P l o t 79 i x LIST OF ABBREVIATIONS DNA d e o x y r i b o n u c l e i c a c i d RNA r i b o n u c l e i c a c i d DNase d e o x y r i b o n u c l e a s e RNase r i b o n u c l e a s e PDase p h o s p h o d i e s t e r a s e NP p - n i t r o p h e n o l DNP 2 , 4 - d i n i t r o p h e n o l Tp-NP p - n i t r d p h e n y l thymidine 3'-phosphate Tp-DNP 2 , 4 - d i n i t r o p h e n y l thymidine 3'-phosphate NP-pT p - n i t r o p h e n y l thymidine 5'-phosphate DMT- d i - p - m e t h o x y t r i t y l - or di-p-methoxyphenylphenylmethyl-DEAE- d i e t h y l a m i n o e t h y l -DCC d i c y c l o h e x y l c a r b o d i i m i d e DMF dimethylformamide EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e T r i s tris(hydroxymethyl)aminomethane TES N - t r i s (hydroxymethyl)methyl-2-aminoethanesulphonic a c i d MES 2-(N-morpholino)ethanesulphonic a c i d HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l p h o n i c a c i d PIPES p i p e r a z i n e - N , N ' - b i s ( 2 - e t h a n e s u l p h o n i c a c i d ) nm nanometer O.D. o p t i c a l d e n s i t y ATPase a d e n o s i n e t r i p h o s p h a t a s e X ACKNOWLEDGEMENTS The author wishes t o express h i s s i n c e r e thanks and a p p r e c i a t i o n t o Dr. S.H. Zbarsky f o r h i s guidance, encourage-ment and c r i t i c i s m d u r i n g the course of t h i s r e s e a r c h . Thanks are a l s o due t o Dr. G.M. Tener and t o Dr. M. Smith f o r ad v i c e on some aspects o f the syntheses and t o Dr. I. Hynie f o r h i s many h e l p f u l d i s c u s s i o n s . The author i s extremely g r a t e f u l t o h i s w i f e f o r t y p i n g the manuscript. I t i s a p l e a s u r e t o acknowledge the p e r s o n a l support of the M e d i c a l Research C o u n c i l i n the form o f s t u d e n t s h i p s . x i NUCLEASE NOMENCLATURE There i s a l a c k o f c o n s i s t e n c y i n t h e naming o f n u c l e o l y t i c enzymes (8,22,113). A t p r e s e n t t h e r e a r e two main systems o f no m e n c l a t u r e i n use. The f i r s t system has been most g e n e r a l l y used t o d e s c r i b e enzymes from mammalian t i s s u e s ( 8 ) . The n u c l e a s e s a r e c l a s s i f i e d a c c o r d i n g t o t h e i r s p e c i f i c i t y i n h y d r o l y z i n g p o l y n u c l e o t i d e . c h a i n s , and t h e numerals I and I I a r e used t o d e s i g n a t e t h o s e enzymes w h i c h cause t h e r e l e a s e o f 5 ' - p h o s p h o r y l - and 3 1 - p h o s p h o r y l -t e r m i n a t e d p r o d u c t s r e s p e c t i v e l y , e.g. DNase I , RNase I I e t c . Thus p h o s p h o d i e s t e r a s e I I i s t h a t enzyme w h i c h a t t a c k s p o l y r i b o - and p o l y d e o x y r i b o n u c l e o t i d e s e x o n u c l e o l y t i c a l l y c a u s i n g t h e r e l e a s e o f n u c l e o s i d e 3'-phosphates. The second system was d e v e l o p e d by Lehman (22) t o enumerate th e p l e t h o r a o f n u c l e o l y t i c a c t i v i t i e s found i n E. c o l i . I n t h i s system t h e numerals I , I I , I I I e t c . r e f e r t o t h e t e m p o r a l o r d e r i n wh i c h t h e n u c l e a s e s were e n c o u n t e r e d . Thus E. c o l i e x o n u c l e a s e I I r e f e r s t o t h e second e x o n u c l e a s e i d e n t i f i e d i n E. c o l i . L a s k o w s k i (7,113) has s u g g e s t e d a number o f c r i t e r i a f o r t h e c l a s s i f i c a t i o n o f t h e s e w i d e s p r e a d enzymes. 1 INTRODUCTION H i s t o r i c a l The e x i s t e n c e of enzymes which h y d r o l y z e n u c l e i c a c i d s was r e p o r t e d by A r a k i (1) and by Iwanoff (2) i n 1903, although Levene has a t t r i b u t e d the f i r s t o b s e r v a t i o n of t h i s a c t i v i t y t o workers i n the l a b o r a t o r i e s of K o s s e l , Salkowski and Schutzenberger over 90 y e a r s ago (3). Iwanoff demonstrated the c a p a b i l i t y of s e v d r a l moulds t o l i q u i f y g e l s c o n t a i n i n g n u c l e i c acid, and he a s c r i b e d t h i s a c t i v i t y , which was d i s t i n g u i s h a b l e from the pro t e a s e a c t i v i t y o f t h e moulds, t o enzymes he c a l l e d " n u c l e a s e s " (2), In the i n t e r v e n i n g y ears much of the work which e l u c i d a t e d the b a s i c i d e a s of n u c l e i c a c i d s t r u c t u r e was accomplished i n the l a b o r a t o r i e s of Levene and Thannhauser. These workers r o u t i n e l y used e x t r a c t s o f v a r i o u s t i s s u e s i n the d e t e r m i n a t i o n of s t r u c t u r e and i s o l a t i o n of components of n u c l e i c a c i d s . The use of e x t r a c t s of i n t e s t i n a l mucosa was of g r e a t h i s t o r i c a l importance i n t h i s r e s p e c t s i n c e i t l e d d i r e c t l y t o the i s o l a t i o n of n u c l e o s i d e s and n u c l e o t i d e s from DNA (4). In s e v e r a l papers i n 1911 Levene and Medigreceanu l a i d the f o u n d a t i o n f o r present-day i d e a s c o n c e r n i n g the h y d r o l y s i s of n u c l e i c a c i d s by enzymes of d i f f e r e n t s p e c i f i c i t i e s (3,5,6). These workers observed t h a t when y e a s t and thymus n u c l e i c a c i d s were t r e a t e d w i t h i n t e s t i n a l j u i c e t h e r e o c c u r r e d a) a r a p i d f a l l i n the o p t i c a l r o t a t i o n of the s o l u t i o n and b) a r a p i d l o s s of p r e c i p i t a b i l i t y of n u c l e i c a c i d by s t r o n g m i n e r a l a c i d . They a l s o p o i n t e d out the e x i s t e n c e of enzymes w i t h d i f f e r e n t s p e c i f i c i t i e s i n d i f f e r e n t glands and concluded " t h a t t h e r e e x i s t i n the organism s e v e r a l enzymes, which a c t harmoniously l e a d i n g t o the d i s i n t e g r a t i o n of the n u c l e i c a c i d s " (6). Levene b e l i e v e d (4,6) t h a t t h r e e k i n d s of enzymes were i n v o l v e d i n the breakdown of n u c l e i c a c i d s -n u c l e i n a s e , n u c l e o t i d a s e and n u c l e o s i d a s e . The e x i s t e n c e o f the l a t t e r two enzymes has been confirmed i n a v a r i e t y o f t i s s u e s but the p r e s e n t c l a s s i f i c a t i o n o f n u c l e o l y t i c enzymes (7,8) would i n c l u d e both endo- and exonucleases i n the term " n u c l e i n a s e N u c l e i c A c i d Metabolism i n I n t e s t i n a l Mucosa The c l a s s i c a l s t u d i e s o f Leblond and Stevens (9) have shown t h a t i n t e s t i n a l mucosa i s an a c t i v e l y p r o l i f e r a t i n g t i s s u e w i t h a h i g h m i t o t i c a c t i v i t y . T h i s f i n d i n g was a l s o i n d i c a t e d by o b s e r v a t i o n t h a t the r a t e of i n c o r p o r a t i o n of such n u c l e i c a c i d p r e c u r s o r s as g l y c i n e , formate, adenine and phosphate i n t o the n u c l e i c a c i d s o f i n t e s t i n e was ve r y h i g h and was comparable to t h a t of r e g e n e r a t i n g r a t l i v e r (10). T h i s very a c t i v e n u c l e i c a c i d metabolism i s a p p a r e n t l y c l o s e l y r e l a t e d t o the r a p i d r e g e n e r a t i o n of i n t e s t i n a l e p i t h e l i a l c e l l s i n : t h e C r y p t s of Lieberkuhn (11). Newly d i v i d e d e p i t h e l i a l c e l l s move along the basement membrane toward the t i p s of the v i l l i , where having completed t h e i r l i f e c y c l e , they are sloughed o f f i n t o the lumen (9,11,12,13).Leblond and coworkers (14,15) l a b e l l e d the DNA of 32 3 r a p i d l y d i v i d i n g c r y p t c e l l s u s i n g P or H-thymidine and by autoradiography e s t i m a t e d the l i f e - s p a n of r a t and mouse i n t e s t i n a l e p i t h e l i a l c e l l s t o be between one and two days (16). The r a p i d t u r n o v e r of e p i t h e l i u m p o s s i b l y f u n c t i o n s to m a i n t a i n 3 the s u r f a c e of the v i l l u s i n p e r f e c t r e p a i r d e s p i t e i t s f r a g i l i t y and the severe treatment i t r e c e i v e s from the mechanical and chemical a c t i o n of the gut contents (17). C o n t r o l of Nuclease A c t i v i t y i n v i v o The s t u d i e s of A l l f r e y and Mirsky (18) have i n d i c a t e d i t i s l i k e l y t h a t a l l t i s s u e s posseas an a c t i v e machinery f o r degrading n u c l e i c a c i d molecules. I t i s probable t h a t t h i s enzymatic apparatus, i f allowed to f u n c t i o n under optimal c o n d i t i o n s , would completely hydrolyze the c e l l u l a r components, of DNA and RNA. Since t h i s does not appear to occur except under extreme c o n d i t i o n s of c e l l l y s i s and death, mechanisms must e x i s t to keep t h i s powerful n u c l e o l y t i c c a p a c i t y under c o n t r o l . Some i n d i c a t i o n s of how t h i s may be p o s s i b l e are now known from s t u d i e s w i t h b a c t e r i a l and mammalian systems. The work of de Duve and co-workers (19) has shown t h a t the a c i d nucleases of mammalian c e l l s are l o c a l i z e d i n the lysosomes which presumably renders them innocuous toward c e l l u l a r n u c l e i c a c i d s . Many other enzymes are a l s o contained i n these s m a l l p a r t i c l e s i n c l u d i n g a c t i v i t i e s a g a i n s t p r o t e i n (cathepsin) and carbohydrate . (3-galactosidase) s u b s t r a t e s . Roth (20) has shown t h a t many animal t i s s u e s c o n t a i n a n a t u r a l i n h i b i t o r a g a i n s t a l k a l i n e RNase I I and t h i s i n h i b i t o r e f f e c t i v e l y masks the a c t i v i t y of the enzyme t o a high degree i n l i v e r . A l s o Lindberg (21) has i s o l a t e d and p u r i f i e d two i n h i b i t o r s from c a l f spleen which are capable of s p e c i f i c a l l y i n h i b i t i n g DNase I from t h a t t i s s u e . I t i s p o s s i b l e t h a t these i n h i b i t o r s may c u r t a i l the a c t i v i t i e s of a l k a l i n e RNase I I and DNase I i n the c e l l i n v i v o . In E. c o l i i t i s known t h a t 4 endonuclease I i s unable t o a t t a c k d o u b l e - s t r a n d e d DNA -presumably the conformation e x i s t i n g i n the c e l l (22). The mechanism by which messanger RNA i s " s t a b i l i z e d " i n the c e l l i s , as y e t , i n c o m p l e t e l y understood. Yanofsky has suggested t h a t w h i l e ribosomes are a t t a c h e d t o the messenger RNA, the l a t t e r i s a f f o r d e d some p r o t e c t i o n from the a c t i o n of n u c l e a s e s (23,24). I t i s a l s o p o s s i b l e t h a t by b i n d i n g t o o t h e r substances, c e l l u l a r n u c l e i c a c i d s are p r o t e c t e d from the a c t i o n of n u c l e a s e s . For i n s t a n c e , attachment of h i s t o n e t o DNA might p r e v e n t a t t a c k by n u c l e o l y t i c enzymes through blockage of e s s e n t i a l a c t i v e or b i n d i n g s i t e s (22,25). However t o assume t h a t c e l l u l a r n u c l e a s e s are dormant or l a t e n t i n v i v o , would r e l e g a t e these enzymes t o a s c a v e n g i n g r o l e i n dead or l y s e d c e l l s i.e. a means whereby the n u c l e i c a c i d s of these c e l l s are degraded t o p r o v i d e p r e f e r r e d s u b s t r a t e s f o r n u c l e i c a c i d s y n t h e s i s i n l i v i n g c e l l s . F u n c t i o n of Nucleases i n v i v o In r e c e n t y ears i t has become g e n e r a l l y accepted t h a t n u c l e a s e s , e s p e c i a l l y DNases p a r t i c i p a t e i n some manner i n the i n t r a c e l l u l a r metabolism of g e n e t i c a l l y and m e t a b o l i c a l l y a c t i v e n u c l e i c a c i d s (26). In 1952 A l l f r e y and M i r s k y compared the " a c i d " DNase content of v a r i o u s t i s s u e s of c a l f , h o r s e , c h i c k e n , mouse and r a t and observed a c o r r e l a t i o n between the n uclease c o n t e n t of a p a r t i c u l a r t i s s u e and i t s c a p a c i t y f o r p r o l i f e r a t i o n or r e g e n e r a t i o n (18). Deoxyribonucleases have a l s o been i m p l i c a t e d i n h o s t DNA s y n t h e s i s f o l l o w i n g i n f e c t i o n of b a c t e r i a (27) and animals (28) by v i r u s e s ; i n g e n e t i c r ecombination of DNA molecules (29) ; i n the r e s t r i c t i o n process i n b a c t e r i a (30) where a f t e r i n f e c t i o n by bacteriophage, the incoming DNA i s degraded and v i r u s r e p l i c a t i o n i s blocked; and i n the r e p a i r of DNA which has been damaged by u l t r a v i o l e t i r r a d i a t i o n or chemical mutagens (31). P i o n e e r i n g work by Kornberg and co-workers using h i g h l y p u r i f i e d enzymes i n v i t r o has i n d i c a t e d t h a t nucleases can profoundly a f f e c t the s t r u c t u r e and a c t i v i t y of the primer i n DNA s y n t h e s i s . Thus Richardson, S c h i l d k r a u t and Kornberg (32) have shown t h a t E. c o l i endonuclease I or p a n c r e a t i c DNase can cleave the DNA primer to produce a d d i t i o n a l 3'-hydroxyl groups and i n t h i s way the primer supports more r a p i d and "extensive DNA s y n t h e s i s . Again i t has been shown by many workers, e s p e c i a l l y those i n Romberg's l a b o r a t o r y , t h a t h i g h l y p u r i f i e d p r e p a r a t i o n s °f c o ± 1 DNA polymerase - the enzyme which c a t a l y z e s the sy n t h e s i s of DNA - c o n t a i n ..'exonuclease I I a c t i v i t y which can not be removed without d e s t r o y i n g the s y n t h e t i c a c t i v i t y of the enzyme (33). The c o n t r o l ' o f s y n t h e s i s of poly p e p t i d e chains on ribosomal templates may i n v o l v e the removal of i n f o r m a t i o n a l RNA molecules by s p e c i f i c RNA depolymerases. Both potassium-a c t i v a t e d phosphodiesterase (RNase I I ) (34) and p o l y n u c l e o t i d e phosphorylase (35,36) have been suggested as candidates f o r messenger RNA degradation. However both of these enzymes are 3' exonucleases and t h i s presents an embarrassing enzymological s i t u a t i o n s i n c e i t has r e c e n t l y been shown t h a t degradation of messenger RNA occurs i n a 5'—>31 d i r e c t i o n along w i t h t r a n s c r i p t i o n and t r a n s l a t i o n (24,37)-. 6 Nucleases have a l s o proved extremely u s e f u l t o o l s i n the study of the primary s t r u c t u r e of n u c l e i c a c i d molecules (38,39). In g e n e r a l , the e l u c i d a t i o n of the s t r u c t u r e of RNA has met w i t h more success than t h a t of DNA, due, perhaps, to the extremely l a r g e s i z e of DNA and the l i m i t e d a v a i l a b i l i t y of degrading enzymes w i t h a h i g h degree of s p e c i f i c i t y and p u r i t y . One of the b e s t demonstrations of the u s e f u l n e s s of n u c l e a s e s as b i o c h e m i c a l t o o l s was g i v e n by H o l l e y and co-workers (40) when they determined the complete primary s t r u c t u r e of a l a n i n e t r a n s f e r RNA from y e a s t . N u c l e o l y t i c Enzymes i n I n t e s t i n e While Levene (6) c o n s i d e r e d t h a t " n u c l e o t i d a s e " was the predominant a c t i v i t y i n i n t e s t i n a l e x t r a c t s , K l e i n (41) showed t h a t h y d r o l y s i s of DNA by i n t e s t i n a l e x t r a c t s i n the presence of a r s e n a t e , which i n h i b i t e d the a l k a l i n e phosphatase a c t i v i t y , l e d t o f o r m a t i o n of a p p r e c i a b l e q u a n t i t i e s of d e o x y r i b o n u c l e o t i d e s by p h o s p h o d i e s t e r a s e a c t i o n . Schmidt and Thannhauser (42.) a l s o encountered t h i s a l k a l i n e p h o s p h o d i e s t e r a s e i n attempts to p u r i f y a l k a l i n e phosphatase. Having e x t e n s i v e l y p u r i f i e d the phosphomonoesteirase they were unable t o remove t r a c e s of a p h o s p h o d i e s t e r a s e which s p e c i f i c a l l y c l e a v e d the i n t e r n u c l e o t i d e bonds of r e l a t i v e l y low m o l e c u l a r weight r i b o - and d e o x y r i b o n u c l e o t i d e s (42). In 1951 Cohn and V o l k i n (43) h y d r o l y s e d l i v e r RNA s u c c e s s i v e l y w i t h c r y s t a l l i n e RNase and w i t h an i n t e s t i n a l e x t r a c t i n the presence of a r s e n a t e and they a p p l i e d the products of d i g e s t i o n t o an anion-exchange column. 7 E l u t i o n of the column y i e l d e d a p p r e c i a b l e q u a n t i t i e s of the f o u r 5' r i b o n u c l e o t i d e s . Although the authors d i d not d i s c u s s the f i n d i n g , t h i s experiment showed t h a t the a l k a l i n e p h o s p h o d i e s t e r a s e a c t i v i t y of i n t e s t i n a l e x t r a c t s l e d t o the f o r m a t i o n of 5' mononucleotides. R a z z e l l has e x t e n s i v e l y p u r i f i e d t h i s enzyme, c a l l e d p h o s p h o d i e s t e r a s e I, from hog k idney (44), w h i l e the enzyme from i n t e s t i n e has been separated from phosphomonoesterase and p u r i f i e d i n t h i s l a b o r a t o r y (45). P h o s p h o d i e s t e r a s e I I In an important r e p o r t p u b l i s h e d i n 1953, Heppel and Hilmoe d e s c r i b e d the h y d r o l y s i s of RNA and RNA "core" - the l i m i t p o l y n u c l e o t i d e s produced by RNase a c t i o n , which are not d i a l y s a b l e a g a i n s t water, by f i v e p a r t i a l l y p u r i f i e d f r a c t i o n s of s p l e e n and i n t e s t i n e (46). These f r a c t i o n s d i f f e r e d i n . t h e i r r a t e s of h y d r o l y s i s of RNA and RNA " c o r e " , i n t h e i r maximal a c t i v i t y a t d i f f e r e n t pH v a l u e s , i n t h e i r i o n i c requirements and i n t h e i r t h e r m o s t a b i l i t y . The authors p r o v i s i o n a l l y concluded t h a t these a c t i v i t i e s r e p r e s e n t e d d i f f e r e n t enzymes. Hilmoe' l a t e r (47) p u r i f i e d e x t e n s i v e l y a f r a c t i o n of s p l e e n which h y d r o l y s e d RNA "core" at a r a p i d r a t e . The p u r i f i e d enzyme, which he c a l l e d s p l e e n p h o s p h o d i e s t e r a s e , had an optimum pH of 6.6 and was i n h i b i t e d by a l a r g e number of d i v a l e n t c a t i o n s n o t a b l y Cu , Hg , and Zn . RNA, RNA " c o r e " and o l i g o n u c l e o t i d e s b e a r i n g e i t h e r a 3' h y d r o x y l or monoester phosphate were completely degraded to 3 1 mononucleotides. S u b s t r a t e s b e a r i n g 5' phosphomonoester groups were r e s i s t a n t t o the enzyme. A l k y l 8 e s t e r s of b o t h p u r i n e and p y r i m i d i n e 3'- phosphates were a l s o h y d r o l y s e d by the enzyme. R a z z e l l and Khorana (48)/ u s i n g an assay based o n the h y d r o l y s i s of the p - n i t r o p h e n y l e s t e r of thymidine 3'—phosphate, f u r t h e r p u r i f i e d the s p l e e n enzyme and confirmed some of the r e s u l t s o b t a i n e d by Hilmoe. In a d d i t i o n , these workers t e s t e d the a c t i v i t y of the enzyme a g a i n s t s e v e r a l d i , t r i , and t e t r a n u c l e o t i d e s , and found t h a t the enzyme h y d r o l y s e d o l i g o n u c l e o t i d e s by stepwise a t t a c k from the end b e a r i n g the 5' h y d r o x y l group and possessed no d e t e c t a b l e endonuclease a c t i v i t y (48). In 1968/ B e r n a r d i and B e r n a r d i (49) d e s c r i b e d a new p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h i s enzyme from s p l e e n , u s i n g as s u b s t r a t e the l i m i t p o l y n u c l e o t i d e s o b t a i n e d from DNA by the a c t i o n o f h i g h l y p u r i f i e d DNase I I . They showed t h a t the enzyme r a p i d l y h y d r o l y s e d r i b o - and d e o x y r i b o 3'-phospho-o l i g o n u c l e o t i d e s , w h i l e n o n - s p e c i f i c p h o s p h o d i e s t e r a s e s u b s t r a t e s l i k e b i s - ( p - n i t r o p h e n y l ) phosphate were o n l y s l o w l y degraded and f o r t h i s r e a s o n they suggested t h a t the enzyme might be more s u i t a b l y c a l l e d s p l e e n exonuclease r a t h e r than s p l e e n p h o s p h o d i e s t e r a s e (47,48). The f i n a l p r e p a r a t i o n of these workers was more s t a b l e than t h a t of R a z z e l l and Khorana (48) and was f r e e from c o n t a m i n a t i n g phosphomonoesterase, DNase, RNase and adenosine deaminase a c t i v i t i e s . The presence of a phosphodiesterase a c t i v e a t a c i d pH In i n t e s t i n e was f i r s t i n d i c a t e d by the experiments o f Heppel and Hilmoe (46). R a z z e l l confirmed the presence of t h i s enzyme c a l l e d p h o s p h o d i e s t e r a s e I I i n many t i s s u e s i n c l u d i n g i n t e s t i n e (50) . However i n t h i s t i s s u e the a c t i v i t y of p h o s p h o d i e s t e r a s e I was 7 times g r e a t e r than t h a t of p h o s p h o d i e s t e r a s e I I . The i n t r a c e l l u l a r l o c a t i o n of p h o s p h o d i e s t e r a s e I I i s s t i l l i n d i s p u t e . R a z z e l l (50) r e p o r t e d t h a t the enzyme was l o c a t e d i n both m i t o c h o n d r i a l and s o l u b l e f r a c t i o n s of l i v e r and kidney homogenates and suggested t h a t perhaps the s o l u b l e p o r t i o n had been r e l e a s e d from the m i t o c h o n d r i a d u r i n g homogenization. Hunter and Korner (51) have a l s o r e p o r t e d the presence of an exonuclease i n r a t l i v e r c e l l sap which appears to be s i m i l a r t o t h a t d e s c r i b e d by R a z z e l l (50). These workers (51) used a v a r i e t y of n a t u r a l and s y n t h e t i c r a d i o a c t i v e l y l a b e l l e d r i b o n u c l e i c a c i d s as s u b s t r a t e s and concluded t h a t the enzyme p r e f e r e n t i a l l y a t t a c k s molecules l a c k i n g secondary s t r u c t u r e from the 5' end. In a paper p u b l i s h e d i n 196 8 van Dyck and Wattiaux (52) d e s c r i b e d the d e t e r m i n a t i o n of a c i d exonuclease a c t i v i t y i n r a t l i v e r u s i n g DNA "core" as s u b s t r a t e . These workers f r a c t i o n a t e d l i v e r homogenates by d i f f e r e n t i a l c e n t r i f u g a t i o n and concluded t h a t the enzyme was a s s o c i a t e d w i t h the l i g h t m i t o c h o n d r i a l or lysosomal f r a c t i o n . They a t t r i b u t e d t h i s disagreement wi t h the r e s u l t s of R a z z e l l (50) t o the use of d i f f e r e n t s u b s t r a t e s i n the two s t u d i e s . However a very r e c e n t r e p o r t by E r e c i n s k a e t a l . (53) d e s c r i b e d the 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 p h o s p h o d i e s t e r a s e I I i n r a t l i v e r u s i n g Tp-NP as s u b s t r a t e . C o n t r a r y t o the r e s u l t s of R a z z e l l (50), these workers found t h a t the a c t i v i t y of the enzyme was l o c a l i z e d l a r g e l y i n the lysosomal f r a c t i o n w i t h low s p e c i f i c a c t i v i t y i n the f i n a l supernatant f r a c t i o n a f t e r c e n t r i f u g a t i o n (53). 10 Hence i t seems t h a t the d i f f e r e n t c o n c l u s i o n s c o n c e r n i n g the l o c a l i z a t i o n of ph o s p h o d i e s t e r a s e I I i n l i v e r (50,52,53) can not be e x p l a i n e d i n terms of the d i f f e r e n t s u b s t r a t e s used (51). P r e l i m i n a r y experiments i n t h i s l a b o r a t o r y i n d i c a t e d t h a t most of the phosp h o d i e s t e r a s e I I a c t i v i t y of i n t e s t i n a l homogenates was l o c a t e d i n the f i n a l s upernatant f r a c t i o n a f t e r d i f f e r e n t i a l c e n t r i f u g a t i o n (54). N e i t h e r the 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 ph o s p h o d i e s t e r a s e I I i n i n t e s t i n e nor i t s i s o l a t i o n and p u r i f i c a t i o n from t h a t source has been p u b l i s h e d . B r i g h t w e l l and Tappel (55) have i n v e s t i g a t e d the s u b c e l l u l a r d i s t r i b u t i o n s i n r a t l i v e r of the enzymes which h y d r o l y s e NP-pT and b i s -( p - n i t r o p h e n y l ) phosphate a t pH 5.2. The enzyme h y d r o l y s i n g NP-pT most r a p i d l y a t pH 5.2 was p r e s e n t i n lysosomes. Enzymes h y d r o l y s i n g b i s - ( p - n i t r o p h e n y l ) phosphate were p r e s e n t i n the lysosomes and c e l l sap of r a t l i v e r , the pH optima being pH 5.2 f o r the lysosomal enzyme and pH 6.6 f o r the s o l u b l e enzyme. Hsu and Tappel (56) showed t h a t s i x i n t r a c e l l u l a r h y d r o l a s e s known to be a s s o c i a t e d w i t h lysosomes i n r a t l i v e r were p r e s e n t i n r a t i n t e s t i n a l mucosa. The s p e c i f i c a c t i v i t i e s of the mucosal enzymes were comparable t o those of the l i v e r . However d i f f e r e n t i a l c e n t r i f u g a t i o n of mucosal homogenates i n d i c a t e d t h a t the p a r t i c l e s from the mucosa were more heterogeneous i n s i z e than those of the l i v e r , b e a r i n g a c l o s e r resemblance t o kidney lysosomes (57). The P r e s e n t I n v e s t i g a t i o n In view of the d i f f e r e n t r e s u l t s o b t a i n e d by van Dyck and Wattiaux (52) and by E r e c i n s k a e t a l . (53) on the one hand i n 11 l i v e r and by Razzell (50) on the other i n l i v e r and kidney, i t seemed desirable to investigate the 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 phosphodiesterase i n i n t e s t i n e . Both the p-nitrophenyl and 2,4-dinitrophenyl esters of thmidine 3'-phosphate were synthesized and an assay based on that employed by Razzell and Khorana (48) was used throughout the study. on OH N0a The p-nitrophenol can be measured spectrophotometrically at 400 nm i n a l k a l i n e medium and the 2,4-dinitrophenol i n a s i m i l a r manner at 360 nm i n neutral or a l k a l i n e media. I n i t i a l experiments indicated that an enzyme capable of hydrolysing Tp-NP indeed existed i n i n t e s t i n a l extracts. This a c t i v i t y seemed to reside within the e p i t h e l i a l c e l l s since mucosal scrapings which had been washed with buffered sucrose y i e l d e d further a c t i v i t y on; homogenization. I t was also seen that 12 the p h o s p h o d i e s t e r a s e I I a c t i v i t y c o u l d be r e l e a s e d o s m o t i c a l l y from crude p r e p a r a t i o n s of e p i t h e l i a l c e l l s . The enzyme a c t i v i t y of suspensions and homogenates of e p i t h e l i a l c e l l s i n an h y p o t o n i c medium (water) i n c r e a s e d 61% and 38% r e s p e c t i v e l y upon s t a n d i n g f o r 96 hours a t 4° w h i l e no such i n c r e a s e s were observed f o r p r e p a r a t i o n s i n an i s o t o n i c medium (0.9% sodium c h l o r i d e ) . In a d d i t i o n the a c t i v i t y o f the enzyme i n both h y p o t o n i c and i s o t o n i c p r e p a r a t i o n s was r e d i s t r i b u t e d i n f a v o u r o f the s o l u b l e p o r t i o n of the homogenate or suspension d u r i n g t h i s p e r i o d o f time. F i r s t , attempts t o f r a c t i o n a t e mucosal homogenates were hampered by mucous p r e s e n t i n the i n t e s t i n e which gave homogenates a j e l l y - l i k e c o n s i s t e n c y . In some cases the i n t e r f e r e n c e due to the mucous was such t h a t no sediment a t low speed c o u l d be o b t a i n e d . While many attempts were made to e l i m i n a t e the i n t e r f e r e n c e due to t h i s mucous, i t always remained a hampering f a c t o r i n the f r a c t i o n a t i o n experiments. Homogenization i n 6% dextran-Krebs-Ringer phosphate pH 7.4 as recommended by C l a r k and Porteous (58) gave q u i t e r e p r o d u c i b l e r e s u l t s , however, w i t h good r e c o v e r y of the p a r t i c u l a t e c e l l components. S e v e r a l f r a c t i o n a t i o n s u s i n g d i f f e r e n t homogenizing media and d i f f e r e n t c e l l d i s r u p t i o n methods were attempted w i t h g e n e r a l l y s i m i l a r r e s u l t s . In a l l cases most of the a c t i v i t y o f p h o s p h o d i e s t e r a s e I I appeared i n the supernatant from the f i n a l high-speed c e n t r i f u g a t i o n , w h i l e the s p e c i f i c a c t i v i t y was g e n e r a l l y h i g h e s t i n the " m i t o c h o n d r i a l " and "lysosomal" f r a c t i o n s . In most of the experiments cytochrome o x i d a s e , a c i d phosphatase and glucose-6-phosphatase were employed as enzyme 13 markers f o r m i t o c h o n d r i a , lysosomes and microsomes r e s p e c t i v e l y . While the d i s t r i b u t i o n of a c t i v i t y o f phosp h o d i e s t e r a s e I I was not i d e n t i c a l t o t h a t of any of the markers, i t bore most s i m i l a r l y t o t h a t of a c i d phosphatase. However the p r o p o r t i o n of a c i d phosphatase a c t i v i t y i n the supernatant a f t e r f i n a l high-speed c e n t r i f u g a t i o n was never as g r e a t as t h a t of phos p h o d i e s t e r a s e I I . S i n c e some evidence was o b t a i n e d by the f r a c t i o n a t i o n experiments t h a t p h o s p h o d i e s t e r a s e was, a t l e a s t , p a r t l y l y s o s o m a l , attempts were made t o c o n f i r m t h i s u s i n g sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . When homogenates or homogenates from which the n u c l e i had been removed (by low-speed c e n t r i f u g a t i o n ) were c e n t r i f u g e d i n a sucrose g r a d i e n t most of the p h o s p h o d i e s t e r a s e I I a c t i v i t y remained a t the top of the g r a d i e n t . The a c t i v i t y of a c i d phosphatase was g e n e r a l l y found a t a p o i n t f u r t h e r down i n the g r a d i e n t along with a s m a l l e r amount of pho s p h o d i e s t e r a s e I I a c t i v t y . T h i s s m a l l e r peak of phos p h o d i e s t e r a s e I I a c t i v i t y behaved i n a manner s i m i l a r t o t h a t of a c i d phosphatase when the time and speed of c e n t r i f u g a t i o n were v a r i e d . I f the p a r t i c l e s remaining i n the supernatant a f t e r removal o f the n u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n a t r 105,000 x g and then a p p l i e d t o the sucrose g r a d i e n t , e s s e n t i a l l y the same r e s u l t was o b t a i n e d - most of the phosph o d i e s t e r a s e II a c t i v i t y was found a t the top of the g r a d i e n t . An attempt t o prepare p u r i f e d lysosomes from i n t e s t i n a l mucosa was not very s u c c e s s f u l and although the s p e c i f i c a c t i v i t i e s of p h o s p h o d i e s t e r a s e II and a c i d phosphatase i n t h i s p r e p a r a t i o n were 2.9 and 5.3 times g r e a t e r than the c o r r e s p o n d i n g v a l u e s i n 14 the homogenate, the r e c o v e r i e s of these a c t i v i t i e s from the homogenate were onl y 0.4% and 0.6% r e p e c t i v e l y . The p o s s i b i l i t y t h a t two enzymes capable of h y d r o l y s i n g Tp-NP and Tp-DNP e x i s t e d i n the i n t e s t i n e - one s o l u b l e and the o t h e r p a r t i c u l a t e was a l s o c o n s i d e r e d . T h i s seems u n l i k e l y i n view of the f a c t t h a t a " n u c l e a r " , a " l i g h t m i t o c h o n d r i a l " and a supernatant f r a c t i o n a l l e x h i b i t e d s i m i l a r pH a c t i v a t i o n p r o f i l e s w i t h optima c l o s e t o 7.0. Some o t h e r p r o p e r t i e s of the enzyme a c t i v i t y were a l s o determined. The M i c h a e l i s c o n s t a n t was 4.5 x 10~ 4 M w i t h Tp-NP as s u b s t r a t e s a t 37°C. The p h o s p h o d i e s t e r a s e a c t i v i t y e x h i b i t e d an unusual temperature p r o f i l e w i t h h i g h e s t a c t i v i t y c l o s e t o 6 0 ° C . S e v e n t y t h r e e p e r c e n t of the enzyme a c t i v i t y remained a f t e r h e a t i n g a t 55°C f o r 1 hour. The a c t i v a t i o n energy f o r the r e a c t i o n was found t o be 14.63 k i l o c a l o r i e s / m o l e . The i n h i b i t i o n by copper r e p o r t e d by Hilmoe (47) and by R a z z e l l and Khorana (48) f o r s p l e e n p h o s p h o d i e s t e r a s e was confirmed f o r the i n t e s t i n a l enzyme but no a c t i v a t i o n by EDTA c o u l d be o b t a i n e d . On the b a s i s of the r e s u l t s o b t a i n e d i t has been concluded t h a t a p h o s p h o d i e s t e r a s e a c t i v i t y s i m i l a r t o t h a t observed i n bovine s p l e e n (47,48) r a t l i v e r (50,51,52>53), r a t and hog kidney (50) and salmon t e s t i s (59) i s p r e s e n t i n i n t e s t i n a l mucosa of r a t s . The enzyme i s most a c t i v e a t a pH c l o s e t o 7 and appears t o be p r e s e n t i n the f i n a l supernatant f r a c t i o n a f t e r c e n t r i f u g a t i o n of mucosal homogenates. Some evidence t h a t the enzyme might be o r i g i n a l l y p r e s e n t i n lysosomes was o b t a i n e d . 15 MATERIALS AND METHODS Si n c e a l a r g e number of n u c l e a s e s are capable of h y d r o l y s i n g p h o s p h o d i e s t e r bonds (8) , i t i s obvious t h a t , f o r a p a r t i c u l a r assay, the nature of the s u b s t r a t e used w i l l be a l l - i m p o r t a n t i n d e t e r m i n i n g which enzyme a c t i v i t y i s being measured. T h i s i s e s p e c i a l l y t r u e f o r the assay of PDase a c t i v i t y and the m u l t i p l i c i t y of enzyme a c t i v i t i e s so f a r encountered has r e s u l t e d i n the use of a v a r i e t y of n a t u r a l and s y n t h e t i c s u b s t r a t e s . Hilmoe (46,47,60) has used RNA "core", the l i m i t p o l y n u c l e o t i d e s formed by e x h a u s t i v e d i g e s t i o n of RNA by p a n c r e a t i c RNase, t o d e t e c t PDase II a c t i v i t y i n s p l e e n . These o l i g o n u c l e o t i d e s which are about 3 t o 5 n u c l e o t i d e s long (61), c o n s i s t of p u r i n e n u c l e o t i d e r e s i d u e s and a t e r m i n a l p y r i m i d i n e r e s i d u e having a phosphomonoester group on i t s 3'-hydroxyl group. However B e r n a r d i and B e r n a r d i (62) have shown t h a t t h i s s u b s t r a t e i s h y d r o l y s e d by s p l e e n RNase and so i s not s u i t a b l e f o r the assay of PDase I I . These workers (49) recommend the use of DNA " c o r e " , prepared by the a c t i o n of h i g h l y p u r i f i e d s p l e e n DNase II on DNA (63) , as the s u b s t r a t e of c h o i c e f o r assay of PDase II ( a c i d e x o n u c l e a s e ) . Among the e a r l i e s t s y n t h e t i c s u b s t r a t e s used t o d e t e c t PDase a c t i v i t y were d i p h e n y l phosphate (64) and b i s - ( p - n i t r o p h e n y l ) phosphate (65), but these compounds are h y d r o l y s e d by a number of d i f f e r e n t enzymes (55,66,67,68). Khorana and co-workers have used w i t h g r e a t success the p - n i t r o p h e n y l e s t e r s of n u c l e o s i d e phosphates as s u b s t r a t e s f o r PDases I and I I (44,48,50,69). 16 B e r n a r d i and G r i f f e (66) have suggested t h a t Tp-NP can be h y d r o l y s e d by h i g h l y p u r i f i e d p r e p a r a t i o n s of DNase, but t h i s f i n d i n g has r e c e n t l y been q u e s t i o n e d (6 8). Other s u b s t r a t e s which have been used t o determine PDase II a c t i v i t y i n c l u d e Tp-DNP (59/70), thymidine 3'-0-methoxy f l u o r e s c e i n phosphate (71), and the a l k y l e s t e r s of n u c l e o s i d e 3 1-phosphates (60). The assay of PDase I I u s i n g Tp-NP and Tp-DNP i s q u i c k and easy and these s u b s t r a t e s were employed throughout the study. At the time the study was begun i t was not p o s s i b l e t o o b t a i n these compounds commercially so i t was necessary to s y n t h e s i z e them i n the l a b o r a t o r y . Tp-NP i s now a v a i l a b l e from Raylo Chemicals L t d . , (Edmonton) and was purchased from t h i s company i n the l a t t e r stages of the work. S y n t h e s i s of S u b s t r a t e s R e a c t i o n s o l v e n t s were d r i e d over c a l c i u m h y d r i d e or L i n d e M o l e c u l a r Sieve (72). Other r e a c t i o n m a t e r i a l s were d r i e d i n a d e s s i c a t o r over phosphorus p e n t o x i d e . E v a p o r a t i o n of s o l v e n t s was c a r r i e d out on a r o t a r y e v aporator a t 30°C except when anhydrous c o n d i t i o n s were r e q u i r e d i n which case, an o i l pump equipped w i t h a Dry-Ice t r a p were used. S e p a r a t i o n of thymidine and 5'-0-DMT thymidine, was c a r r i e d out by t h i n l a y e r chromatography on Merck S i l i c a Gel F254 pr e - c o a t e d p l a t e s w i t h CHCI3:MeOH (9:1,v/v) (73) as s o l v e n t ( s o l v e n t 1 ) . Descending paper chromatography of n u c l e o t i d e s and r e a c t i o n mixtures on Whatman 40 or Whatman 31 ( f o r l a r g e - s c a l e work) sheets was c a r r i e d out u s i n g the f o l l o w i n g s o l v e n t systems; s o l v e n t 2, isopropanol-ammonia-water (7:1:2, v/v/v.) (70,72); s o l v e n t 3, 17 b u t a n o l - a c e t i c acid-water (5:2:3, v/v/v) (72); s o l v e n t 4, i s o b u t y r i c a c i d - 1 M NH^OH (5:3, v/v) (70). The n u c l e o t i d e s and n u c l e o t i d e e s t e r s were d e t e c t e d by view i n g the chromatograms under a s h o r t wavelength u l t r a v i o l e t l i g h t (257.3 nm) i n a Chromato-Vue c a b i n e t . A. S y n t h e s i s of Tp-NP: The method o f s y n t h e s i s o f t h i s s u b s t r a t e was e s s e n t i a l l y t h a t o f Borden and Smith (72). The s t a r t i n g m a t e r i a l , 5'-0-DMT thymidine was s y n t h e s i z e d by the method o f S c h a l l e r e t a l . (74). Thymidine (2.43 g. 10 mMole) was suspended i n 10 ml of dry p y r i d i n e and DMT c h l o r i d e (3.5 g, 10.33 mMole) was added. A s o l u -t i o n was o b t a i n e d by g e n t l e shaking and was allowed t o stand a t room temperature f o r 4 hours. One ml of methanol was added and the s o l u t i o n was c o n c e n t r a t e d t o a gum which was d i s s o l v e d i n 20 ml o f ch l o r o f o r m . T h i s c h l o r o f o r m s o l u t i o n was e x t r a c t e d s e v e r a l times w i t h 10 ml p o r t i o n s o f water t o remove unreacted thymidine, d r i e d w i t h sodium s u l p h a t e , and then evaporated t o y i e l d a brown gum which was d i s s o l v e d i n 200 ml of hot benzene. Cyclohexane was c a r e -f u l l y added t o t h i s s o l u t i o n u n t i l a f a i n t t u r b i d i t y appeared and t h i s was allowed t o stand f o r 12 hours d u r i n g which time a p r e c i p i t a t e o f 5'-O-DMT thymidine came down. T h i s was c o l l e c t e d by f i l t r a t i o n . Y i e l d 4.6 g, 85%. T h i n l a y e r chromatography i n s o l v e n t 1 i n d i c a t e d the presence o f s m a l l amounts of thymidine. T h i s was removed i n the f o l l o w i n g way. The crude product was d i s s o l v e d i n a minimal volume of e t h a n o l and was added dropwise t o 2 l i t r e s o f 2% NH 40H a t 4°. The f i n e p r e c i p i t a t e of 5'-O-DMT 18 thymidine was allowed t o stand o v e r n i g h t and was then c o l l e c t e d by f i l t r a t i o n on a s i n t e r e d - g l a s s f i l t e r . The p r e c i p i t a t e was d r i e d oyer phosphorus pentoxide and r e c r y s t a l l i z e d from benzene: cyclohexane as d e s c r i b e d above. The product was c h r o m a t o g r a p h i c a l l y homogeneous i n s o l v e n t 1 and the y i e l d was 3.9 g (72%). P-nitropheny1 phosphate (disodium s a l t , 2 mMole) was d i s s o l v e d i n 5 ml of water and passed through a s m a l l (10 cm x 1 cm) column of Dowex-50 (H + form). One ml of p y r i d i n e was added t o the e l u a t e and the s o l u t i o n was evaporated t o dryness. Repeated a d d i t i o n s of dry p y r i d i n e f o l l o w e d by e v a p o r a t i o n under anhydrous c o n d i t i o n s removed l a s t t r a c e s of water by entrainment. T h i s anhydrous p y r i d i n i u m s a l t of p - n i t r o p h e n y l phosphate (2 mMole) was then d i s s o l v e d i n 5 ml of DMF and 2.5 ml of p y r i d i n e c o n t a i n i n g 125 mg of dry Dowex-50 (H + form), f o l l o w e d by the a d d i t i o n of 1 mMole of 5'-0-DMT thymidine and 5 mMole DCC. The r e a c t i o n v e s s e l was covered w i t h aluminum f o i l and shaken f o r 2 days, a f t e r which more DCC was added (1 mMole) and the shaking process c a r r i e d on •f o r a f u r t h e r 2 days. One ml of water was added and the mixture allowed to stand f o r 24 hours a f t e r which the s o l v e n t was removed by e v a p o r a t i o n and 50 ml of e t h a n o l added. The mixture was f i l t e r e d and the f i l t r a t e passed onto a D E A E - c e l l u l o s e column (26 cm x 20 cm; carbonate form) which had been packed i n 50% e t h a n o l . The column was washed s u c c e s s i v e l y w i t h 50% e t h a n o l (500 ml), water (250 ml) and 0.1 M ammonium b i c a r b o n a t e (500 ml). E l u t i o n was c a r r i e d out u s i n g a l i n e a r g r a d i e n t of 50% e t h a n o l ( 1 l i t r e ) and 0.1 M ammonium b i c a r b o n a t e i n 50% e t h a n o l and the o p t i c a l d e n s i t y of the e l u a t e was monitored a t 260 nm. One l a r g e peak of 19 u l t r a v i o l e t a b s o r b i n g m a t e r i a l and a number of s m a l l e r ones were observed and 5*-0-DMT thymidine 3 1 ( p - n i t r o p h e n y l ) phosphate was i d e n t i f i e d i n the l a r g e peak by i t s spectrum.and by i t s Rf i n s o l v e n t 2 which was 0.80 (7.2). The s o l v e n t was removed by e v a p o r a t i o n and the r e s i d u e t r e a t e d w i t h 10 ml of 80% a c e t i c a c i d . A f t e r f o u r hours the orange-red s o l u t i o n was evaporated and the r e s i d u e taken up i n 10 ml of water, which was e x t r a c t e d w i t h e t h e r (10 m l ) . The aqueous phase, from which l a s t t r a c e s of e t h e r were removed by a p p l i c a t i o n of a p a r t i a l vacuum, was passed onto a D E A E - c e l l u l o s e column (26 cm x 2.0 cm; carbonate form), which was washed w i t h 250 ml of water. E l u t i o n was c a r r i e d out u s i n g a l i n e a r g r a d i e n t system of water (1 l i t r e ) and 0.1 M N H 4 H C O 3 ( 1 l i t r e ) . Twenty ml f r a c t i o n s were c o l l e c t e d every 6 minutes and the O.D. of each f r a c t i o n was determined a t 267 nm. A s i n g l e l a r g e peak c o n t a i n i n g the Tp-NP was e l u t e d i n f r a c t i o n s 35 - 75. These f r a c t i o n s were combined and the s o l u t i o n thus o b t a i n e d was c o n c e n t r a t e d by e v a p o r a t i o n . The p r o d u c t was f i n a l l y i s o l a t e d a f t e r f r e e z e - d r y i n g and the y i e l d was 320 mg (71%). The n u c l e o t i d e was c h r o m a t o g r a p h i c a l l y homogenous i n s o l v e n t 2 w i t h a Rf v a l u e of 0.74 w h i l e NP-pT (purchased from Raylo Chemicals Ltd.) e x h i b i t e d an Rf of 0.57 i n the same s o l v e n t . The Rf v a l u e s o b t a i n e d by Borden and Smith (72) f o r these compounds were 0.72 and 0.58 r e s p e c t i v e l y . The f i n a l p r o d u c t behaved i d e n t i c a l l y i n s o l v e n t s 2 and 3 w i t h a sample of Tp-NP k i n d l y donated by Dr. W.E. R a z z e l l . I t s e x t i n c t i o n c o e f f i c i e n t a t 267 nm was 16,. 800 which d i f f e r s from the v a l u e r e p o r t e d by Turner and Khorana (112) which was 15,400. 20 In l a t e r experiments Tp-NP which became a v a i l a b l e from Raylo Chemicals L t d . was used. T h i s m a t e r i a l was found t o have an e x t i n c t i o n c o e f f i c i e n t of 14,800 a t 267 nm and a Rf i n s o l v e n t 2 of 0.76. B. S y n t h e s i s of Tp-DNP: Tp-DNP was prepared u s i n g a procedure f i r s t d e s c r i b e d by Borden and Smith (72) f o r the 5* e s t e r and l a t e r more f u l l y e l a b o r a t e d by von T i g e r s t r o m and Smith (70) . The method i n v o l v e s condensation of thymidine 3'-phosphate w i t h 2 , 4 - d i n i t r o p h e n o l i n the presence of DCC. Thymidine 3'-phosphate was prepared by the method of Tener (75) which i n v o l v e s p h o s p h o r y l a t i o n of 5'-O-DMT thymidine u s i n g 2-cyanoethyl phosphate. Cyanoethyl phosphate (Ba s a l t , 4 mMole) was suspended i n 10 ml water and Dowex 50 (H + form) added u n t i l the s a l t d i s s o l v e d . The s o l u t i o n was passed through a s m a l l (5 cm x 1 cm) column c o n t a i n i n g Dowex 50 (H + form). Ten ml of p y r i d i n e were added to i the e l u a t e and the s o l u t i o n was evaporated t o dryness. 5-0- DMT thymidine (2 mMole) was added t o the r e s i d u e and the a d d i t i o n of 5 ml p o r t i o n s of dry p y r i d i n e f o l l o w e d by e v a p o r a t i o n was repeated s e v e r a l times t o remove l a s t t r a c e s of water. The anhydrous gummy r e s i d u e was f i n a l l y d i s s o l v e d i n 8 ml of dry p y r i d i n e and DCC (3.34 g, 16 mMole) was added. The r e a c t i o n mixture was allowed t o stand f o r 52 hours. E i g h t ml of water were added and the mixture evaporated t o g i v e a t h i c k suspension from which l a s t t r a c e s of p y r i d i n e were removed by repeated a d d i t i o n and e v a p o r a t i o n of s m a l l (5 ml) p o r t i o n s of water. The r e s i d u e was suspended i n 30 ml of 80% a c e t i c a c i d and r e f l u x e d g e n t l y f o r 30 minutes a f t e r which the a c e t i c a c i d was removed by e v a p o r a t i o n . Twenty ml of water were added and e v a p o r a t i o n of t h i s removed l a s t t r a c e s of water by entrainment. T h i r t y f i v e ml of 1 N l i t h i u m hydroxide were added and the s o l u t i o n a g a i n r e f l u x e d f o r 25 minutes. A f t e r c o o l i n g the p r e c i p i t a t e was removed by f i l t r a t i o n . Dowex 50 (H + form) was then added t o the f i l t r a t e to r a i s e the pH t o about 4, a f t e r which the s o l u t i o n was passed through a s m a l l Dowex 50 (H + form) column t o ensure the f o r m a t i o n of the f r e e a c i d . The pH of the e l u a t e was a d j u s t e d t o 7.4 w i t h s a t u r a t e d barium hydroxide and the s o l u t i o n c o n c e n t r a t e d by e v a p o r a t i o n t o a s m a l l volume. The barium s a l t of thymidine 3"-phosphate was p r e c i p i t a t e d by the a d d i t i o n of 2 volumes of e t h a n o l . The n u c l e o t i d e was c o l l e c t e d by f i l t r a t i o n and was washed s u c c e s s i v e l y w i t h 50% e t h a n o l , e t h a n o l , acetone and e t h e r . The y i e l d was 0.83 g (81%) and the product was c h r o m a t o g r a p h i c a l l y homogenous i n s o l v e n t s 2 and 3. Thymidine 3'-phosphate (510 mg) was converted t o the f r e e a c i d i n the u s u a l manner. The n u c l e o t i d e s o l u t i o n was reduced by e v a p o r a t i o n t o about 10 ml and t r i e t h y l a m i n e (1 ml) was added a f t e r which the s o l u t i o n was taken t o drynes s . The r e s i d u e was d i s s o l v e d i n 20 ml of DMF and 2 , 4 - d i n i t r o p h e n o l (10 mMole) and DCC (20 mMole) were added. A p r e l i m i n a r y experiment, performed on a s m a l l s c a l e i n d i c a t e d t h a t the r e a c t i o n was complete a f t e r 4 hours, s i n c e no thymidine 3'-phosphate c o u l d be d e t e c t e d chromatographically. a f t e r t h i s time. A t the end of t h i s p e r i o d 1 ml of water was added and the mixture l e f t f o r a f u r t h e r hour. The suspension was c o n c e n t r a t e d by e v a p o r a t i o n and the r e s i d u e e x t r a c t e d w i t h t h r e e p o r t i o n s (30 ml each) of water. Excess 2 , 4 - d i n i t r o p h e n o l was removed by s e v e r a l e x t r a c t i o n s w i t h e t h e r . T r a c e s o f e t h e r were removed from the aqueous s o l u t i o n under a p a r t i a l vacuum and i t was then passed onto a D E A E - c e l l u l o s e column (34 cm x 2.5 cm, carbonate form). The column was washed wit h 500 ml of water and Tp-DNP was e l u t e d u s i n g a g r a d i e n t of water (2 l i t r e s ) and 0.1 M NH^HCO^ (2 l i t r e s ) . Twenty ml f r a c t i o n s were c o l l e c t e d every 5 minutes and a l a r g e peak of m a t e r i a l which absorbed l i g h t a t 267 nm was e l u t e d i n f r a c t i o n s 55-72. T h i s m a t e r i a l was found t o be the product by i t s m o b i l i t y i n s o l v e n t 3. These f r a c t i o n s were combined and c o n c e n t r a t e d by e v a p o r a t i o n , the pH of the s o l u t i o n b e i n g m a i n t a i n e d around 4 by the a d d i t i o n of Dowex 50 (H + form). A f t e r f i l t r a t i o n t o remove the r e s i n the s o l u t i o n was l y o p h i l i z e d t o g i v e a f a i n t l y y e l l o w powder. Paper chromatography of the prod u c t i n s o l v e n t 3 i n d i c a t e d the presence of s m a l l amounts of thymidine 3 *-phosphate, 2 , 4 - d i n i t r o p h e n o l and two slow-running f l u o r e s c e n t compounds which were u n i d e n t i f i e d . The remainder of the crude product was chromatographed on Whatman 31 paper i n s o l v e n t 3. Tp-DNP was e l u t e d from the paper With water and r e c o v e r e d by l y o p h i l i z a t i o n . The y i e l d was 310 mg, 61%. The Rf v a l u e s of the product i n s o l v e n t s 3 and 4 were 0.49 and 0.76 r e s p e c t i v e l y . The l a t t e r v a l u e agrees w i t h t h a t o b t a i n e d by von T i g e r s t r o m and Smith (70). The n u c l e o t i d e migrated 3.6 cm a f t e r 48 hour chromatography i n s o l v e n t 5. A v a l u e of 4.9 cm was o b t a i n e d by von T i g e r s t r o m and Smith (7 0). Chromatography i n s o l v e n t 2 l e d t o e x t e n s i v e breakdown of the e s t e r due t o the a l k a l i n i t y of the s o l v e n t . The molar e x t i n c t i o n c o e f f i c i e n t a t the a b s o r p t i o n maximum (265 nm) was 20,420 i n good agreement wit h the v a l u e of 20,200 o b t a i n e d by von T i g e r s t r d m and Smith (70). Enzyme Assays Phos p h o d i e s t e r a s e I I : A. With'Tp-NP as s u b s t r a t e : The assay was c a r r i e d out r o u t i n e l y a t 37° i n s m a l l t e s t tubes (70 mm x 8 mm) i n a manner s i m i l a r t o t h a t d e s c r i b e d by R a z z e l l and Khorana (48) . The i n c u b a t i o n mixture c o n t a i n e d 30 uMole ammonium s u c c i n a t e , 1 uMole Tp-NP and an a l i q u o t of enzyme s o l u t i o n i n a t o t a l volume of 0.30 ml a t pH 6.1. Where neces s a r y the enzyme s o l u t i o n was d i l u t e d so t h a t the r e l e a s e of p - n i t r o p h e n o l was l i n e a r w i t h time up to 45 minutes. The K M f o r -4 the enzyme was l a t e r found t o be 4.5 x 10 M and t h i s f i n d i n g allowed the amount of s u b s t r a t e t o be reduced i n l a t e r experiments t o 0.15 uMole per r e a c t i o n tube. The r e a c t i o n was stopped by adding 2.7 ml of 0.1 N NaOH and the o p t i c a l d e n s i t y of the r e s u l t i n g s o l u t i o n was measured a t 400 nm - the a b s o r p t i o n peak of p - n i t r o p h e n o l . B. With Tp-DNP as s u b s t r a t e : The assay was c a r r i e d out i n spectrophotometer micro-c u v e t t e s c o n t a i n i n g 100 uMole of ammonium s u c c i n a t e , 0.5 uMole Tp-DNP and 10 u l i t r e s of enzyme s o l u t i o n i n a t o t a l volume of 1.0 ml a t pH 6.1. The r e l e a s e of 2 , 4 - d i n i t r o p h e n o l was 24 measured continuously a t 360 nm u s i n g a G i l f o r d Model 2000 r e c o r d i n g spectrophotometer a t 37°. The molar e x t i n c t i o n c o e f f i c i e n t s f o r p - n i t r o p h e n o l i n 0.1 N NaOH and 2 , 4 - d i n i t r o p h e n o l i n water were determined t o be 17,600 and 13,900 r e s p e c t i v e l y . These v a l u e s are s l i g h t l y s m a l l e r than those r e p o r t e d by von T i g e r s t r o m and Smith (70). Only i n s i g n i f i c a n t changes i n the a b s o r p t i o n of 2 , 4 - d i n i t r o p h e n o l occur above pH 5 so t h a t a f i x e d - t i m e assay f o r PDase I I can a l s o be performed u s i n g Tp-DNP as s u b s t r a t e , the r e a c t i o n b e i n g stopped w i t h 0.1 N NaOH o r , b e t t e r , 0.1 M Na 2C02« S i n c e i n t e s t i n a l mucosa was b e i n g f r a c t i o n a t e d w i t h . a view t o l o c a l i z i n g the PDase I I a c t i v i t y , i t was c o n s i d e r e d necessary t o employ some enzyme markers on o r d e r t o i d e n t i f y the v a r i o u s f r a c t i o n s o b t a i n e d . A c c o r d i n g l y the a c t i v i t i e s of a c i d phosphatase, cytochrome o x i d a s e , glucose-6-phosphatase, a l k a l i n e phosphatase and s u c c i n i c dehydrogenase were used as markers f o r lysosomes (56,76,77), m i t o c h o n d r i a (76,78,79,80), microsomal fragments of endoplasmic r e t i c u l u m (76,81), microsomal fragments of brush b o r d e r e p i t h e l i u m (82,83,84,85) and m i t o c h o n d r i a (79,86, 87) r e s p e c t i v e l y . A c i d phosphatase: The assay was performed i n a manner s i m i l a r t o t h a t f o r PDase I I , by measuring the p - n i t r o p h e n o l l i b e r a t e d from 8 mMole p - n i t r o p h e n y l phosphate i n a 0.1 M sodium a c e t a t e b u f f e r a t pH 5.0. R e a c t i o n was f o r 20 minutes a t 37° (55). 25 Cytochrome o x i d a s e : T h i s a c t i v i t y was measured a c c o r d i n g t o the method of C o o p e r s t e i n and Lazarow (88) as d e s c r i b e d by Appelmans e t a l . ( 7 6 ) . Horse h e a r t cytochrome c was purchased from Calbiochem (Los A n g e l e s ) . Reduced cytochrome c was prepared by adding 0.2 ml of a f r e s h l y prepared 1.2 M s o l u t i o n of sodium h y d r o s u l p h i t e t o 60 - 5 ml of a 1.7 x 10 M s o l u t i o n of cytochrome c i n 0.03 M potassium phosphate b u f f e r . The pH of the s o l u t i o n was 7.4. One-t e n t h ml of a s u i t a b l y d i l u t e d t i s s u e sample was added t o 3 ml of reduced cytochrome c i n a spectrophotometer c u v e t t e a t room temperature. The decrease i n o p t i c a l d e n s i t y a t 550 nm was measured f o r 2-3 min, a f t e r which a blank r e a d i n g was o b t a i n e d f o l l o w i n g the a d d i t i o n of a few c r y s t a l s of potassium f e r r i c y a n i d e t o completely o x i d i z e the cytochrome. The c a l c u l a t i o n s were performed i n the manner d e s c r i b e d by C o o p e r s t e i n and Lazarow (88). Glucose-6-phosphatase: Glucose-6-phosphate was purchased from Sigma Chemical Co. The assay was performed a t 37° i n an i n c u b a t i o n mixture of 1 ml which c o n t a i n e d 8 mMole glucose-6-phosphate i n 0.05 M i m i d a z o l e -HC1 b u f f e r and a s u i t a b l e d i l u t i o n of enzyme s o l u t i o n . The pH of the s o l u t i o n was 6.5. The r e a c t i o n was stopped by the a d d i t i o n of 1 ml of 10% (w/v) t r i c h l o r a c e t i c a c i d . The s o l u t i o n s were c e n t r i f u g e d and the i n o r g a n i c phosphate was determined i n the supernatant u s i n g B a r t l e t t ' s m o d i f i c a t i o n (89) of the method of F i s k e and Subbarow (90). 26 A l k a l i n e phosphatase: The assay medium co n t a i n e d 25 uMole of g l y c i n e b u f f e r , 2.5 uMole M g C l 2 , 0.5 uMole Z n C l 2 , 9.0 uMole p - n i t r o p h e n y l phosphate and an enzyme i n a volume of 0.6 ml a t pH 9.2 (85). The r e a c t i o n was allowed t o proceed f o r 45 minutes a t 37° and was stopped by the a d d i t i o n of 2.4 ml of 0.1 N NaOH." The l i b e r a t e d p - n i t r o p h e n o l was e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y . S u c c i n i c dehydrogenase: T h i s a c t i v i t y as measured by the r e d u c t i o n of 2-( p - i o d o p h e n y l ) - 3 - ( p - n i t r o p h e n y l ) - 5 - p h e n y l t e t r a z o l i u m h y d r o c h l o r i d e was determined by C l a r k and Porteous *• m o d i f i c a t i o n (58) of the method of Pennington (86). The a c t i v i t y o f s u c c i n i c dehydrogenase was o b t a i n e d by measuring the O.D. of the formazan formed. DNase and RNase were assayed i n the manner d e s c r i b e d by de Duve e t a l . (77). U n i t s and C a l c u l a t i o n s For PDase I I , a c i d phosphatase, a l k a l i n e phosphatase and glucose-6-phosphatase, one u n i t of a c t i v i t y i s d e f i n e d as the amount of enzyme c a u s i n g the h y d r o l y s i s of- 1 uMole of s u b s t r a t e i n 1 hour under the c o n d i t i o n s d e s c r i b e d . One u n i t of cytochrome oxidase a c t i v i t y i s d e f i n e d as the amount of enzyme which causes the l o g a r i t h m of the c o n c e n t r a t i o n of reduced cytochrome c t o decrease by one unit/minute/100 ml of i n c u b a t i o n mixture (88). The a b s o l u t e u n i t s of a c t i v i t y f o r DNase, RNase and s u c c i n i c dehydrogenase were not c a l c u l a t e d and measurement of these 27 a c t i v i t i e s was based on the o p t i c a l d e n s i t i e s o b t a i n e d under the c o n d i t i o n s of assay. Because of t h i s the u n i t s of a c t i v i t y of these enzymes are a r b i t r a r y and are expressed i n r e l a t i v e terms as a percentage of a p a r t i c u l a r v a l u e . P r o t e i n was determined a c c o r d i n g t o the method of Lowry e t a l . (91) w i t h bovine serum albumin as s t a n d a r d . The s p e c i f i c a c t i v i t i e s o f PDase I I , a c i d phosphatase, glucose-6-phosphatase, a l k a l i n e phosphatase and cytochrome oxidase were d e f i n e d as u n i t s of enzyme/mg p r o t e i n . T o t a l a c t i v i t y was determined by m u l t i -p l y i n g the s p e c i f i c a c t i v i t i e s o f a f r a c t i o n by the t o t a l amount of p r o t e i n ( i n mg) i n t h a t f r a c t i o n . P r e p a r a t i o n of I n t e s t i n a l Mucosal Homogenates Male W i s t a r r a t s weighing 180-200 g were o b t a i n e d from the Zoology V i v a r i u m of the U n i v e r s i t y of B r i t i s h Columbia. The r a t s were s t a r v e d f o r 24 hours p r i o r t o s a c r i f i c e , but were allowed water ad l i b i t u m d u r i n g t h i s p e r i o d . Each animal was stunned by a blow on the head and d e c a p t i t a t e d . The i n t e s t i n e was q u i c k l y removed onto an i c e - c o l d g l a s s s u r f a c e and c u t i n t o p i e c e s about 10 cm l o n g . A hypodermic s y r i n g e f i l l e d w i t h i c e -c o l d 0.9% sodium c h l o r i d e was used t o remove extraneous matter (mucous, food p a r t i c l e s ) from the lumen of the p i e c e s of gut b e f o r e these were s l i t open on the g l a s s s u r f a c e e p i t h e l i a l -s i d e upwards. A microscope s l i d e was drawn a c r o s s the s u r f a c e and the mucosal s c r a p i n g s thus o b t a i n e d were p l a c e d i n a p r e -weighed beaker which was kept on i c e . About 2-2.5 g of t i s s u e was o b t a i n e d per r a t . Before homogenization the s c r a p i n g s were 28 washed by g e n t l e s t i r r i n g i n a s m a l l volume of the a p p r o p r i a t e medium and r e c o v e r e d by c e n t r i f u g a t i o n f o r 5 minutes a t 12,10 0 x g. The t y p e s o f s u s p e n s i o n medium used were: i 0.25 M s u c r o s e i n 1 mM EDTA. pH 7.4 (77) i i 0.25 M s u c r o s e i n 5 mM EDTA. pH 7.4 (58,87) i i i 0.3 M m a n n i t o l (92) and i v 6% d e x t r a n - K r e b s - R i n g e r phosphate pH 7.4 ( 5 8 ) . A 1:10 (w/v) s u s p e n s i o n of t i s s u e was p r e p a r e d and t h i s was homogenized i n a P o t t e r - E l v e h j e m t y p e homogenizer e q u i p p e d w i t h a l o o s e l y - f i t t i n g T e f l o n p e s t l e ( A r t h u r H. Thomas Co., P h i l a d e l p h i a ) . The s u s p e n s i o n was p l a c e d i n t h e s m o o t h - w a l l e d g l a s s v e s s e l o f t h e homogenizer, w h i c h was k e p t i n a b e a k e r o f i c e . The h o m o g e n i z a t i o n was c a r r i e d o ut by f o r c i n g t h e t i s s u e p a s t t h e p e s t l e w h i c h was m o t o r - d r i v e n a t a speed of 400 r.p.m. One complete up-and-down movement of t h e homogenizer i s c a l l e d one pass and each h o m o g e n i z a t i o n c o n s i s t e d of 4-6 p a s s e s . The homogenate thus formed was f i l t e r e d q u i c k l y t h r o u g h 4 l a y e r s o f a gauze t o remove mucous and p i e c e s o f smooth m u s c l e . R a p i d passage of t h e m a t e r i a l t h r o u g h t h e f i l t e r was a i d e d by s t r o k i n g t h e gauze w i t h a c l e a n g l a s s r o d . F r a c t i o n a t i o n of Homogenates by D i f f e r e n t i a l C e n t r i f u g a t i o n D i f f e r e n t i a l c e n t r i f u g a t i o n was p e r f o r m e d u s i n g a S o r v a l l RC2-B c e n t r i f u g e e q u i p p e d w i t h a SS-34 r o t o r and a Beckman Model L u l t r a c e n t r i f u g e e q u i p p e d w i t h a S p i n c o 30 r o t o r . The s u c r o s e and m a n n i t o l f i l t r a t e s were f r a c t i o n a t e d u s i n g t h e f o l l o w i n g p r o c e d u r e w h i c h i s l o o s e l y based on t h a t o f de Duve e t a l . (77). The f i l t r a t e was f i r s t c e n t r i f u g e d f o r 5 minutes a t 7 55 x g. The s u p e r n a t a n t was removed c a r e f u l l y u s i n g a hypodermic s y r i n g e e q u i p p e d w i t h a number 18 n e e d l e and t h e l o o s e l y - p a c k e d , f l o c c u l e n t s ediment was resuspended i n a volume o f medium e q u a l t o t h a t o f t h e s u p e r n a t a n t . R e s u s p e n s i o n of t h e sediment was c a r r i e d o u t i n t h e homogenizer d e s c r i b e d above, t h e p e s t l e b e i n g g e n t l y r o t a t e d by hand. The resuspended sediment was c e n t r i f u g e d a t 755 x g f o r 5 m i n u t e s as b e f o r e . The s u p e r n a t a n t was removed and t h e sediment was washed f o r a second t i m e . A w e l l - p a c k e d p e l l e t was o b t a i n e d by c e n t r i f u g a t i o n a t 12,100 x g f o r 5 m i n u t e s . The twice-washed sediment was f i n a l l y t a k e n up i n a s m a l l volume of medium and was c a l l e d f r a c t i o n I . The f i n a l d i l u t i o n o f f r a c t i o n I as e x p r e s s e d i n g o f o r i g i n a l wet t i s s u e / m l was 1:4. The s u p e r n a t a n t from t h e t h r e e c e n t r i f u -g a t i o n s were combined and c e n t r i f u g e d a t 2445 x g f o r 15 m i n u t e s . The sediment t h u s o b t a i n e d was washed t w i c e w i t h t h e a p p r o p r i a t e medium and resuspended such t h a t i t s f i n a l d i l u t i o n was 1:2. T h i s was c a l l e d f r a c t i o n I I . The t h r e e s u p e r n a t a n t s were a g a i n combined and were f r a c t i o n a t e d t o y i e l d s u c c e s s i v e l y f r a c t i o n s I I I and IV w h i c h were o b t a i n e d by c e n t r i f u g a t i o n a t 15,9 00 x g and 105,000 x g f o r 20 minutes and 120 m i n u t e s r e s p e c t i v e l y . F r a c t i o n I I I was washed j u s t once i n a few ml of medium and f r a c t i o n IV was n o t washed i n o r d e r t o reduce t h e d i l u t i o n o f the f i n a l s u p e r n a t a n t w h i c h was c a l l e d f r a c t i o n V. The f i n a l d i l u t i o n s o f f r a c t i o n s I I I , IV and V were 1:2, 1:4 and a p p r o x i m a t e l y 1:15 r e s p e c t i v e l y , e x p r e s s e d as g of o r i g i n a l t i s s u e / m l . 30 The f i l t e r e d homogenate i n 6% dextran-Krebs Ringer phosphate (58) was t r e a t e d as f o l l o w s . The p r e p a r a t i o n was c e n t r i f u g e d a t 19 35 x g f o r 5 minutes and the b u f f - c o l o u r e d supernatant was c a r e f u l l y removed. The sediment was r e d i s p e r s e d i n a volume of 6% dextran-Krebs Ringer phosphate equal t o t h a t of the supernatant and the suspension was c e n t r i f u g e d as b e f o r e f o r 5 minutes a t 1935 x g. The sediment from t h i s c e n t r i f u g a t i o n which a c c o r d i n g t o C l a r k and Porteous (58) c o n s i s t s of e p i t h e l i a l c e l l - " g h o s t s " was resuspended i n enough 0.25 M sucrose - 5 mM EDTA. pH 7.4 to g i v e a 1:10 (w/v) p r e p a r a t i o n based on the o r i g i n a l wet weight of t i s s u e . The s u s p e n s i o n was then homogenized w i t h 4 passes of the homogenizer as d e s c r i b e d b e f o r e . The e p i t h e l i a l - c e l l "ghost" homogenate (58) thus o b t a i n e d was f r a c t i o n a t e d a c c o r d i n g t o the procedure o u t l i n e d above f o r sucrose and m a n n i t o l homogenates and f r a c t i o n s i_ through v were o b t a i n e d i n t h i s manner. In a number of experiments a more p r o t r a c t e d f r a c t i o n a t i o n of the "ghost" homogenate was c a r r i e d out. A g r e a t e r number of c e l l u l a r f r a c t i o n s were o b t a i n e d as f o l l o w s , f r a c t i o n 1 (480 x g f o r 10 m i n u t e s ) ; f r a c t i o n 2 (1085 x g f o r 15 minutes); f r a c t i o n 3 (1935 x g f o r 20 minutes); f r a c t i o n 4 (7710 x g f o r 25 minutes); f r a c t i o n 5 (17,300 x g f o r 30 minutes); f r a c t i o n 6 (105,000 x g f o r 120 minutes); and f r a c t i o n 7 ( f i n a l s u p e r n a t a n t ) . Each of the f r a c t i o n s was washed twice except 5 which was washed o n l y once and 6 which was unwashed. The f i n a l d i l u t i o n s of f r a c t i o n s 1 and 6, expressed as g o r i g i n a l wet weight of t i s s u e / ml were 1:4 w h i l e those of f r a c t i o n s 2 through 5 were 1:2. 31 The d i l u t i o n of the f i n a l s upernatant was approximately 1:20. The enzyme content of each f r a c t i o n was completely r e l e a s e d by f r e e z i n g and thawing the samples 10 times (76) , or by making the assay i n c u b a t i o n medium 0.1% (v/v) w i t h r e s p e c t t o T r i t o n X-100 (77). The p r o t e i n c o n t e n t and the s p e c i f i c and t o t a l a c t i v i t i e s of both PDase I I and a c i d phosphatase were determined i n a l l the f r a c t i o n s o b t a i n e d and i n a d d i t i o n most f r a c t i o n s were assayed f o r cytochrome oxidase and glucose-6-phosphatase. In the f i r s t experiments the r e c o v e r y of a l l these a c t i v i t i e s from the t i s s u e homogenate was very v a r i a b l e and q u i t e i r r e p r o d u c i b l e . T h i s was e s p e c i a l l y t r u e of the r e c o v e r i e s from the s u c r o s e and m a n n i t o l homogenates. The f o l l o w i n g method which was adopted as a g e n e r a l procedure was used t o o b t a i n a v a l u e f o r the t o t a l a c t i v i t i e s i n the homogenate. When the f i r s t sediment was o b t a i n e d by low-speed c e n t r i f u g a t i o n , a p o r t i o n of the supernatant was removed and subsequently assayed w i t h the o t h e r f r a c t i o n s . The sum of the t o t a l a c t i v i t i e s , o b t a i n e d s e p a r a t e l y , f o r the low-speed sediment ("nuclear f r a c t i o n " ) and the supernatant ("cytoplasmic f r a c t i o n " ) was taken t o be r e p r e s e n t a t i v e of the whole t i s s u e and r e c o v e r i e s were c a l c u l a t e d from t h i s t o t a l v a l u e (77). The r e c o v e r y of p r o t e i n and such s t a b l e enzymes as P D a s e i l l and a c i d phosphatase v a r i e d between 92 and 110 p e r c e n t and were q u i t e r e p r o d u c i b l e . A p p a r e n t l y the g e l a t i n o u s mucous which p r e s e n t s d i f f i c u l t i e s i n the f r a c t i o n a t i o n of homogenates of i n t e s t i n a l mucosa (58,78,84,85,87,92), a l s o i n t e r f e r e s w i t h assay procedures i n these crude p r e p a r a t i o n s . 32 Sucrose D e n s i t y G r a d i e n t C e n t r i f u g a t i o n C e n t r i f u g a t i o n was c a r r i e d out u s i n g a Beckman L2-65B u l t r a c e n t r i f u g e equipped w i t h a SW 25.1 r o t o r . L i n e a r d e n s i t y g r a d i e n t s were prepared from 12% t o 50% (w/v) sucrose u s i n g a B u c h l e r p o l y s t a l t i c pump and g r a d i e n t : maker. One r a t was used per experiment and an e p i t h e l i a l - c e l l "ghost" homogenate was prepared i n the u s u a l way. Unbroken c e l l s and n u c l e i were removed by a s i n g l e low-speed c e n t r i f u g a t i o n (755 x g f o r 10 minutes) and the p a r t i c l e s i n the supernatant were o b t a i n e d by c e n t r i f u g a t i o n a t 105,000 x g f o r 120 minutes. The high-speed supernatant was d i s c a r d e d and the sediment g e n t l y resuspended i n 0.25 M s u c r o s e -1 mM EDTA. pH 7.4. Three ml of t h i s s uspension were g e n t l y layered on--.top of the sucrose g r a d i e n t . The t o t a l volume i n the tube ( g r a d i e n t and t i s s u e sample) was 29-32 ml. The samples thus prepared were c e n t r i f u g e d , a t d i f f e r e n t i n t e g r a t e d f o r c e s (77,93), f o r e i t h e r a) 3 hours at 25,000 r.p.m. b) 2 hours a t 15,000 r.p.m. or c) 1 1/2 hours a t 10,000 r.p.m. A f t e r c e n t r i f u g a t i o n a h o l e was punched i n the bottom of the tubes and 1 ml f r a c t i o n s were c o l l e c t e d v i s u a l l y i n s m a l l t e s t - t u b e s (70 mm x 8 mm) u s i n g a Bu c h l e r c o l l e c t i n g d e v i c e . An a i r p r e s s u r e of 3 p . s . i . was used t o f a c i l i t a t e f low of the dense sucrose suspension through the c o l l e c t i n g n e e d l e . The l i n e a r i t y of the sucrose g r a d i e n t i n a sample to which no t i s s u e suspension had been added was checked u s i n g a r e f r a c t o m e t e r . P r e p a r a t i o n of " P u r i f i e d " Lysosomes from I n t e s t i n a l Mucosa " P u r i f i e d " lysosomes were prepared a c c o r d i n g t o a procedure based on A l p e r ' s a d a p t a t i o n (94) of the method of Sawant e t a l . (95). The procedure i s o u t l i n e d i n F i g . l . The i n i t i a l c e n t r i f u g a t i o n s t o o b t a i n f r a c t i o n s 1,2,3,and 4 were performed u s i n g a S o r v a l l RC2-B c e n t r i f u g e equipped w i t h a GSA r o t o r . The g r a d i e n t c e n t r i f u g a t i o n was done u s i n g a Beckman L2-65B u l t r a c e n t r i f u g e and a SW 25.1 r o t o r and the remaining c e n t r i f u g i n g was performed u s i n g a SS-34 r o t o r i n the S o r v a l l c e n t r i f u g e . 34 Homogenate i n 6% d e x t r a n -Krebs-Ringer phosphate pH 7.4 2000,x g f o r 10 min sediment (1) " c e l l - g h o s t s " rehomogenized i n 0.25 M sucrose - 5 mM EDTA PH 7.4 J 650 x g f o r 10 min supernatant ( d i s c a r d ) sediment rehomogenized i n sucrose-EDTA supernatant 650 x g f o r 10 min sediment ( d i s c a r d ) supernatant (2) 16,300 x g j f o r 10 min (3) sediment resuspended i n 0.4 M sucrose 16,300 x g l f o r 10 min j supernatant ( d i s c a r d ) (4) sediment supernatant ( d i s c a r d ) resuspended i n 7 ml 0.4 M sucrose l a y e r e d over 12 ml 0.7 M sucrose (bottom) and 10 ml 0.6 M sucrose (middle) 14,422 x g f o r 30 min (5) sediment resuspended i n 0.7 M sucrose 5900 x g ( f o r 30 min supernatant 17,300 x g j f o r 20 min sediment (8) microsomes sediment (9) "mi t o c h o n d r i a" supernatant (6) supernatant ( d i s c a r d ) 17,300 x g f o r 20 min sediment (7) "lysosomes" supernatant ( d i s c a r d ) F i g . 1. " P u r i f i c a t i o n " of lysosomes from r a t i n t e s t i n a l mucosa. E p i t h e l i a l s c r a p i n g s from 5 r a t s were used i n the experiment. Samples from the numbered f r a c t i o n s were removed and assayed f o r PDase I I , a c i d phosphatase and p r o t e i n . 35 RESULTS I n t r o d u c t o r y . - I t was i n i t i a l l y found t h a t c r u d e homogenates o f mucosal s c r a p i n g s c o n t a i n e d an a c t i v i t y w h i c h was c a p a b l e of h y d r o l y z i n g Tp-NP. S u b s e q u e n t l y , an homogenate was p r e p a r e d i n 0.25 M s u c r o s e - 1 mM EDTA. pH 7.4, c e n t r i f u g e d a t 105,000 x g f o r 1 hour and PDase I I a c t i v i t y was found i n t h e s u p e r n a t a n t s o l u t i o n . The e f f e c t o f d i l u t i o n of t h i s f r a c t i o n on t h e time-dependent h y d r o l y s i s o f Tp-NP and Tp-DNP i s shown i n F i g . 2. I t was found t h a t t h e r a t e of h y d r o l y s i s o f Tp-DNP by t h i s s u p e r n a t a n t f r a c t i o n was g r e a t e r t h a n t h a t o f Tp-NP. T h i s has a l s o been r e p o r t e d by Smi t h and co-workers f o r s p l e e n PDase (70) and salmon t e s t i s PDase (59). Under s t a n d a r d assay c o n d i t i o n s t h e s p e c i f i c a c t i v i t i e s o f t h e p r e p a r a t i o n w i t h Tp-NP and Tp-DNP as s u b s t r a t e s were 0.220 and 0.417 r e s p e c t i v e l y . A l t h o u g h t h e assay of PDase I I w i t h Tp-DNP as s u b s t r a t e was q u i c k e r and more c o n v e n i e n t t h a n t h a t e m p l o y i n g Tp-NP, t h e l i m i t e d a v a i l a b i l i t y o f t h e d i n i t r o p h e n y l e s t e r n e c e s s i t a t e d t h e use of Tp-NP, w h i c h was a t hand i n g r e a t e r amounts, i n most of t h e e x p e r i m e n t s . Some of the p r o p e r t i e s o f PDase I I were d e t e r m i n e d w i t h Tp-DNP as s u b s t r a t e . . . I n t e s t i n a l O r i g i n of t h e P h o s p h o d i e s t e r a s e I I A c t i v i t y E a r l y i n t h e work i t was n e c e s s a r y t o e s t a b l i s h t h a t t h e enzyme found i n i n t e s t i n a l p r e p a r a t i o n s d i d n o t . a r i s e from an organ whose s e c r e t i o n e n t e r s t h e i n t e s t i n e . To t h i s end t h e TIME OF INCUBATION (min) F i g . 2. E f f e c t of time on the h y d r o l y s i s o f Tp-NP (A) and Tp-DNP (B) by the PDase I I a c t i v i t y of r a t i n t e s t i n a l mucosa. A supernatant f r a c t i o n , prepared as d e s c r i b e d i n the t e x t , was used as a source of enzyme. R e s u l t s are g i v e n f o r the u n d i l u t e d supernatant ( A , ) , and f o r a two f o l d d i l u t i o n (•, ) and a f o u r f o l d d i l u t i o n ) of the o r i g i n a l supernatant. 37 f o l l o w i n g experiments were c a r r i e d out. A 1:10 (w/v) suspension of mucosal s c r a p i n g s from 3 r a t s was prepared i n 0.25 M s u c r o s e - 1 mM EDTA, pH 7.4. The t i s s u e was s t i r r e d g e n t l y w i t h a g l a s s rod f o r 30 sec and c e n t r i f u g e d a t 12,000 x g f o r 10 min. The supernatant s o l u t i o n was removed and the sediment was homogenized i n a volume of f r e s h sucrose-EDTA equal to t h a t of the supernatant. Assays f o r PDase I I , a c i d phosphatase, cytochrome o x i d a s e , glucose-6-phosphatase and p r o t e i n were c a r r i e d out on the o r i g i n a l suspension f l u i d and on the homogenized sediment. The r e s u l t s are shown i n Table I. I t can be seen t h a t even a f t e r t h i s a p p a r e n t l y g e n t l e treatment of the mucosal t i s s u e , a p p r e c i a b l e amounts of a c i d phosphatase, PDase I I and p r o t e i n were p r e s e n t i n the supernatant f r a c t i o n . These a c t i v i t i e s c o u l d not be sedimented by c e n t r i f u g a t i o n a t 105,000 x g f o r 30 min. Since no cytochrome oxidase or g l u c o s e -6-phosphatase a c t i v i t y was found i n the supernatant i t i s probable t h a t the s t i r r i n g treatment d i d not damage the t i s s u e s u f f i c i e n t l y t o r e l e a s e m i t o c h o n d r i a and/or microsomes. Two e x p l a n a t i o n s f o r the appearance of the c o n s i d e r a b l e amounts of PDase I I , a c i d phosphatase and p r o t e i n i n the supernatant were c o n s i d e r e d . i ) the a c t i v i t i e s found i n the supernatant p o r t i o n of the suspension might have been p r e s e n t i n the lumen of the i n t e s t i n e when the animals were k i l l e d . T h i s does not seem very l i k e l y s i n c e the p i e c e s of i n t e s t i n e were thoroughly washed w i t h i s o t o n i c s a l i n e b e f o r e the mucosa was scraped o f f . TABLE I D i s t r i b u t i o n o f some enzyme a c t i v i t i e s i n a suspension of r a t i n t e s t i n a l mucosa  A c i d Cytochrome Glucose-6-Component of P r o t e i n PDase I I Phosphatase Oxidase Phosphatase suspension mg % u n i t s . % u n i t s % u n i t s % u n i t s % Supernatant 114.7 33 21.8 23 213.3 40 0.0 0 1.1 0.4 Sediment 237.0 67 73.5 77 322.3 60 291.5 100 296.3 99.6 39 i i ) the s o f t , f r a g i l e e p i t h e l i a l t i s s u e might have s u f f e r e d enough damage to r e l e a s e some of the c e l l c o n s t i t u e n t s , r e s u l t i n g i n the l i b e r a t i o n o f s u b s t a n t i a l amounts of PDase I I , a c i d phosphatase and p r o t e i n . The l a t t e r p o s s i b i l i t y was c o n s i d e r e d more l i k e l y and i t was t e s t e d by c a r r y i n g out the f o l l o w i n g experiment. The mucosal s c r a p i n g s from 2 r a t s were washed as f o l l o w s . A 1:10 (w/v) sus p e n s i o n was prepared i n 0.25 M sucrose - 1 mM EDTA and a f t e r g e n t l e s t i r r i n g was c e n t r i f u g e d a t 27,000 x g f o r 10 min. The supernatant was s e t a s i d e and the sediment resuspended i n a volume o f su c r o s e - EDTA e q u a l t o the supernatant. The susp e n s i o n was c e n t r i f u g e d as b e f o r e and the supernatant was a g a i n removed and s t o r e d . The procedure was repeated a t h i r d t i m e and the t h r i c e washed sediment was f i n a l l y suspended i n sucrose - EDTA and homogenized. The homogenate was c e n t r i f u g e d a t 27,000 x g f o r 10 min to g i v e a supernatant and c e l l d e b r i s f r a c t i o n s . The PDase I I , a c i d phosphatase and p r o t e i n contents of these f r a c t i o n s were measured as w e l l as those i n the t h r e e washes o b t a i n e d p r e v i o u s l y . The r e s u l t s are g i v e n i n Ta b l e I I . I t was shown t h a t as the mucosal s c r a p i n g s were washed, p r o g r e s s i v e l y l e s s and l e s s p r o t e i n , PDase I I and a c i d phosphatase were found i n the washings. On homogenization, however, i n c r e a s e d amounts of PDase I I , a c i d phosphatase and p r o t e i n were r e c o v e r e d i n the supernatant f r a c t i o n . T h i s was an i n d i c a t i o n t h a t the PDase I I a c t i v i t y o f i n t e s t i n a l e x t r a c t s was i n t r a c e l l u l a r and was not due to an enzyme r e l e a s e d i n t o the lumen of the i n t e s t i n e from another organ. Presumably the 40 TABLE I I The PDase I I and a c i d phosphatase a c t i v i t i e s and the p r o t e i n content of the s u c c e s s i v e washings and homogenate* of a sample of r a t i n t e s t i n a l mucosa P r o t e i n PDase I I A c i d Phosphatase F r a c t i o n mg % u n i t s % u n i t s % 1 s t Washing 37.2 18. 8 10.4 21. 8 84.1 31.2 2nd Washing 31.2 15. 7 4.1 8. 6 19.0 7.1 3rd Washing 22.8 11. 5 4.1 8. 6 8.0 3.0 Supernatant 40.8 20. 5 13.8 28. 9 50.8 18.8 * Sediment 66.4 33. 5 15.3 32. 1 107.6 39.9 198.2 47.7 269.5 The homogenate comprises the supernatant and sediment f r a c t i o n s 41 a p p r e c i a b l e amounts of a c i d phosphatase and PDase I I found i n the i n i t i a l washings of i n t e s t i n a l s c r a p i n g s were due t o r u p t u r e of the t i s s u e d u r i n g i s o l a t i o n . L o c a l i z a t i o n of Phosphodiesterase I I A c t i v i t y i n I n t e s t i n a l T i s s u e A. A c t i v a t i o n Experiments S i n c e van Dyck and Wattiaux (52) and E r e c i n s k a e t a l . (53) have r e p o r t e d t h a t PDase I I i n the r a t l i v e r i s a s s o c i a t e d w i t h lysosomes and R a z z e l l has i n d i c a t e d (50) t h a t t h i s might a l s o be t r u e f o r hog kidney, i t was of immediate i n t e r e s t t o see i f t h i s was the case f o r i n t e s t i n a l PDase I I . De Duve and co-workers (96) have shown t h a t one of the c l a s s i c p r o p e r t i e s of lysosomal enzymes i s t h a t they can become " a c t i v a t e d " by osmotic or mechanical means. " A c t i v a t i o n " occurs when the lysosomal membrane breaks and the enzyme i s r e l e a s e d i n t o the s o l u b l e p o r t i o n of the suspension. Suspensions and homogenates of mucosal s c r a p i n g s ( 1 g/ 10 ml) were prepared i n both water and 0.154 M sodium c h l o r i d e both of which had been a d j u s t e d t o pH 7.0 by the a d d i t i o n of a s m a l l amount of sodium b i c a r b o n a t e . Each of the f o u r p r e p a r a -t i o n s was d i v i d e d e q u a l l y i n t o p o r t i o n s , one of which was assayed f o r PDase I I a t zero time and the o t h e r a f t e r s t a n d i n g f o r 96 hours a t 4 a. Before assay each p o r t i o n was c e n t r i f u g e d a t 105,000 x g f o r 1 hour t o g i v e a supernatant and a sediment f r a c t i o n . The assay r e s u l t s are g i v e n i n Tables I I I and IV. I t was observed t h a t i n both of the p r e p a r a t i o n s t r e a t e d w i t h the h y p o t o n i c medium (water) t h e r e was an i n c r e a s e i n the t o t a l 42 TABLE I I I E f f e c t o f medium t o n i c i t y on the PDase I I a c t i v i t y of suspensions and homogenates of r a t i n t e s t i n a l mucosa Treatment T o t a l A c t i v i t y (Units) Zero Time A f t e r 96 Hr Suspended i n water 35.3 57.0 Homogenized i n water 36.5 50.5 Suspended i n i s o t o n i c s a l i n e 44.0 41.5 Homogenized i n i s o t o n i c s a l i n e 43.6 46.3 43 TABLE IV R e d i s t r i b u t i o n of PDase I I a c t i v i t y between the s o l u b l e and p a r t i c u l a t e components of homogenates and suspensions prepared i n media of d i f f e r i n g t o n i c i t y P r e p a r a t i o n F r a c t i o n Zero Time A f t e r 96 Hr u n i t s % u n i t s % Suspension Supernatant 17 .9 50 .7 35. 3 61. 9 i n water Sediment 17 .4 49 .3 21. 7 38. 1 Homogenate Supernatant 24 .4 66 .9 34. 7 68. 7 i n water Sediment 12 .1 33 .1 15. 8 31. 3 Suspension i n Supernatant 17 .7 40 .2 29. 7 71. 6 i s o t o n i c s a l i n e Sediment 26 .3 59 .8 11. 8 28. 4 Homogenate i n Supernatant 25 .0 57 .3 32. 2 69. 6 i s o t o n i c s a l i n e Sediment 18 .6 42 .7 14. 1 30. 4 a c t i v i t y of PDase I I a f t e r s t a n d i n g , f o r 96 h r (Table I I I ) . T h i s i n c r e a s e amounted t o 61% f o r t h e s u s p e n s i o n i n w a t e r and 38% f o r t h e homogenate i n w a t e r . I n t h e same p e r i o d o f t i m e t h e r e was l i t t l e o r no i n c r e a s e i n t h e t o t a l a c t i v i t y o f PDase I I i n the p r e p a r a t i o n s suspended and homogenized i n i s o t o n i c s a l i n e . I t was a l s o seen ( T a b l e IV) t h a t t h e r e was a r e d i s t r i b u t i o n o f t h e PDase I I a c t i v i t y i n f a v o u r o f t h e s o l u b l e p o r t i o n o f each o f t h e f o u r p r e p a r a t i o n s . T h i s was most a p p a r e n t when th e mucosa was suspended r a t h e r t h a n homogenized i n t h e media (Table IV) . These f i n d i n g s a r e c o m p a t i b l e w i t h t h e p r e m i s e t h a t t h e a c t i v i t y o f PDase I I i n i n t e s t i n e i s i n t r a c e l l u l a r and p r o b a b l y r e s i d e s i n some o r g a n e l l e s i n c e homogenates o f i n t e s t i n a l mucosa, w h i c h a r e presumably s u s p e n s i o n s o f a l l t h e c e l l o r g a n e l l e s , c o u l d a l s o be o s m o t i c a l l y a c t i v a t e d . Some of t h e o t h e r f i n d i n g s a r e q u i t e p u z z l i n g and can n o t be e x p l a i n e d i n terms of t h e r e a s o n i n g o u t l i n e d above. F o r i n s t a n c e , t h e i n i t i a l t o t a l a c t i v i t e s o f PDase I I i n t h e p r e p a r a t i o n s i n i s o t o n i c s a l i n e were about 23% h i g h e r t h a n t h o s e o b t a i n e d w i t h t h e p r e p a r a t i o n s i n w a t e r (Table I I I ) . A g a i n i t was n o t e d (Table IV) t h a t , a f t e r s t a n d i n g f o r 96 h r , t h e se d i m e n t s from t h e p r e p a r a t i o n s i n w a t e r had h i g h e r a c t i v i t i e s t h a n t h o s e a t z e r o t i m e w h i l e t h e v a l u e s f o r t h e sediments i n s a l i n e d e c r e a s e d . These r e s u l t s w i l l be d i s c u s s e d l a t e r . I n a n o t h e r e x p e r i m e n t t h e a c t i v i t i e s o f t h r e e known l y s o s o m a l enzymes (56) and PDase I I were measured i n homogenates of i n t e s t i n a l mucosa under t h e a c t i v a t i n g c o n d i t i o n s d e s c r i b e d 45 by Appelraans e t a l . (76) and by de Duve e t a l . (77). The r e s u l t s are g i v e n i n Table V. I t was seen t h a t the f o u r enzymes were a c t i v a t e d t o d i f f e r e n t e x t e n t s by d i f f e r e n t treatments. De Duve e t a l . (77) found t h a t the a c t i v i t y o f a c i d phosphatase i n homogenates of l i v e r i n c r e a s e d about 5 times under a c t i v a t i n g c o n d i t i o n s whereas i n t h i s case (Table V) the a c t i v i t y of t h i s enzyme o n l y i n c r e a s e d 1.6 times. S i m i l a r low degrees of a c t i v a -t i o n were observed f o r RNase and PDase I I . I t i s p o s s i b l e t h a t these enzymes had a l r e a d y become a c t i v a t e d t o some e x t e n t d u r i n g the p r e p a r a t i o n of the homogenate. B. D i f f e r e n t i a l C e n t r i f u g a t i o n of M u c o s a l - C e l l Homogenates I n i t i a l attempts were made to f r a c t i o n a t e mucosal t i s s u e by a method based on t h a t of de Duve e t a l . (77) u s i n g homogenates i n 0.25 M s u c r o s e - 1 mM EDTA. The r e s u l t s of the enzyme d e t e r m i n a t i o n s on the f r a c t i o n s o b t a i n e d from such an experiment are g i v e n i n Tab l e V I . The mucosal s c r a p i n g s from 2 r a t s were used i n the experiment. The d a t a show t h a t although o n l y 0.6% o f the p r o t e i n s were r e c o v e r e d i n F r a c t i o n I the s p e c i f i c a c t i v i t i e s o f PDase I I , a c i d phosphatase and cytochrome oxidase were a l l h i g h e s t i n t h i s f r a c t i o n w h i l e the t o t a l a c t i v i t i e s of these enzymes were h i g h e r i n ot h e r f r a c t i o n s . F r a c t i o n I I c o n t a i n e d 72% of the r e c o v e r e d cytochrome oxidase a c t i v i t y w h i l e f r a c t i o n IV c o n t a i n e d 66% of the r e c o v e r e d glucose-6-phosphatase a c t i v i t y , so t h a t i t i s l i k e l y t h a t these two f r a c t i o n s are composed l a r g e l y of m i t o c h o n d r i a and microsomes r e s p e c t i v e l y . While approximately 80% of the PDase I I and a c i d phosphatase 46 TABLE V E f f e c t of a c t i v a t i n g c o n d i t i o n s on the a c t i v i t i e s o f PDase I I and some lysosomal enzymes i n an homogenate.* P r e p a r a t i o n DNase RNase A c i d Phosphatase PDase I I U n t r e a t e d homogenate 100 .100 100 100 B l e n d o r - t r e a t e d homogenate a 431 100 103 97 Homogenate + T r i t o n X-100 b 646 186 160 113 * A c t i v i t i e s are expressed as a percentage of the v a l u e s i n the u n t r e a t e d homogenate a T r e a t e d w i t h a Sorvall-Omni Mixer f o r 3 min a t a r h e o s t a t s e t t i n g of 50 b Homogenate was made 0.5% wi t h r e s p e c t t o T r i t o n X-100 TABLE VI D i s t r i b u t i o n of the a c t i v i t i e s o f PDase I I , a c i d phosphatase, cytochrome oxidase and glucose-6-phosphatase i n s u b c e l l u l a r f r a c t i o n s of an homogenate of r a t i n t e s t i n a l mucosa P r o t e i n PDase I I A c i d Phosphatase Cytochrome Oxidase Glucose-6-Phosphatase F r a c t i o n T o t a l S.A. T.A. mg u n i t s u n i t s % T.A. S.A. u n i t s T.A. u n i t s Homogenate 271. .9 100 0. .29 78. ,9 100 0. .94 255. .6 100 0. .79 214. .4 100 0. .77 209. .4 100 I 1. .5 0. 6 1. .34 2. .0 2 .5 3. .60 5. .4 2 .1 6. .27 9. .4 4. 4 0. .0 0. .0 0. 0 I I 16. .5 6. 1 0. .67 11. .1 14 .1 1. .34 22. .1 8 .6 6. .01 99, .2 46. 3 . 1. 01 16, .7 8. 0 I I I 46. .9 17. 2 0. .33 15. .5 19 .2 1. .13 53. .0 20 .7 0. .53 24, .9 11. 6 0. .94 44. .1 21. 1 IV 62. .5 23. 0 0. .13 8. .1 10 .3 0. .90 56. .3 22 .0 0 , .06 3, .6 1. 7 1. .86 116, .3 55. 5 V 161. .3.. ,. 59. 0 0. .25 39. ,9 50 .6 0. .62 100. .7 39 .4 0. .0 0. .0 0. 0 0. .0 0. .0 0. 0 Recovery 105. 9% 97 .1% 92 .8% 64. 0% 84. 6% a S.A. = S p e c i f i c A c t i v i t y (Units per mg Prot e i n ) b T.A. = T o t a l A c t i v i t y 48 a c t i v i t i e s were re c o v e r e d i n f r a c t i o n s I I I through V the d i s t r i -b u t i o n of the a c t i v i t i e s w i t h i n these f r a c t i o n s was not i d e n t i c a l . Although the experiment was c a r r i e d out s e v e r a l times i t was i n v a r i a b l y found t h a t the percentage of PDase II a c t i v i t y p r e s e n t i n the f i n a l supernatant ( f r a c t i o n V) was g r e a t e r than t h a t of a c i d phosphatase. The low r e c o v e r y of cytochrome oxidase i s i n agreement w i t h the r e s u l t s of Robinson (78). I t s h o u l d be p o i n t e d out t h a t although the experiment was repeated many times, problems were always encountered i n o b t a i n i n g a low-speed sediment ( f r a c t i o n I ) . On s e v e r a l o c c a s i o n s i t was not p o s s i b l e t o o b t a i n t h i s sediment because of the l a r g e q u a n t i t i e s of mucous p r e s e n t a f t e r homogenization. The use of 0.3 M m a n n i t o l as recommended by Takesue and Sato (92) f o r homogenization and f r a c t i o n a t i o n of mucosal t i s s u e d i d not a l l e v i a t e the problem of the mucous and y i e l d e d no b e t t e r r e s u l t s than those d e s c r i b e d above. In o r d e r t o minimize the i n t e r f e r e n c e due to mucous i n the i s o l a t i o n of low-speed sediments from r a t i n t e s t i n a l homogenates, a procedure developed by C l a r k and Porteous (58) was employed i n the subsequent f r a c t i o n a t i o n experiments. The method i n v o l v e s d i s p e r s i n g the t i s s u e by homogenization i n 6% dextran-Krebs Ringer phosphate, pH 7.4, f o l l o w e d by f i l t r a t i o n through n y l o n gauze and c e n t r i f u g a t i o n of the f i l t r a t e a t low speed. C l a r k and Porteous r e p o r t e d t h a t the e p i t h e l i a l - c e l l "ghosts" they prepared c o n t a i n e d a l l of the DNA and most of the a c t i v i t i e s o f such p a r t i c u l a t e enzymes as a c i d phosphatase, a l k a l i n e phosphatase, i n v e r t a s e , ATPase and s u c c i n i c dehydrogenase which 49 were p r e s e n t i n the o r i g i n a l homogenate (58). However o n l y 52% of the t o t a l p r o t e i n , 53-57% of the RNA and s m a l l amounts of such s o l u b l e enzymes as l a c t a t e dehydrogenase (10%) and phosphoglucose isomerase (2%) p r e s e n t i n the o r i g i n a l homogenate were r e c o v e r e d i n the e p i t h e l i a l - c e l l "ghosts" (58). Since the "ghosts", which were s o - c a l l e d because they l a c k the a c t i v i t i e s mentioned above, appeared t o be m o r p h o l o g i c a l l y i n t a c t , as seen by l i g h t microscopy, C l a r k and Porteous s t a t e d t h a t most of the l o s s e s c o u l d be e x p l a i n e d by leakage of c e l l - s a p d u r i n g homogenization (58). These same workers showed t h a t most of the mucous which i n t e r f e r e d w i t h the f r a c t i o n a t i o n of mucosal homogenates by c o n v e n t i o n a l methods was removed by the i n i t i a l homogenization i n d e x t r a n and t h a t subsequent d i f f e r e n t i a l c e n t r i f u g a t i o n of e p i t h e l i a l - c e l l "ghost" homogenates i n s u c r o s e -EDTA y i e l d e d c l e a n e r f r a c t i o n s as judged by l i g h t microscopy (58) >. S i n c e one of the o b j e c t s of the p r e s e n t i n v e s t i g a t i o n was t o see whether PDase I I a c t i v i t y was l o c a l i z e d i n a s p e c i f i c p a r t i c u l a t e f r a c t i o n , the use o f t h i s method even w i t h i t s l i m i t a t i o n s seemed j u s t i f i e d . E p i t h e l i a l - c e l l "ghosts" were i s o l a t e d from the i n t e s t i n a l mucosal s c r a p i n g s of 2 r a t s a f t e r homogenization i n 6% dextran-Krebs-Ringer phosphate, pH 7.4. The r e c o v e r i e s o f p r o t e i n , PDase I I and a c i d phosphatase from the homogenate i n d e xtran-Krebs-Ringer phosphate b u f f e r are g i v e n i n Table V I I . The r e c o v e r i e s of cytochrome o x i d a s e and glucose-6-phosphatase from t h i s homogenate were 93.3% and 71.7% r e s p e c t i v e l y . The balance of the glucose-6-phosphatase a c t i v i t y was r e c o v e r a b l e 50 TABLE VII Recovery of p r o t e i n and the a c t i v i t i e s o f PDase I I and a c i d phosphatase from a dextran-phosphate homogenate d u r i n g the p r e p a r a t i o n of e p i t h e l i a l - c e l l "ghosts" from r a t i n t e s t i n a l mucosa F r a c t i o n P r o t e i n mg % PDase. u n i t s I I % . A c i d Phosphatase u n i t s % Homogenate i n 6% dextran-Krebs-Ringer phosphate 298.2 100 70.1 100 202.1 100 E p i t h e l i a l - c e l l "ghosts" 168.1 56.4 23.3 33.2 66.3 32.8 Dextran-phosphate su p e r n a t a n t 122.5 41.1 49.7 70.9 127.4 63.0 Recovery 97.5% 104.1% 95.8% i n a sediment o b t a i n e d a f t e r v e r y high-speed c e n t r i f u g a t i o n of the d e x t r a n s u p e r n a t a n t . Only about 33% of the a c t i v i t i e s o f PDase I I and a c i d phosphatase of the dextran-phosphate homogenate was r e c o v e r e d i n the e p i t h e l i a l - c e l l "ghost" f r a c t i o n (Table V I I ) . C l a r k and Porteous r e c o v e r e d up to 8 3% of the a c i d phosphatase of the o r i g i n a l homogenate i n the "ghost" p r e p a r a -t i o n ( 5 8 ) . In s e v e r a l e p i t h e l i a l - c e l l "ghost" p r e p a r a t i o n s o b t a i n e d from dextran-phosphate homogenates by very g e n t l e homogenization, the amount of a c i d phosphatase a c t i v i t y which was r e c o v e r e d was never g r e a t e r than 54%. However i t i s p o s s i b l e t h a t the d i s p a r i t y o f r e s u l t s can be e x p l a i n e d by the use of d i f f e r e n t s u b s t r a t e s f o r the measurement of a c i d phosphatase a c t i v i t y . C l a r k and Porteous (58) employed ^-glycerophosphate as s u b s t r a t e w h i l e p - n i t r o p h e n y l phosphate was used i n t h i s study and N e i l and Horner (97) have shown t h a t an enzyme which had marked p - n i t r o p h e n y l phosphatase a c t i v i t y but l i t t l e 3-glycerophosphatase a c t i v i t y e x i s t e d i n the supernatant f r a c t i o n o f homogenates of l i v e r from a v a r i e t y o f animals. I t i s p o s s i b l e t h a t the same enzyme i s p r e s e n t i n i n t e s t i n e . T h i s w i l l be d i s c u s s e d more f u l l y l a t e r . The f r a c t i o n a t i o n of e p i t h e l i a l - c e l l "ghosts" was accomplished w i t h o u t the d i f f i c u l t i e s a s s o c i a t e d w i t h crude mucosal p r e p a r a t i o n s and the d i s t r i b u t i o n o f enzyme a c t i v i t i e s i n the f r a c t i o n s o b t a i n e d from such an experiment i s shown i n T a b l e V I I I . I t was shown t h a t f r a c t i o n i , which might be expected t o c o n t a i n mostly n u c l e i and c e l l d e b r i s as w e l l as some unbroken c e l l s , was h e a v i l y contaminated w i t h m i t o c h o n d r i a TABLE VIII D i s t r i b u t i o n of the a c t i v i t i e s of PDase I I , acid phosphatase, cytochrome oxidase and glucose-6-phosphatase i n subcellular fractions of an homogenate of e p i t h e l i a l - c e l l "ghosts" Fr a c t i o n Homogenate 168.1 100 0*14 23.3 100 0.39 66.3 100 1.07 180.5 100 0.92 154.5 100 i 38.6 23.0 0.09 3.5 15.0 0.30 11.6 17.5 2.45 94.6 52.4 1.25 48.2 31.2 i i 17.8 10.6 0.07. 1.2 5.2 0.33 5.9 8.9 2.46 43,7 24,2 0.06 1.1 0.7 i i i 28.9 17.2 0.12 3.5 15.0 0.84 24.3 36.7 0.14 4.0 2.2 1.31 37.9 24.5 i v 19.7 11.7 0.11 2.1 9.0 0.71 14.0 21.1 0.12 2.3 1.3 2.72 53.5 34.6 v 61.1 36.3 0.23 14.1 60.5 0.23 14.1 21.3 0.0 0.0 0.0 0.0 0.0 0.0 Recovery 98.8% 104.7% 105.5% 80.1% 91.0% a S.A.= S p e c i f i c A c t i v i t y (Units per mg Protein) b T.A.= Tot a l A c t i v i t y FRACTION NO. I II in iv v I 1 " I — I 1 • •• B i_r 50 3 3 r r 100 i ii III iv y_ 1 — i 1 1 2 £ 2 > u -1 < 1 y JO u o a. 13 0 0 3 f LU > - 2 < 2 i LU DC -Ii i JO OL 50 100 TOTAL PROTEIN (%) F i g . 3 . The d i s t r i b u t i o n o f PDase I I ( A ) , a c i d phosphatase ( B ) , cytochrome o x i d a s e (C), and g l u c c s e - 6 - p h o s p h a t a s e (D) i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d from an homogenate o f e p i t h e l i a l - c e l l " g h o s t s " . The r e s u l t s a r e the same as t h o s e shown i n T a b l e V I I I . R e l a t i v e s p e c i f i c a c t i v i t y = % t o t a l enzyme a c t i v i t y / % t o t a l p r o t e i n f o r a p a t r i c u l a r f r a c t i o n . The s u b c e l l u l a r f r a c t i o n s L—»v are r e p r e s e n t e d on t h e a b c i s s a e by t h e i r p r o t e i n c o n t e n t s i n t h e o r d e r i n which they were i s o l a t e d (from l e f t t o r i g h t ) . and microsomes, as i n d i c a t e d by the h i g h a c t i v i t i e s of cytochrome o x i d a s e and glucose-6-phosphatase r e s p e c t i v e l y . However i t was p o s s i b l e to remove these contaminating a c t i v i t i e s by washing f r a c t i o n i_ 8-10 times w i t h sucrose-EDTA. F r a c t i o n l i a p p a r e n t l y c o n t a i n e d mostly m i t o c h o n d r i a w i t h h i g h s p e c i f i c and t o t a l a c t i v i t i e s of cytochrome oxidase and s m a l l amounts of PDase I I and a c i d phosphatase. Most of the a c i d phosphatase a c t i v i t y was r e c o v e r e d i n f r a c t i o n s i i i and i v and the same was t r u e f o r glucose-6-phosphatase. However s i n c e the s p e c i f i c and t o t a l a c t i v i t i e s of a c i d phosphatase were h i g h e s t i n f r a c t i o n i i i and those of glucose-6-phosphatase were h i g h e s t i n f r a c t i o n i v i t appeared t h a t f r a c t i o n s i i i and i v c o n s i s t l a r g e l y of lysosomes and microsomes r e s p e c t i v e l y . Most of the PDase I I (60.5%) was r e c o v e r e d i n the f i n a l supernatant f r a c t i o n a f t e r high-speed c e n t r i f u g a t i o n compared with o n l y 21.3% of the a c i d phosphatase a c t i v i t y . These r e s u l t s are p r e s e n t e d i n a more i l l u s t r a t i v e manner i n F i g . 3 u s i n g the diagrammatic method employed by de Duve e t a l . (77). A f i n a l f r a c t i o n a t i o n of e p i t h e l i a l - c e l l "ghosts" was attempted u s i n g a more p r o t r a c t e d d i f f e r e n t i a l c e n t r i f u g a t i o n procedure. A g r e a t e r number of f r a c t i o n s was o b t a i n e d , w i t h the o b j e c t of d e t e r m i n i n g whether the p a t t e r n of d i s t r i b u t i o n of PDase I I and a c i d phosphatase was s i m i l a r i n the p a r t i c u l a t e f r a c t i o n s i s o l a t e d from the e p i t h e l i a l - c e l l "ghost" homogenate. An experiment was c a r r i e d u s i n g the e p i t h e l i a l - c e l l "ghosts" o b t a i n e d from 2 r a t s and the d i s t r i b u t i o n of PDase II and a c i d phosphatase i n the f r a c t i o n s o b t a i n e d are shown i n Table 55 TABLE IX D i s t r i b u t i o n of the a c t i v i t i e s o f PDase I I and a c i d phosphatase i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d by p r o t r a c t e d d i f f e r e n t i a l c e n t r i f u g a t i o n of an homogenate of e p i t h e l i a l - c e l l "ghosts" P r o t e i n PDase I I A c i d Phosphatase a b F r a c t i o n T o t a l S.A. T.A. S.A. T.A. mg % u n i t s % u n i t s % Homogenate 181, .4 100 0, .16 28. .4 100. 0, .39 70, .1 100 1 51, .5 28. .4 0. .10 5. .3 18. .7 Q. .28 14, .2 2 0. .3 2 9, .2 5. .1 0. .10 0. .9 3, .2 0, .46 4, .2 5. .9. 3 8. .8 4. .9 0. .11 1. .0 3. .5 Of. 64 5, .6 8. .0. 4 8. .8 4. .9 0. .14 1. .2 4. .2 0. .89 7. .7 11. .0 5 13. .3 7. .3 0. .07 0. .9 3. .2 0. .72 9. .6 13. .7 6 17. .2 9. ,5 0. .05 0. .9 3. .2 0. .54 9. .3 13. ,3 7 77. .7 42. .8 0. .21 16. .0 56. .3 0. .28 21. .9 31. .3 Recovery 10 2. ,9% 92. .3% 103. .5% S.A.= S p e c i f i c A c t i v i t y (Units per mg P r o t e i n ) b T.A.= T o t a l A c t i v i t y 56 FRACTION NO. U U L U Q _ C O 1 2 3 4 5 6 1 i i i—i—r 3 r >- 2 > t— 0 3 L U ^ 21-L U cm 0 B 50 100 TOTAL PROTEIN (%) F i g . 4. The d i s t r i b u t i o n of PDase I I (A) and a c i d phosphatase (B) i n s u b c e l l u l a r f r a c t i o n s o b t a i n e d by p r o t r a c t e d f r a c t i o n a -t i o n of an homogenate of e p i t h e l i a l - c e l l "ghosts". The r e s u l t s are the same as those shown i n Table IX. For f u r t h e r d e t a i l s see the t e x t and the legend f o r F i g . 3. IX and F i g . 4. Although most of the PDase I I was r e c o v e r e d i n the f i n a l s upernatant, the s m a l l p o r t i o n which was p a r t i c u l a t e seemed t o occur i n those f r a c t i o n s which had the h i g h e s t s p e c i f i c a c t i v i t y f o r a c i d phosphatase. These data suggest t h a t t h i s p o r t i o n of PDase I I a c t i v i t y of e p i t h e l i a l - c e l l "ghosts" was l y s o s o m a l . C. Sucrose D e n s i t y G r a d i e n t C e n t r i f u g a t i o n of M u c o s a l - C e l l Homogenates T h i s technique was used i n an attempt t o c o n f i r m the r e s u l t s o b t a i n e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . I n i t i a l attempts t o f r a c t i o n a t e e p i t h e l i a l - c e l l "ghost" homogenates u s i n g t h i s method were hampered by a l a r g e amount of q u i c k l y sedimenting m a t e r i a l which i n t e r f e r e d w i t h the procedure. A l s o most of the PDase I I i n the g r a d i e n t was found c l o s e t o the top of the g r a d i e n t and l i t t l e a c t i v i t y elsewhere even a f t e r prolonged c e n t r i f u g a t i o n . I t i s l i k e l y t h a t t h i s a c t i v i t y was due to the l a r g e amounts of " s o l u b l e " enzyme i n the p r e p a r a t i o n . The i n t e r f e r i n g p a r t i c u l a t e m a t e r i a l s e t t l e d i n the bottom of the c e n t r i f u g a t i o n tube and made i t i m p o s s i b l e to c o l l e c t f r a c t i o n s from the g r a d i e n t . I t was found t h a t t h i s g e l a t i n o u s f r a c t i o n c o u l d be removed by c e n t r i f u g i n g the homogenate of e p i t h e l i a l -c e l l "ghosts" f o r 10 min a t 755 x g. The p a r t i c l e s remaining i n the supernatant a f t e r t h i s treatment were sedimented by c e n t r i f u g a t i o n a t 105,000 x g f o r 1 hour and were then s u b j e c t e d t o s u c r o s e - d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . In order t o observe the behaviour of the c e l l u l a r p a r t i c l e s under d i f f e r i n g 5 8 i n t e g r a t e d f o r c e s ( g min) (93) t h r e e e x p e r i m e n t s were c a r r i e d o u t i n w h i c h b o t h t h e t i m e and speed of c e n t r i f u g a t i o n were v a r i e d . The t o t a l a c t i v i t y of each f r a c t i o n was c a l c u l a t e d i n a r b i t r a r y u n i t s and was e x p r e s s e d as a p e r c e n t a g e o f t h e most a c t i v e f r a c t i o n / o b t a i n e d . I n t h e f i r s t e x p e r i m e n t t h e c e n t r i f u g a t i o n was f o x 3 h r a t 25,000 r.p.m. and t h e d i s t r i b u t i o n , o f t h e a c t i v i t i e s o f PDase I I , a c i d p h o s p h a t a s e , s u c c i n i c dehydrogenase and g l u c o s e - 6 - p h o s p h a t a s e i n t h e f r a c t i o n s o b t a i n e d from t h e g r a d i e n t i s shown i n F i g . 5. The p a r t i c l e s w h i c h were c l o s e s t t o t h e bottom o f t h e tube a f t e r c e n t r i f u g a t i o n appeared t o be m i t o c h o n d r i a s i n c e t h e s e b r o w n - c o l o u r e d p a r t i c l e s e x h i b i t e d t h e h i g h e s t a c t i v i t y of s u c c i n i c dehydrogenase ( 8 7 ) . The d i s t r i b u t i o n o f a c i d p h o s p h atase was r a t h e r d i f f u s e and s p r e a d t h r o u g h o u t t h e g r a d i e n t . I n most of t h e e x p e r i m e n t s p e r f o r m e d under t h e s e c o n d i t i o n s , one l a r g e peak o f a c i d p h o s p hatase a c t i v i t y was f o u n d c o m p r i s i n g f r a c t i o n s 5-16 a p p r o x i m a t e l y . However on t h r e e s e p a r a t e o c c a s i o n s i n c l u d i n g t h e one d e p i c t e d i n F i g . 5 an a d d i t i o n a l s m a l l e r peak o f a c t i v i t y was found about o n e - t h i r d o f t h e way down t h e g r a d i e n t . The p a r t i c u l a t e PDase I I a c t i v i t y i n t h e s u c r o s e - g r a d i e n t , was, however, a s s o c i a t e d w i t h o n l y t h e l a r g e r . o f t h e two peaks o f a c i d p hosphatase a c t i v i t y ( F i g . 5 ) . The l a r g e amount o f PDase I I a c t i v i t y found a t t h e t o p o f t h e s u c r o s e - g r a d i e n t c o n f i r m e d t h e r e s u l t s o f t h e e x p e r i m e n t s u s i n g d i f f e r e n t i a l c e n t r i f u g a t i o n , where more t h a n 50% o f t h i s a c t i v i t y was found i n t h e s o l u b l e p o r t i o n o f i n t e s t i n a l mucosal homogenates. The v a r i o u s i l l - d e f i n e d bands o b s e r v a b l e i n t h e t o p h a l f o f t h e s u c r o s e g r a d i e n t a f t e r c e n t r i f u g a t i o n ( F i g . 5) a r e due t o 59 Ol l 1 ' ' 1 1 1 1 5 9 13 17 21 25 29 F R A C T I O N N O . * i i . — i 1 1 1 0 2 4 6 6-7 C M from bottom of tube 6-7 6 4 C M 2 0 F i g . 5. S u c r o s e d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n c f s u b c e l l u l a r p a r -t i c l e s of r a t i n t e s t i n a l mucosa. The d i s t r i b u t i o n s of PDase I I ( — } , a c i d p h o s p h atase (——) / s u c c i n i c dehydrogenase ( ) , and g l u c o s e - 6 -p h o s p hatase ( ) a r e shown a l o n g w i t h t h e r e f r a c t i v e i n d i c e s (• -) of t h e f r a c t i o n s . The r e f r a c t i v e i n d i c e s o f 50% and 12% s u c r o s e s t a n d a r d s a r e i n d i c a t e d by t h e r e s p e c t i v e p o s i t i o n s of x and y on th e r i g h t hand o r d i n a t e . The enzyme a c t i v i t i e s a r e e x p r e s s e d as p e r c e n t a g e s o f t h e h i g h e s t a c t i v i t y found f o r each enzyme. A schema-t i c d r a w i n g o f t h e c e n t r i f u g e tube a f t e r c e n t r i f u g a t i o n i s a l s o shown a l o n g w i t h t he d i s t a n c e s of t h e bands from t h e bottom o f t h e t u b e . C e n t r i f u g a t i o n was f o r 3 h r a t 25,000 r.p.m. F o r f u r t h e r d e t a i l s see the t e x t . I S 60 d i f f e r e n t p o p u l a t i o n s of membrane f r a g m e n t s . These have been shown by Dr. I . Hynie i n t h i s l a b o r a t o r y t o be m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e under t h e e l e c t r o n m i c r o s c o p e . I n a second e x p e r i m e n t t h e i n t e g r a t e d f o r c e of c e n t r i f u -g a t i o n was r e d u c e d by s p i n n i n g t h e g r a d i e n t f o r 2 h r e t 15,000 r.p.m. The d i s t r i b u t i o n p a t t e r n c f PDase I I was compared w i t h t h o s e o f a l k a l i n e p h o s p h a t a s e , c l u c o s e - 6 - p h o s p h a t a s e and a c i d p h o s p h a t a s e i n t h e f r a c t i o n s o b t a i n e d from t h e g r a d i e n t a f t e r c e n t r i f u g a t i o n and t h e s e r e s u l t s a r e shown i n F i g . 6A. A peak w h i c h c o n t a i n e d a c t i v i t y o f each of t h e enzymes was found c l o s e t o t h e bottom o f the t u b e . S i n c e o t h e r e x p e r i m e n t s had r e v e a l e d - t h a t m i t o c h o n d r i a sediment i n t h i s r e g i o n under t h e s e c e n t r i f u -g a t i o n c o n d i t i o n s , i t was t h o u g h t t h a t t h e m u l t i p l e enzyme a c t i v i t i e s o b s e r v e d i n t h e r e g i o n were due t o enzyme wh i c h was a d s o r b e d on t h e m i t o c h o n d r i a . I t was a l s o seen t h a t t h e a c i d p h o s p h a t a s e a c t i v i t y was s p r e a d o v e r a wide a r e a i n t h e s u c r o s e g r a d i e n t . However as was o b s e r v e d i n t h e p r e v i o u s e x p e r i m e n t t h e PDase I I a c t i v i t y c o - s e d i m e n t s w i t h t h e f r o n t o f a c i d p h o s p h a t a s e a c t i v i t y and i t i s l i k e l y t h a t t h e p a r t i c l e s t o w h i c h b o t h t h e s e enzymes a r e bound a r e s i m i l a r t o some e x t e n t . I n the f i n a l e x p e r i m e n t the c e n t r i f u g a t i o n was f o r 1.5 hr a t 10,000 r.p.m. The a c t i v i t i e s o f PDase I I , a c i d p h o s p h a t a s e , s u c c i n i c dehydrogenase and a l k a l i n e p hosphatase were d e t e r m i n e d i n t h e f r a c t i o n s o b t a i n e d from the g r a d i e n t and t h e s e a r e shown i n F i g . 6B. As i n t h e p r e v i o u s two e x p e r i m e n t s the m i t o c h o n d r i a o f t h e t i s s u e sample were found c l o s e t o t h e bottom of the tube a f t e r c e n t r i f u g a t i o n . I n marked c o n t r a s t t o t h e p a t t e r n s seen i n 61 100 ->-> u < 1 5 B6TTOM BOTTOM F R A C T I O N N O . F i g s . 6A and 6B. Sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n of sub-c e l l u l a r p a r t i c l e s of r a t i n t e s t i n a l mucosa. The d i s t r i b u t i o n s of PDase I I ( -) , a c i d phosphatase ( ) , a l k a l i n e phosphatase (• -) / glucose-6-phosphatase (• •) and s u c c i n i c dehydrogenase (. .) a r e shown. The enzyme a c t i v i t i e s are expressed as p e r -centages of the h i g h e s t a c t i v i t y o b t a i n e d f o r each enzyme. C e n t r i f u g a t i o n i n A was f o r 2 hr a t 15,000 r.p.m. and i n B f o r 1.5 hr a t 10,000 r.p.m. For f u r t h e r d e t a i l s see the t e x t and the c a p t i o n f o r F i g . 5. F i g . 6A, almost no a c i d phosphatase or PDase II was p r e s e n t i n the i n t e r m e d i a t e p o r t i o n of the g r a d i e n t . However i n t h i s case as i n the o t h e r s the p a r t i c u l a t e p o r t i o n of the PDase I I a c t i v i t y co-sedimented w i t h the "lysosomal" a c i d phosphatase a c t i v i t y . Taken t o g e t h e r the t h r e e experiments seemed t o i n d i c a t e t h a t as the i n t e g r a t e d f o r c e of c e n t r i f u g a t i o n was v a r i e d the a c t i v i t i e s of a c i d phosphatase and PDase I I behaved i n a manner t o suggest t h a t they were c o n t a i n e d i n p a r t i c l e s w i t h s i m i l a r or i d e n t i c a l s i z e s and d e n s i t i e s . D. P r e p a r a t i o n of P u r i f i e d Lysosomes from I n t e s t i n a l Mucosa Sin c e i t was found from the d i f f e r e n t i a l and d e n s i t y -g r a d i e n t c e n t r i f u g a t i o n experiments, d e s c r i b e d above, t h a t the PDase I I a c t i v i t y of i n t e s t i n a l mucosa homogenates was a t l e a s t p a r t l y lysosomal i t was of i n t e r e s t t o determine whether the a c t i v i t y of t h i s enzyme i n c r e a s e d i n p a r a l l e l with the " p u r i f i c a t i o n " of lysosomes. Mucosal s c r a p i n g s from 5 r a t s were processed a c c o r d i n g t o the scheme o u t l i n e d i n F i g . 1. Samples were removed a t the p o i n t s i n d i c a t e d and assayed f o r p r o t e i n , PDase I I and a c i d phosphatase. These r e s u l t s are g i v e n i n Table X and i t can be seen o n l y 0.14% of the p r o t e i n content of the homogenate of e p i t h e l i a l - c e l l "ghosts" was re c o v e r e d i n the " p u r i f i e d lysosomes" ( f r a c t i o n 7 ) . A l s o d u r i n g the p u r i f i c a t i o n the s p e c i f i c a c t i v i t y of PDase I I p a r a l l e l e d t h a t of a c i d phosphatase and the h i g h e s t s p e c i f i c a c t i v i t i e s f o r both enzymes were observed i n f r a c t i o n 7. However o n l y 0.44% and 0.72% of the 63 TABLE X The PDase I I and a c i d phosphatase a c t i v i t i e s o f a number of f r a c t i o n s o b t a i n e d d u r i n g an attempt t o p u r i f y lysosomes from r a t i n t e s t i n a l mucosa P r o t e i n PDase I I A c i d Phosphata se F r a c t i o n Number * T o t a l S.A R e l a t i v e . a S.A. T.A • S * A • R e l a t i v e S.A. T.A. mg u n i t s u n i t s 1 741 0.09 1.00 69.7 0.38 1.00 278 2 576 0.12 1.33 66.8 0.46 1.21 266 3 96 0.14 1.55 14.0 0.89 2.34 85 4 57 0.17 1.88 9.7 0.46 1.21 26 5 30 0.13 1.44 4.0 0.64 1.68 19 6 3 0.13 1.44 0.4 1.67 4.40 5 7 1 0.26 2.88 0.3 2.00 5.26 2 8 14 0.21 2.33 2.9 1.07 2.82 15 9 2 3 0.10 1.11 2.4 0.52 1.37 12 * The numbers r e f e r t o the f r a c t i o n numbers i n F i g .1. a S.A. = S p e c i f i c A c t i v i t y (Units per mg P r o t e i n ) b R e l a t i v e S.A. = R e l a t i v e S p e c i f i c A c t i v i t y which i s based on the s p e c i f i c a c t i v i t y o f f r a c t i o n 1 which was taken t o be 1.00 . c T.A. = T o t a l A c t i v i t y r e s p e c t i v e amounts o f PDase I I and a c i d phosphatase p r e s e n t i n the o r i g i n a l homogenate were r e c o v e r e d i n t h i s p u r i f i e d p a r t i c u l a t e f r a c t i o n . These r e s u l t s agree w i t h those o f p r e v i o u s experiments i n which i t was shown t h a t o n l y s m a l l amounts of PDase I I a c t i v i t y were found i n the p a r t i c u l a t e f r a c t i o n s . N e v e r t h e l e s s the h i g h e s t s p e c i f i c a c t i v i t y f o r PDase I I was found i n a p a r t i c u l a t e f r a c t i o n which was presumed t o be lysosomal (94) . However s i n c e no e l e c t r o n - m i c r o s c o p i c examination of the f r a c t i o n was made the m o r p h o l o g i c a l nature of the p a r t i c l e s p r e s e n t was not known. The low r e c o v e r i e s of a c i d phosphatase and PDase I I from the o r i g i n a l homogenate may j u s t r e f l e c t a poor i s o l a t i o n of lysosomes. The r e s u l t s of a l l these f r a c t i o n a t i o n experiments w i l l be examined more c l o s e l y l a t e r . Some P r o p e r t i e s of Phosphodiesterase I I A c t i v i t y of Rat I n t e s t i n e A. pH and I o n i c Requirements A number of experiments v/ere c a r r i e d out t o determine the optimum pH f o r the PDase I I a c t i v i t y of r a t i n t e s t i n e i n  v i t r o . In order t o measure the a c t i v i t y of the enzyme over a wide range o f pH, s e v e r a l b u f f e r systems were used. In the f i r s t s e r i e s of experiments PDase I I a c t i v i t y i n a supernatant f r a c t i o n , prepared as d e s c r i b e d f o r f r a c t i o n V (Table VI) was assayed i n c i t r a t e , phosphate, and g l y c i n e b u f f e r s of v a r y i n g pH. In a second group of experiments the PDase I I a c t i v i t y of a " n u c l e a r " and a "lysosomal" f r a c t i o n prepared i n the manner d e s c r i b e d f o r f r a c t i o n s i_ and i i i (Table VIII) was determined i n the f o l l o w i n g b u f f e r s - borax-NaOH, T r i s - H C l , TES-NaOH, 100r >-I— > I— U < lOOr >-> h-u < 50 8 10 12 pH OF MEDIUM F i g . 7. PDase I I a c t i v i t y of a supernatant f r a c t i o n (A) and a "lysosomal" f r a c t i o n (B) as a f u n c t i o n of pH. The a c t i v i t y was determined under standard assay c o n d i t i o n s w i t h Tp-NP as s u b s t r a t e except t h a t the f o l l o w i n g b u f f e r s were used. For A the b u f f e r s were 0.06 M c i t r a t e (•), 0.02 M phosphate (n) and 0.06 M g l y c i n e (•). For B the b u f f e r s a t a c o n c e n t r a t i o n of 0.05 M were borax-NaOH (o), T r i s - H C l (o) , TES-NaOH (•), MES-NaOH (A) and ammonium a c e t a t e (•). In both cases the a c t i v i t y i s expressed as a percentage of the optimum v a l u e o b t a i n e d . For the p r e p a r a t i o n of the f r a c t i o n s see the t e x t . The r e s u l t s o b t a i n e d w i t h a " n u c l e a r " f r a c t i o n were i d e n t i c a l t o t h o s e . i n B and are not r e p o r t e d s e p a r a t e l y . 66 TABLE XI Comparison of the optimum pH and a c t i v i t y of PDase I I i n a v a r i e t y o f b u f f e r s .Buffer Optimum pH A c t i v i t y PIPES 6.85 100 MES 6.45 98 TES 7.0 93 HEPES 6.95 92 S u c c i n a t e 6.05 97 Imidazole 6.85 — a The c o n c e n t r a t i o n of each b u f f e r was 0.05 M. k The a c t i v i t y i s expressed as a percentage of the s p e c i f i c a c t i v i t y o b t a i n e d i n PIPES b u f f e r which was 0.154. The a c t i v i t y i n i m i d a z o l e b u f f e r was not d i r e c t l y comparable s i n c e i t was o b t a i n e d i n a separate experiment. 67 MES-NaOH and ammonium a c e t a t e . No d i f f e r e n c e c o u l d be d e t e c t e d i n the pH dependence of the PDase I I a c t i v i t y of the two f r a c t i o n s . The v a r i a t i o n of PDase I I a c t i v i t y w i t h pH as d e t e r -mined i n these experiments i s shown i n F i g . 7 and i t was found t h a t i n both cases the optimum pH f o r PDase I I a c t i v i t y was c l o s e t o 7. I t seems l i k e l y t h e r e f o r e t h a t the PDase I I a c t i v i t y i n v a r i o u s f r a c t i o n s o b t a i n e d from mucosal c e l l homogenates was •'>. due to one enzyme, although the p o s s i b i l i t y e x i s t s t h a t two (or more) enzymes were, p r e s e n t i n each f r a c t i o n i n s i m i l a r • p r o p o r t i o n s . T h i s l a t t e r s u g g e s t i o n should not be r u l e d out e n t i r e l y s i n c e B e r n a r d i e t a l . (98) have shown t h a t a v a r i e t y of n u c l e a s e a c t i v i t i e s can be p u r i f i e d from s p l e e n by a g e n e r a l i z e d procedure. In a t h i r d s e r i e s of experiments the PDase I I a c t i v i t y i n a supernatant f r a c t i o n ( f r a c t i o n V; Table VI) was measured i n a number of b u f f e r s which are most e f f e c t i v e around n e u t r a l i t y . These i n c l u d e d s e v e r a l z w i t t e r i o n i c b u f f e r s d e s c r i b e d by Good e t a l . (99), i m i d a z o l e - H C l and ammonium s u c c i n a t e . The r e s u l t s « are shown i n Tabl e XI and i t was noted t h a t the optimum pH v a l u e s i n the z w i t t e r i o n i c b u f f e r and i m i d a z o l e - H C l v a r i e d between 6.45 and 7.0 w h i l e i n the s u c c i n a t e b u f f e r the optimum pH was 6.05. T h i s l a t t e r o b s e r v a t i o n i s more i n agreement wi t h the optimum pH of 5.9 i n s u c c i n a t e b u f f e r r e p o r t e d by R a z z e l l and Khorana (48) f o r s p l e e n p h o s p h o d i e s t e r a s e . Except where i n d i c a t e d , i n the remainder of the experiments t o be d e s c r i b e d i n t h i s s e c t i o n a supernatant f r a c t i o n was used as a source o f the enzyme and was prepared i n the manner d e s c r i b e d f o r 68 < 01 I : I I 6-2 6-6 70 pH OF MEDIUM F i g . 8. PDase II a c t i v i t y i n phosphate buffers of d i f f e r e n t concentration and pH. The a c t i v i t y i n a supernatant f r a c t i o n , prepared as described i n the text, was determined under standard assay conditions except that phosphate buffers at the following concentrations were used: 0.006 M (a), 0.06 M (•) and 0.6 M ( A ) . The PDase II a c t i v i t y i s expressed as a percentage of the optimum value obtained i n 0.006 M' phosphate. 69 p i » » i ' • I I I 0-2 0-4 0-6 0-8 [ S U C C I N A T E ] F i g . 9. E f f e c t o f c o n c e n t r a t i o n of s u c c i n a t e b u f f e r on the a c t i v i t y of PDase I I . The a c t i v i t y i n a supernatant f r a c t i o n , prepared as d e s c r i b e d i n the t e x t , was determined under standar d assay c o n d i t i o n s u s i n g Tp-DNP as s u b s t r a t e , except t h a t the c o n c e n t r a t i o n of b u f f e r was v a r i e d as i n d i c a t e d . The a c t i v i t y i s expressed as a percentage of the maximum v a l u e o b t a i n e d . 70 f r a c t i o n V (Table VI) except t h a t EDTA was omitted from the homogenizing medium. The e f f e c t of c o n c e n t r a t i o n of b u f f e r on the a c t i v i t y of PDase I I i s shown i n F i g s . 8 and 9 wit h phosphate and s u c c i n a t e b u f f e r s r e s p e c t i v e l y . I t was seen t h a t , g e n e r a l l y , the a c t i v i t y of PDase I I was i n c r e a s e d a t lower c o n c e n t r a t i o n s of b u f f e r . I t i s u n l i k e l y t h a t the dramatic decrease i n PDase I I a c t i v i t y i n the presence of c o n c e n t r a t i o n s of s u c c i n a t e b u f f e r h i g h e r than 0.1 M can be e x p l a i n e d by the removal of an e s s e n t i a l metal s i n c e as shown below the a c t i v i t y of the enzyme was u n a f f e c t e d i n the presence of 10 mM EDTA. The decrease i n PDase I I a c t i v i t y observed a t very low c o n c e n t r a t i o n s of s u c c i n a t e may be a r t i f a c t u a l s i n c e the b u f f e r i n g c a p a c i t y of the medium must be q u i t e s m a l l i n t h i s r e g i o n . The products of PDase I I a c t i v i t y are a c i d i c and c o u l d have lowered the pH of the i n c u b a t i o n medium c o n s i d e r a b l y . The e f f e c t of v a r i o u s i o n s on the a c t i v i t y of PDase I I i s shown i n Table X I I . Most i n t e r e s t i n g of these, perhaps, i s the e f f e c t of C u + + . . A t a c o n c e n t r a t i o n of 8.3 mM t h i s c a t i o n i n h i b i t e d i n t e s t i n a l PDase I I 47% w h i l e Hilmoe has r e p o r t e d (47) t h a t s p l e e n PDase was i n h i b i t e d 95% i n the presence of o n l y 1 mM C U C I 2 and t h i s s t r o n g i n h i b i t i o n has r e c e n t l y been confirmed w i t h a p r e p a r a t i o n of PDase I I from salmon t e s t i s (59). I t a l s o seemed t h a t the a c t i v i t y of PDase I I from i n t e s t i n e was much l e s s s e n s i -t i v e than the s p l e e n enzyme to d i v a l e n t c a t i o n s g e n e r a l l y . The e f f e c t of EDTA on the PDase I I a c t i v i t y of i n t e s t i n e was a l s o markedly d i f f e r e n t from t h a t r e p o r t e d f o r the s p l e e n enzyme. (47). 71 TABLE XII E f f e c t of a number of d i v a l e n t c a t i o n s and of EDTA on the PDase II a c t i v i t y o f r a t i n t e s t i n a l mucosa C o n c e n t r a t i o n A c t i v i t y none — 100 C a C l 2 0.83 • 95 C a C l 2 8.3 101 M g C l 2 0.83 105 M g C l 2 8.3 . 101 Z n C l 2 0.83 95 Z n C l 2 8.3 65 CuS0 4 0.83 85 CuS0 4 8.3 53 H g C l 2 0.83 37 H g C l 2 8.3 0 EDTA 1.0 104 EDTA 10.0 107 72 Hilmoe showed (47) t h a t the a c t i v i t y of s p l e e n PDase c o u l d be i n c r e a s e d 1.9 times i n the presence of 10 mM EDTA whereas i n the p r e s e n t experiments w i t h PDase I I from i n t e s t i n e , EDTA had l i t t l e o r no e f f e c t (Table X I I ) . B. E f f e c t s of Temperature: The r a t e of h y d r o l y s i s o f Tp-NP by the PDase I I a c t i v i t y p r e s e n t i n i n t e s t i n e was g r e a t e s t a t a temperature c l o s e t o 60°. T h i s i s i l l u s t r a t e d i n F i g . 10 and i t was shown a l s o t h a t the PDase I I a c t i v i t y was i n a c t i v a t e d q u i c k l y a t temperatures h i g h e r than 60°. In view of the h i g h optimum temperature o b t a i n e d f o r PDase I I i t was of i n t e r e s t t o determine i f the enzyme was s t a b l e t o these h i g h temperatures e s p e c i a l l y i n view of the known temperature s t a b i l i t y of o t h e r PDases (4£,59). A sup-e r n a t a n t f r a c t i o n ( f r a c t i o n V, Table VI) was heated a t 55° and a t i n t e r v a l s a l i q u o t s were removed from the i n c u b a t i o n v e s s e l , c o o l e d on i c e f o r e x a c t l y 5 min and assayed f o r PDase I I a c t i v i t y i n the standa r d assay d e s c r i b e d i n "Methods". The r e s u l t s a r e shown i n F i g . 11 and i t was observed t h a t a f t e r h e a t i n g f o r 1 hr 0 a t 55 , approximately 73% of the PDase I I a c t i v i t y o f t h i s . s u p e r n a t a n t f r a c t i o n remained. The curve o b t a i n e d i n F i g . 11 appeared t o have a b i p h a s i c nature and t h i s might be e x p l a i n e d by e i t h e r o f ltwo p o s s i b i l i t i e s : a) Two enzymes capable of h y d r o l y z i n g Tp-NP but d i f f e r i n g i n t h e r m o s t a b i l i t y c o u l d have been p r e s e n t i n i n t e s t i n a l e x t r a c t s , and the break i n the curve observed a f t e r h e a t i n g f o r 25 min c o u l d have been due to the d e s t r u c t i o n of one of these a c t i v i t i e s . 73 J i J i I I I i L_ 0 20 40 60 80 TEMPERATURE °C F i g . 10. E f f e c t o f t e m p e r a t u r e on t h e a c t i v i t y o f PDase I I . The a c t i v i t y i n a s u p e r n a t a n t f r a c t i o n ( f r a c t i o n V, T a b l e VI) was d e t e r m i n e d under s t a n d a r d a s s a y c o n d i t i o n s w i t h Tp-NP as s u b s t r a t e , e x c e p t t h a t t h e t e m p e r a t u r e o f i n c u b a t i o n was v a r i e d as i n d i c a t e d . Time o f i n c u b a t i o n was 15 m i n u t e s . The a c t i v i t y o f PDase I I i s e x p r e s s e d as a p e r c e n t a g e o f t h e maximum v a l u e o b t a i n e d . F i g . 11. Rate of heat i n a c t i v a t i o n of PDase I I . For experiment d e t a i l s see the t e x t . The enzyme a c t i v i t y i s expressed as a percentage of the zero time sample. b) The b r e a k i n t h e c u r v e m i g h t be due t o t h e r m a l d e s t r u c t i o n o f an i n h i b i t o r o f PDase I I . E x p e r i m e n t s d e s c r i b e d p r e v i o u s l y showed t h a t PDase I I from v a r i o u s f r a c t i o n s , o f i n t e s t i n a l t i s s u e had s i m i l a r pH a c t i v a t i o n p r o f i l e s ( F i g . 7). T h i s might be t a k e n as e v i d e n c e f o r t h e e x i s t e n c e i n i n t e s t i n e o f o n l y one enzyme a c t i v e towards Tp-NP. On t h e o t h e r hand, t h e r e i s no s u p p o r t i n g e v i d e n c e f o r t h e p r e s e n c e o f an i n h i b i t o r o f PDase I I i n i n t e s t i n e . C. D e t e r m i n a t i o n o f K^, V m a x and A c t i v a t i o n Energy: The e f f e c t o f i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n on PDase I I o f i n t e s t i n e was d e t e r m i n e d w i t h Tp-NP as s u b s t r a t e . The r e s u l t s of t h e s e e x p e r i m e n t s a r e shown i n F i g . 12. The K M o f t h e s u p e r n a t a n t f r a c t i o n a t 37 ° was c a l c u l a t e d from t h e -4 L i n e w e a v e r - B u r k (100) p l o t shown i n F i g . 12 t o be 4.5 x 10 M. T h i s v a l u e i s about o n e - s e v e n t h o f t h a t r e p o r t e d f o r s p l e e n PDase by R a z z e l l and Khorana (48). The e f f e c t o f i n c r e a s i n g o 0 s u b s t r a t e c o n c e n t r a t i o n was a l s o d e t e r m i n e d a t 20.5 and 50 w i t h t h e same enzyme p r e p a r a t i o n and t h e K M v a l u e s were d e t e r m i n e d from t h e L i n e w e a v e r - B u r k (100) p l o t s a t each t e m p e r a t u r e . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n T a b l e X I I I and from them i t can be seen t h a t t h e a f f i n i t y of o 0 PDase I I f o r Tp-NP was 2.8 t i m e s g r e a t e r a t 20.5 t h a n a t 50 . S u f f i c i e n t q u a n t i t i e s o f Tp-DNP were n o t a v a i l a b l e t o d e t e r m i n e t h e K M o f t h e enzyme f o r t h i s s u b s t r a t e . The maximum i n i t i a l v e l o c i t i e s o b t a i n e d from t h e 0 0 0 L i n e w e a v e r - B u r k (100) p l o t s a t 20.5 , 37 and 50 a r e shown 0-05 h 0-03 ±: 0-01 50 O V v >• 30 ^y* 10 1 0 i • 1 • -2 -1 ( } V[s] i x TO - 3 2 3 4 I I I I I 0 10 15 20 25 30 [ S ] x 1 0 : F i g . 12. E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the a c t i v i t y o f PDase I I . A l i q u o t s of a supernatant f r a c t i o n ( f r a c t i o n V; Table VI) c o n t a i n i n g 110 yg of p r o t e i n were assayed as d e s c r i b e d i n "Methods" except t h a t the c o n c e n t r a t i o n of Tp-NP was v a r i e d as i n d i c a t e d . The i n i t i a l v e l o c i t i e s are g i v e n i n phosphodiesterase u n i t s . The K M and V m a x were c a l c u l a t e d from the Lineweaver-Burk p l o t shown and were found to be 4.5 x 10~ 4 M and 0.435 yMoles Tp-NP hydrolyzed/hr/mg p r o t e i n r e s p e c t i v e l y . 77 TABLE X I I I E f f e c t of temperature on the K M and V m a x o f PDase I I Temperature 5 l A V max ° c M x 1 0 4 2 0 . 5 2 . 5 5 0 . 1 1 1 37 . 4 . 5 0 . 4 3 5 50 7 . 2 1 . 0 5 7 ymoles Tp-NP hydrolyzed/hour/mg p r o t e i n 78 a l s o i n Tab l e X I I I . The i n t e g r a t e d form of the A r r h e n i u s e q u a t i o n , which g i v e s the r e l a t i o n s h i p between the v e l o c i t y o f r e a c t i o n and the a b s o l u t e temperature, i s as f o l l o w s : E l o g k = - + c o n s t a n t 2.30 3 RT where k i s the r a t e c o n s t a n t and i s p r o p o r t i o n a l t o the V m a x , E i s the a c t i v a t i o n energy, R i s the molar gas c o n s t a n t and T the a b s o l u t e temperature. T h e r e f o r e , a p l o t of l o g V m a x versus 1/T should y i e l d a s t r a i g h t l i n e whose s l o p e i s - E/2.30 3 R. By means of such an A r r h e n i u s p l o t ( F i g . 13) the sl o p e was found o t o be -32 00 K and so the a c t i v a t i o n energy of the enzyme c a t a l y z e d h y d r o l y s i s of Tp-NP was c a l c u l a t e d to be 14.63 k c a l o r i e s / m o l e . 79 80 DISCUSSION 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 the P h o s p h o d i e s t e r a s e I I A c t i v i t y  i n Rat I n t e s t i n a l " Mucosa The demonstration of PDase I I a c t i v i t y i n i n t e s t i n a l t i s s u e c onfirmed the e a r l i e r r e p o r t s of Heppel and Hilmoe (46) and R a z z e l l (50), I t was thought I n i t i a l l y t h a t the PDase I I a c t i v i t y demonstrable i n the s o l u b l e p o r t i o n of suspensions of mucosal t i s s u e might be due to an e x t r a - i n t e s t i n a l enzyme. For i n s t a n c e , i t c o u l d have found I t s way t o the i n t e s t i n a l lumen from the e x c r e t i o n s of the pancreas or g a l l b l a d d e r or i t might have been s e c r e t e d by the b a c t e r i a l f l o r a p r e s e n t In the i n t e s t i n e . However s i n c e f u r t h e r PDase II a c t i v i t y c o u l d be r e l e a s e d by homogenizing well-washed mucosal s c r a p i n g s , i t seemed more l i k e l y t h a t the PDase I I a c t i v i t y , i n i t i a l l y p r e s e n t i n suspensions of the t i s s u e , had been r e l e a s e d from some c e l l s which were damaged when the i n t e s t i n e was scraped. A more d e f i n i t e answer to t h i s problem might be o b t a i n e d by u s i n g germ-free r a t s and/or I s o l a t e d i n t e s t i n a l loops- as: the source of t i s s u e m a t e r i a l . Most of the PDase I I a c t i v i t y of r a t I n t e s t i n a l homogenates was r e c o v e r e d i n a " s o l u b l e " f r a c t i o n after-d i f f e r e n t i a l c e n t r i f u g a t i o n (Tables VI, V I I , V I I I and IX).. I t i s i n t e r e s t i n g i n t h i s r e s p e c t t o compare thes e r e s u l t s wlth. those of o t h e r i n v e s t i g a t o r s who used d i f f e r e n t t i s s u e s and sometimes d i f f e r e n t s u b s t r a t e s . R a z z e l l (50) f r a c t i o n a t e d homogenates of r a t l i v e r and kidney and found, u s i n g Tp-NP as 81 s u b s t r a t e . , 63% and 58% of the PDase I I a c t i v i t y of homogenates of these t i s s u e s i n the f i n a l supernatant a f t e r d i f f e r e n t i a l c e n t r i f u g a t i o n . The r e s u l t s o b t a i n e d w i t h r a t i n t e s t i n e i n t h i s study (Table VI) would appear t o agree w i t h those of R a z z e l l (50). However when R a z z e l l (50) f r a c t i o n a t e d homogenates of hog kidney he found o n l y 23% of the PDase I I a c t i v i t y i n the f i n a l s upernatant w h i l e approximately 37% of the t o t a l a c t i v i t y was r e c o v e r e d i n a " m i t o c h o n d r i a l " f r a c t i o n . R a z z e l l concluded (50) " t h a t the p h o s p h o d i e s t e r a s e I I had been r e l e a s e d p r e f e r e n t i a l l y from the m i t o c h o n d r i a " . In 1968 Hunter and Korner confirmed the presence of a h i g h l y a c t i v e PDase I I i n a c e l l sap p r e p a r a t i o n from r a t l i v e r (51). In the same year van Dyck and Wattiaux (52) used a DNA "core", composed of 3'-phosphodeoxyribonucleotides (63) t o d e t e c t " a c i d exonuclease" (PDase II) a c t i v i t y i n r a t l i v e r . On the b a s i s of d i f f e r e n t i a l and d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n experiments, they concluded t h a t the enzyme was p r e s e n t i n lysosomes. These i n v e s t i g a t o r s a t t r i b u t e d (52) the d i f f e r e n c e s between t h e i r r e s u l t s and those of R a z z e l l (50) to the use of d i f f e r e n t s u b s t r a t e s i n the two s t u d i e s . However, very r e c e n t l y , E r e c i n s k a e t a l . (53) o b t a i n e d r e s u l t s s i m i l a r t o those of van Dyck and Wattiaux (52 ) u s i n g the same t i s s u e ( r a t l i v e r ) and s u b s t r a t e (Tp-NP) which were employed by R a z z e l l (50) . The d i f f e r e n c e s i n r e s u l t s o b t a i n e d by the above authors (50,52,53) are p u z z l i n g and may p o s s i b l y be e x p l a i n e d by s u b t l e d i f f e r e n c e s i n homogenization s i n c e i t i s l i k e l y t h a t s u b c e l l u l a r s t r u c t u r e s g e n e r a l l y (101) and lysosomes i n p a r t i c u l a r (102,103) can be damaged d u r i n g t h i s procedure. 82 Attempts t o reach c o n c l u s i o n s r e g a r d i n g the d i s t r i b u t i o n of PDase I I i n the p a r t i c u l a r case of r a t l i v e r have been c o m p l i c a t e d by the o b s e r v a t i o n s of B r i g h t w e l l and Tappel (55). These workers have r e p o r t e d t h a t t h i s t i s s u e c o n t a i n e d a t l e a s t 2 and pr o b a b l y 3 PDases which were a c t i v e a t a c i d i c pH v a l u e s . The e x i s t e n c e of lysosomes i n i n t e s t i n a l mucosa has been i n d i c a t e d by e l e c t r o n microscopy (104,105), h i s t o c h e m i c a l (105,106) and b i o c h e m i c a l (56) da t a . Since there was good evidence, o u t l i n e d above, t h a t PDase I I i n r a t l i v e r was l o c a l i z e d i n lysosomes, i t seemed p o s s i b l e t h a t PDase I I a c t i v i t y o f i n t e s t i n e might a l s o be ly s o s o m a l . Appelmans and de Duve (96) have c o n v i n c i n g l y shown t h a t a c i d phosphatase i n p a r t i c l e s o b t a i n e d from r a t l i v e r can be " a c t i v a t e d " by making the medium hypoosmotic. I n d i c a t i o n s t h a t PDase I I of r a t i n t e s t i n e c o u l d be o s m o t i c a l l y " a c t i v a t e d " i n suspensions and homogenates (Table I I I ) suggested t h a t the enzyme was indeed c o n t a i n e d i n an> i n t r a c e l l u l a r compartment. Some o t h e r r a t h e r unexpected r e s u l t s were o b t a i n e d i n t h i s experiment. For i n s t a n c e i t might be expected t h a t a f t e r 96 hr a l l of the o s m o t i c a l l y r e l e a s e d PDase I I a c t i v i t y would be i n the supernatant ( s o l u b l e ) f r a c t i o n . While t h i s was t r u e t o a gr e a t e x t e n t , i t was seen t h a t the sediments from the suspension and homogenate i n water showed a s l i g h t i n c r e a s e i n the a c t i v i t y a f t e r 96 hr (Table I V ) . However i t was thought t h a t t h i s was due t o a d s o r p t i o n of the s o l u b l e enzyme on the p a r t i c l e s s i n c e Conchie and Levvy (102) and Levvy and Conchie (103) have shown t h a t t h i s i s a common p r o p e r t y of many "lysosomal" h y d r o l a s e s . 83 A g a i n , a s u b s t a n t i a l d e c r e a s e was o b s e r v e d i n t h e PDase I I a c t i v i t y o f t h e sed i m e n t s from p r e p a r a t i o n s i n i s o t o n i c s a l i n e a f t e r 96 h r . I t was thought t h a t t h i s may have been due t o t h e r e l e a s e of t h e adsorbed enzyme by the s a l t p r e s e n t . However i t seems t h a t such r e a s o n i n g can n o t e x p l a i n a t h i r d r e s u l t . I t was o b s e r v e d (Table I I I ) t h a t s u s p e n s i o n s and homogenates i n i s o t o n i c s a l i n e had a p p r o x i m a t e l y 20% h i g h e r t o t a l PDase I I a c t i v i t y t h a n t h e c o r r e s p o n d i n g p r e p a r a t i o n s i n w a t e r . T h i s i n c r e a s e d a c t i v i t y c o u l d n o t be e x p l a i n e d by p o s t u l a t i n g a s t i m u l a t o r y e f f e c t on t h e enzyme by sodium c h l o r i d e s i n c e t h e PDase I I a c t i v i t y o b s e r v e d i n t h e s u p e r n a t a n t s were i d e n t i c a l f o r b o t h p r e p a r a t i o n s (Table I V ) . I t t h e r e f o r e seems t h a t i n t h e p r e s e n c e o f sodium c h l o r i d e , t h e r e was e i t h e r an i n c r e a s e d amount o f enzyme p r e s e n t i n t h e sediments o r t h e r e was an i n c r e a s e d a c c e s s i b i l i t y o f enzyme t o s u b s t r a t e i n t h e s e d i m e n t s . No adequate e x p l a n a t i o n o f t h e s e r e s u l t s can be g i v e n . L e vvy and co-workers (102,103) and o t h e r s (76,77) have shown t h a t l y s o s o m a l enzymes can a l s o be a c t i v a t e d by m e c h a n i c a l o r c h e m i c a l p r o c e d u r e s . When t h e s e methods were used w i t h homogenates o f r a t i n t e s t i n a l mucosa i t appeared t h a t known l y s o s o m a l enzymes (56) were a c t i v a t e d t o d i f f e r e n t e x t e n t s (Table V ) , w h i l e v e r y l i t t l e a c t i v a t i o n o f PDase I I was o b s e r v e d a t a l l . Hsu and T a p p e l (56) who s t u d i e d t h e d i s t r i b u t i o n o f a number ;of l y s o s o m a l enzymes i n i n t e s t i n e c o n c l u d e d t h a t t h e t i s s u e c o n t a i n e d a p o p u l a t i o n o f lysosomes w h i c h was much more heterogenous t h a n t h a t o f r a t l i v e r . S i n c e lysosomes a r e t h o u g h t t o v a r y i n t h e i r enzymic c o n t e n t (107), i t i s l i k e l y t h a t t h e v a r i a b l e a c t i v a t i o n 84 of the lysosomal enzymes DNase, RNase and acid phosphatase observed i n the present work (Table V) can be explained by the s e l e c t i v e breakage of s p e c i f i c lysosomes during homogenization. If indeed PDase II i s lysosomal as has been suggested by van Dyck and Wattiaux (52) and Erecinska et a l . (53) then the low degree of a c t i v a t i o n observed i n the present work i s puzzling. However c e r t a i n investigators (102,103) have noted the high a c t i v i t y of c e r t a i n lysosomal enzymes which were present i n the f i n a l supernatant a f t e r f r a c t i o n a t i o n of such tissues as mouse spleen and S37 mouse tumor (108), even a f t e r extremely gentle homogenization. I t i s possible that homogenization of i n t e s t i n a l t i s s u e which i s very f r a g i l e could give s i m i l a r r e s u l t s . Therefore, since l i t t l e a c t i v a t i o n of PDase II could be found i n homogenates of i n t e s t i n a l mucosa, using mechanical or chemical methods (Table V), i t i s possible that most of the PDase II a c t i v i t y had already been released by breakage of lysosomes during the course of homogenization. As has been mentioned previously, f r a c t i o n a t i o n of i n t e s -t i n a l mucosal tissue was hampered by the presence of large amounts of mucous which became apparent when the tissue was homogenized. Similar d i f f i c u l t i e s have been reported by others (58,84,85,87,92). Notwithstanding t h i s hindrance, several f r a c -t i o n a t i o n experiments were c a r r i e d out and i n each case the greatest proportion of PDase II a c t i v i t y was found i n the supernatant solution remaining a f t e r the f i n a l c e n t r i f u g a t i o n step (Tables VI, VIII and IX). I t was also found that the portion of PDase II a c t i v i t y which was present i n the p a r t i c u l a t e 85 f r a c t i o n s seemed t o be a s s o c i a t e d w i t h those g r a n u l e s which had the h i g h e s t s p e c i f i c a c t i v i t i e s f o r a c i d phosphatase (Tables VI, V I I I and I X ) . The experiments performed u s i n g sucrose d e n s i t y -g r a d i e n t c e n t r i f u g a t i o n confirmed t h i s i n t e r p r e t a t i o n ( F i g s . 5 and 6 ) . Since the osmotic a c t i v a t i o n experiments i n d i c a t e d t h a t a l a r g e p o r t i o n of the PDase II a c t i v i t y of i n t e s t i n e p r o b a b l y r e s i d e d i n some i n t r a c e l l u l a r g r a n u l e ( s ) and i n view of the evidence, o u t l i n e d above, of the f r a g i l i t y of lysosomes f o r c e r t a i n t i s s u e s , i t seems p o s s i b l e t h a t most of the PDase II a c t i v i t y r e c o v e r e d i n the f i n a l supernatant c o u l d have a r i s e n from lysosomes. Attempts have been made to i n v e s t i g a t e the l o c a l i z a t i o n of PDase II u s i n g c y t o c h e m i c a l methods (109) . Sierakowska e t a l . (110) used a-naphthol thymidine 3'-phosphate as a s u b s t r a t e f o r the enzyme i n the hope t h a t the a-naphthol r e l e a s e d by the a c t i v i t y c o u l d be c o u p l e d with a d i a z o t a t e ( 5 - c h l o r o - O - t o l u i d i n e ) to form a r e d i n s o l u b l e p r e c i p i t a t e . However these experiments were abandoned when i t was found t h a t t h i s s u b s t r a t e was t o t a l l y r e s i s t a n t to the enzyme (110). I t was noted d u r i n g the p r e p a r a t i o n of e p i t h e l i a l - c e l l "ghosts" from mucosal t i s s u e by the method of C l a r k and Porteous (58) t h a t l a r g e amounts of p - n i t r o p h e n y l phosphatase a c t i v i t y were r e c o v e r e d i n the supernatant f r a c t i o n a f t e r c e n t r i f u g a t i o n (Table V I I ) . Although the experiment was repeated many times, more than 50% of the p - n i t r o p h e n y l phosphatase a c t i v i t y of the o r i g i n a l homogenate was always r e c o v e r e d i n the s o l u b l e f r a c t i o n . On the o t h e r hand C l a r k and Porteous have r e p o r t e d (58) t h a t 86 only s m a l l amounts of the 3-glycerophosphatase a c t i v i t y of the o r i g i n a l homogenate were l o s t d u r i n g homogenization and 83% of t h i s a c t i v i t y was r e c o v e r e d i n the e p i t h e l i a l - c e l l "ghost" f r a c t i o n . S i n c e t h i s d i s c r e p a n c y would appear t o be too g r e a t t o be e x p l a i n e d by ex p e r i m e n t a l e r r o r , i t was l i k e l y t h a t , as i n the case of r a t l i v e r (97) , a l a r g e p o r t i o n of the p - n i t r o p h e n y l phosphatase a c t i v i t y of homogenates of i n t e s t i n a l mucosa was due to a s o l u b l e enzyme which i s d i s t i n c t from the lysosomal " a c i d phosphatase". N e i l and Horner (97) r e p o r t e d t h a t an enzyme having marked p - n i t r o p h e n y l phosphatase a c t i v i t y b u t o n l y s l i g h t 3 - g l y c e r o p h o s p h a t a s e a c t i v i t y was p r e s e n t i n the supernatant f r a c t i o n of homogenates of l i v e r from a v a r i e t y of animals i n c l u d i n g r a t . The a c t i v i t y of the s o l u b l e p - n i t r o p h e n y l phospha-t a s e was about f i v e times as g r e a t as t h a t of the s o l u b l e 3 - g l y c e r o p h o s p h a t a s e (97). The r e s u l t s of the experiments performed u s i n g sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n agreed w e l l w i t h the r e s u l t s o f the other f r a c t i o n a t i o n experiments. In g e n e r a l the p a r t i c u l a t e PDase I I a c t i v i t y seemed to sediment i n the sucrose g r a d i e n t along w i t h those p a r t i c l e s which had the h i g h e s t a c i d phosphatase a c t i v i t y . I t should be noted t h a t these c e l l u l a r g r a n u l e s were i s o l a t e d and washed s e v e r a l times b e f o r e b e i n g c e n t r i f u g e d i n the d e n s i t y - g r a d i e n t . So i t i s l i k e l y t h a t the s o l u b l e PDase I I and a c i d phosphatase a c t i v i t i e s which were observed a t the top of the g r a d i e n t ( F i g s . 5 and 6) were r e l e a s e d d u r i n g manipula-t i o n of these f r a g i l e p a r t i c l e s . p r i o r to d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . 87 I t was observed t h a t d u r i n g the attempt t o " p u r i f y " lysosomes not j u s t one but a number of the f i n a l f r a c t i o n s had h i g h a c t i v i t i e s of PDase I I and a c i d phosphatase (Table X ) . Hsu and Tappel (56) r e p o r t e d t h a t lysosomes i n i n t e s t i n e were a heterogenous group of p a r t i c l e s and would appear to resemble the g r a n u l e s from kidney -(57) i n t h i s r e s p e c t . I t i s t h e r e f o r e not. s u r p r i s i n g t h a t a number of the f i n a l f r a c t i o n s o b tained e x h i b i t e d h i g h a c t i v i t i e s f o r a c i d phosphatase and PDase I I s i n c e one would expect to r e c o v e r d i f f e r e n t s i z e g r a n u l e s in. d i f f e r e n t f r a c t i o n s . P r o p e r t i e s of the Phosphodiesterase I I A c t i v i t y The PDase I I a c t i v i t y o f i n t e s t i n e c a t a l y z e d the h y d r o l y -s i s c f Tp-NP and Tp-DNP a t a. pH close, t o n e u t r a l i t y and i n t h i s r e s p e c t i t was s i m i l a r t o s p l e e n PDase (48,70). The p r o p e r t i e s which have been p u b l i s h e d f o r PDase I I have been obtained w i t h h i g h l y p u r i f i e d enzymes from c a l f s p l e e n (47,48) and salmon t e s t i s (59) . Both these enzymes were ir.cst a c t i v e a t pH v a l u e s ++ s l i g h t l y l e s s than 6 and were s t r o n g l y i n h i b i t e d by Cu (48,59) . However w h i l e the s p l e e n enzyme was i n h i b i t e d by such d i v a l e n t c a t i o n s as Mg and Ca- (47) no such i n h i b i t i o n c o u l d be observed i n the case of the salmon enzyme (50). I t would seem t h a t the e f f e c t s of these ions cn the PDase I I a c t i v i t y I n i n t e s t i n e (Table XII) are more i n keeping with, those o f the. salmon enzyme. Using NP-pT as s u b s t r a t e , B r i g h t w e l l and Tappel (.55). have d e s c r i b e d an enzyme a c t i v i t y i n r a t l i v e r lysosomes which -4 they c a l l e d a c i d PDase I. The enzyme had a K of 4 x 10 M 88 was not a f f e c t e d by EDTA, M g + + or C a + + and was i n a c t i v a t e d g r a d u a l l y by h e a t i n g a t 52°. I t i s i n t e r s t i n g t o note t h a t the p r o p e r t i e s of PDase I I from i n t e s t i n e are somewhat s i m i l a r to those d e s c r i b e d above f o r a c i d PDase I. However s i n c e B r i g h t w e l l and Tappel (55) observed two pH optima (at 3.2 and 5.2) and s i n c e the s u b s t r a t e they used was d i f f e r e n t t o t h a t employed i n the p r e s e n t study i t i s d o u b t f u l t h a t the same a c t i v i t y was b e i n g measured i n both t i s s u e s . The PDase I I of i n t e s t i n e was i n a c t i v a t e d r a p i d l y a t temperatures h i g h e r than 60° ( F i g . 10). However between 20° e and 60 the. PDase I I was s t r o n g l y a c t i v a t e d by i n c r e a s i n g temperature w i t h an apparent optimum temperature of 58°. The t h e r m o s t a b i l i t y of the a c t i v i t y appeared t o be g r e a t e r than t h a t of p u r i f i e d PDases from s p l e e n (49) and t e s t i s (59) which q u i c k l y l o s e t h e i r a c t i v i t y a t temperatures g r e a t e r than 50°. The k i n e t i c s o f the heat i n a c t i v a t i o n of PDase I I ( F i g . 11) might l e a d one to b e l i e v e t h a t t h e r e was more than one enzyme capable of h y d r o l y z i n g Tp-NP i n e x t r a c t s o f i n t e s t i n a l mucosa. However the pH a c t i v a t i o n curves of t h r e e s u b c e l l u l a r f r a c t i o n s i s o l a t e d from the i n t e s t i n a l mucosa were almost superimposable (Fig.7) and the optimum pH observed i n each f r a c t i o n was approx-i m a t e l y 6.8 i n d i c a t i n g perhaps t h a t a s i n g l e enzyme was i n v o l v e d . I t would seem, t h e r e f o r e , t h a t , although a v a r i e t y of nuc l e a s e s w i t h o v e r l a p p i n g s p e c i f i c i t i e s e x i s t s i n o t h e r t i s s u e s (55,68, 98), o n l y minimal evidence i s a v a i l a b l e f o r the e x i s t e n c e of such a s i t u a t i o n i n i n t e s t i n e . In t h i s r e s p e c t i t i s i n t e r e s t i n g t o note the r e s u l t s of N e i l and Horner (97,111) who showed t h a t 89 a v a r i e t y of phosphomonoesterases w i t h v e r y s i m i l a r s p e c i f i c i t i e s were p r e s e n t i n many animal t i s s u e s . Using p - n i t r o p h e n y l phosphate and phenyl phosphate as s u b s t r a t e s these i n v e s t i g a t o r s showed t h a t s p e c i f i c phosphomonoesterases a c t i v e a t a c i d i c pH v a l u e s were p r e s e n t i n s e v e r a l s u b c e l l u l a r f r a c t i o n s of t i s s u e homogenates. However i t i s l i k e l y t h a t the enzymes which h y d r o l y z e doubly e s t e r i f i e d p hosphoric a c i d are q u i t e s p e c i f i c (8) and i n t h i s r e s p e c t Tp-NP and Tp-DNP would appear to be l e g i t i m a t e and s p e c i f i c s u b s t r a t e s f o r the assay of PDase I I a c t i v i t y (8,68,70). I t i s to be hoped, however, t h a t the enzyme w i l l e v e n t u a l l y be t e s t e d on such n a t u r a l s u b s t r a t e s as DNA, RNA and the o l i g o -n u c l e o t i d e s o b t a i n e d from both of these. 90 SUMMARY 1. Pho s p h o d i e s t e r a s e II a c t i v i t y , i n the i n t e s t i n a l mucosa of the r a t was i n v e s t i g a t e d u s i n g as s u b s t r a t e s the p - n i t r o p h e n y l and 2 , 4 - d i n i t r o p h e n y l e s t e r s of thymidine 3'-phosphate. 2. S i n c e they were i n i t i a l l y not commercially a v a i l a b l e , p - n i t r o p h e n y thymidine 3'-phosphate and 2 , 4 - d i n t r o p h e n y l thymidine '3'-phosphate were s y n t h e s i z e d . During the l a t t e r stages of the i n v e s t i g a t i o n the p - n i t r o p h e n y l e s t e r became a v a i l a b l e and t h i s s u b s t r a t e was used f o r the remainder of the experiments. 3. Suspensions of. mucosal s c r a p i n g s from r a t i n t e s t i n e c o n t a i n e d s o l u b l e p h o s p h o d i e s t e r a s e I I a c t i v i t y . T h i s a c t i v i t y c o u l d be removed by e x t e n s i v e washing of the mucosal s c r a p i n g s . Homogeni-z a t i o n of the washed s c r a p i n g s r e l e a s e d f u r t h e r p h o s p h o d i e s t e r a s e I I a c t i v i t y . 4. When suspensions of mucosal t i s s u e i n water were allowed to st a n d f o r 96 hr the a c t i v i t y of phosphodiesterase I I i n c r e a s e d by 61% w h i l e w i t h homogenates an i n c r e a s e of 38% was observed. Very l i t t l e change was observed i n p r e p a r a t i o n s t r e a t e d w i t h i s o t o n i c s a l i n e over the same p e r i o d of time. L i t t l e or no a c t i v a t i o n of p h o s p h o d i e s t e r a s e I I a c t i v i t y c o u l d be observed by treatment w i t h mechanical or chemical methods. 5. The 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 phosphodiesterase I I was i n v e s t i g a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n of homogenates of i n t e s t i n a l mucosa t i s s u e . S e v e r a l known enzyme a c t i v i t i e s were used as markers to i d e n t i f y b i o c h e m i c a l l y the f r a c t i o n s o b t a i n e d . The d i s t r i b u t i o n of phosp h o d i e s t e r a s e I I was s i m i l a r but not i d e n t i c a l w i t h t h a t of a c i d phosphatase, the enzyme marker f o r lysosomes. Most of the phosp h o d i e s t e r a s e II a c t i v i t y was found i n the supernatant s o l u t i o n a f t e r the f i n a l c e n t r i f u g a t i o n step and a s m a l l e r amount was found i n the "lysosomal" f r a c t i o n . 6. S e v e r a l experiments employing sucrose d e n s i t y - g r a d i e n t c e n t r i -f u g a t i o n were c a r r i e d out i n order t o c o n f i r m the r e s u l t s o b t a i n e d u s i n g d i f f e r e n t i a l c e n t r i f u g a t i o n . The p a r t i c l e s p r e s e n t i n " n u c l e a r - f r e e " homogenates were c e n t r i f u g e d on sucrose d e n s i t y -g r a d i e n t s a t d i f f e r e n t i n t e g r a t e d f o r c e s (g min). In each of the thr e e experiments performed, the p a r t i c l e s t o which phosphodies-t e r a s e I I were bound a l s o e x h i b i t e d the h i g h e s t a c t i v i t y of a c i d phosphatase. ; 7. An attempt t o p u r i f y lysosomes from i n t e s t i n a l mucosa was o n l y p a r t i a l l y s u c c e s s f u l , o n l y s m a l l amounts of the o r i g i n a l p h o s p h o d i e s t e r a s e I I being r e c o v e r e d i n the " p u r i f i e d " f r a c t i o n . N e v e r t h e l e s s the s p e c i f i c a c t i v i t y of phosp h o d i e s t e r a s e II i n c r e a s e d p a r a l l e l with t h a t of a c i d phosphatase throughout the p u r i f i c a t i o n and the s m a l l q u a n t i t y of " p u r i f i e d lysosomes " i s o l a t e d had the h i g h e s t s p e c i f i c a c t i v i t y of ph s o p h o d i e s t e r a s e I I . 8. The pH a c t i v i t y curves f o r phosphodiesterase I I i n a " n u c l e a r " , a "lysosomal" and a s o l u b l e f r a c t i o n were found t o be markedly s i m i l a r and the optimum pH was c l o s e t o 7. T h i s was found t o be so i n a wide v a r i e t y o f b u f f e r s . However i n s u c c i n a t e b u f f e r , the pH optimum was lower, b e i n g c l o s e t o 6. 9. The phosph o d i e s t e r a s e I I a c t i v i t y of the supernatant s o l u t i o n , remaining a f t e r c e n t r i f u g i n g an homogenate of mucosal t i s s u e , was v i r t u a l l y u n a f f e c t e d by Mg + +, C a + + and e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d . In the presence o f 8.3 mM z i n c c h l o r i d e , copper s u l p h a t e and m e r c u r i c c h l o r i d e the a c t i v i t y was i n h i b i t e d 35%, 47% and 100% r e s p e c t i v e l y . 10. The s o l u b l e p h o s p h o d i e s t e r a s e I I was most a c t i v e a t a temperature of 50°-58° and was r a p i d l y i n a c t i v a t e d a t temperatures above 60°. Heating a sample of the enzyme f o r one hour a t 55° r e s u l t e d i n a l o s s of 27% of the phosph o d i e s t e r a s e I I a c t i v i t y . 11. The M i c h a e l i s c o n s t a n t f o r the s o l u b l e enzyme a t 37° wit h -4 p - n i t r o p h e n y l thymidine 3'-phosphate as s u b s t r a t e was 4.5 x 10 M. 12. The e f f e c t of temperature on the a c t i v i t y of phosphodiesterase I I i n the presence of i n c r e a s i n g c o n c e n t r a t i o n s of s u b s t r a t e was i n v e s t i g a t e d . At 50° the M i c h a e l i s c o n s t a n t s and maximum i n i t i a l v e l o c i t i e s were 2.8 times and 9.5 times g r e a t e r than the © r e s p e c t i v e v a l u e s a t 20.5 . 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