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A study of phosphorylase in neoplastic tissue McBride, John Raymond 1954

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A STUDY OP PHOSFHORYLASE IN NEOPLASTIC TISSUE JOHN RAYMOND MCBRIDE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTSSFOR THE DEGREE OF MASTER OF ARTS i n the Department of BIOLOGY AND BOTANY We 'accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree of MASTER OF ARTS. Members of the Department^ of THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 195ij.. A b s t r a c t Phosphorylase a c t i v i t i e s and glycogen l e v e l s were determined i n t r a n s p l a n t a b l e hepatomas i n w i s t a r r a t s , i n t r a n s -p l a n t a b l e mammary carcinomas i n C ^ B l mice, i n the l i v e r s of these tumor-bearing animals, and i n the l i v e r s of c o n t r o l a n i -mals. In one s e r i e s of experiments the animals were deprived of food f o r twenty-four hours. I n a second s e r i e s of e x p e r i -ments the t i s s u e s were analyzed two hours a f t e r the i n t r a p e r i -t o n e a l i n j e c t i o n of glucose i n t o the 2i^-hour f a s t e d animals. Phosphorylase and glycogen were found to be very low i n both tumors t e s t e d . The i n j e c t i o n of glucose i n t o the f a s t e d animals r e s u l t e d i n marked changes i n phosphorylase and glycogen i n the normal l i v e r but r e l a t i v e l y l i t t l e change i n the tumor enzyme a c t i v i t y or i n the tumor glycogen l e v e l . Evidence was obtained of an impairment i n the g l y c o -genetic f u n c t i o n of the l i v e r i n the tumor-bearing animal. The r e l a t i v e amounts of the a c t i v e and i n a c t i v e forms of phosphor-y l a s e present i n these various t i s s u e s were a l s o a s c e r t a i n e d by means of the c o - f a c t o r a d e n y l i c a c i d . Purines and p y r i m i d i n e s , other than a d e n y l i c a c i d , were found to have no a c t i v a t i n g or i n h i b i t i n g e f f e c t on phos-phorylase a c t i v i t y i n normal l i v e r , tumor-bearing l i v e r and tumor from normal and tumor-bearing w i s t a r r a t s and G^jBl mice. C e l l u l a r f r a c t i o n a t i o n i n d i c a t e d phosphorylase a c t i v i t y ~=tp be present i n both the cytoplasmic and p a r t i c u l a t e f r a c t i o n s , i n normal l i v e r , tumor-bearing l i v e r and tumor from normal and tumor-bearing w i s t a r r a t s and C ^ B l mice. ACKNOWLEDGMENTS I wish to express my g r a t e f u l acknow-ledgment to Dr. E. S. Goranson, Department of Biology and Botany, f o r h i s p a t i e n t guidance and constant encouragement which sustained me throughout the p r e p a r a t i o n of t h i s t h e s i s . My s i n c e r e thanks and a p p r e c i a t i o n are also o f f e r e d to Dr. M. Darrach, Department of Biochemistry, f o r h i s i n v a l u a b l e c o n t r i -b u t i o n of the Novik o f f hepatoma which went so f a r i n making the experimental work f o r t h i s paper p o s s i b l e . My deepest a p p r e c i a t i o n and g r a t i t u d e are f u r t h e r extended to the N a t i o n a l Cancer I n s t i t u t e f o r a s s i s t a n c e of inesti m a b l e value which made p o s s i b l e the completion of the work necessary to compile the r e s u l t s contained i n t h i s t h e s i s . TABLE OP CONTENTS PAGE INTRODUCTION: METHODS; A. Animals '. 7 B. T r a n s p l a n t a t i o n Of Tumors 7 C. Assay Of Phosphorylase 11 D. Determination Of Glycogen Ik. E. Determination Of P r o t e i n N itrogen Io P. Method Of C e l l F r a c t i o n a t i o n 17 RESULTS; A. Phosphorylase A c t i v i t y i n Tissues From Twenty-four Hour Fasted Normal and Tumor-Bearing Rats and Mice. 19 B. Glycogen L e v e l i n Tissues From Twenty-four Hour Fasted Normal and Tumor-Bearing Rats and Mice 25 C. E f f e c t of A d m i n i s t e r i n g Glucose, F o l l o w i n g A Twenty-four Hour Fast , on Phosphorylase A c t i v i t y i n Tissues From Normal and Tumor-Bearing Rats and Mice 25 D. E f f e c t of A d m i n i s t e r i n g Glucose, F o l l o w i n g A Twenty-four Hour Fast, on Glycogen L e v e l i n Tissues From Normal and Tumor-Bearing Rats and Mice 35 E. E f f e c t of A d e n y l i c , U r i d y l i c , C y t i d y l i c and Guanylic A c i d s , i n V i t r o , on Phosphorylase A c t i v i t y i n Tissues From Normal and Tumor-Bearing Rats and Mice 35 F. Phosphorylase A c t i v i t y i n C e l l F r a c t i o n s of Normal and Tumorous Tissues 1|1 DISCUSSION: 2+3 SUMMARY:... IjJ BIBLIOGRAPHY i|8 1 INTRODUCTION E a r l y work by Warburg (1) i n 1920 to 1930 showed an abnormally h i g h aerobic g l y c o l y s i s i n tumors. Another char-a c t e r i s t i c reported by s e v e r a l i n v e s t i g a t o r s (2) (3) (Ij.) i s a low l e v e l of glycogen i n tumors. Weinhouse (5) has suggested two p o s s i b l e explanations f o r the h i g h g l y c o l y t i c r a t e noted i n tumors. F i r s t , i f a disturbance i n the e l e c t r o n t r a n s p o r t a t i o n system e x i s t s , e l e c t r o n s would p i l e up and would have no other choice than to combine wi t h an e l e c t r o n acceptor (such as pyruvate) to form l a c t i c a c i d . Recent repo r t s by P o t t e r (6) and Schneider (7) however, f a i l to show any impairment i n the e l e c t r o n t r a n s p o r t system i n tumors. The second p o s s i b i l i t y i s that one of the r e a c t i o n s l e a d i n g pyruvate i n t o the c i t r i c a c i d c y c l e i s d e f e c t i v e . Weinhouse (8) has reported that the r a t hepatoma and mouse adenocarcinoma were able to o x i d i z e l a b e l l e d glucose to CO2 at a .rate comparable w i t h normal l i v e r . This would i n d i c a t e that these tumors at l e a s t showed no defect I n the c i t r i c a c i d c y c l e . However, P o t t e r and co-workers (9) have reported that the enzymes of the c i t r i c a c i d c y c l e were present i n much -l o i t e r amounts i n r a t hepatoma than i n normal l i v e r , kidney or h e a r t . 2 A high aerobic g l y c o l y t i c rate, however, i s not l i m i t e d to tumors. Weinhouse (10), Dickens and Simer (11) have reported high rates of g l y c o l y s i s i n the embryo, brain, and heart, whether these tissues were malignant or not. I t has also been reported (12) that the rate of g l y c o l y s i s exceeds the rate of u t i l i z a t i o n of 0^  reactants i n the tumor tissues. Olson (13) and Zamecnik (llj.) have reported that i n tissue s l i c e experiments more G3 fragments were d i -verted into protein and l i p i d synthesis i n the hepatoma than i n the normal l i v e r . Olson (15) i n a recent review, has summarized i n the form of a chart (Pig. l ) ; t h e chief differences i n the meta-bolism of carbohydrate i n normal l i v e r and i n that of the hepatoma. The thickness of the arrows indicate the o r i e n t a t i o n of glucose along various pathways i n both the normal l i v e r and i n the hepatoma. In the normal l i v e r , glycogen i s synthesized from blood sugar and other metabolites such as pyruvate, l a c t a t e , constituents of the Kreb cycle and other substrates which enter t h i s cycle,(16). The enzyme hexokinase i s responsible for the introduction of the blood sugar into the metabolic pathway. The glycogen i s then stored i n the l i v e r u n t i l re-secreted as glucose into the blood or degraded to meet normal energy requirements for metabolic functions such as synthesis (17) (18). 3 HEPATOMA GLYCOGEN FRUCTOSE -6 - PO4 II FRUCTOSE l,6-P04 FA CHOL PYRUVATE |^S"C2-FRAG + lOAAa SR f MAL CIT | KREBS ) [ FUM a KE1 ^ s u c c ^ V GLUCOSE LACTATE: I  ACETATE: CO-LIVER FRUCTOSE-6-POA II FRUCTOSE I, 6-P04 PYRUVATE f ;C2-FRAG + lOAAi FA CHOL MAL CIT | KREBS FUM a KETO j^f^^SUCC* ) SR H20 H20 h I n the " f u l l - b l o w n " hepatoma, however, n e a r l y a l l the blood sugar Is d i v e r t e d immediately i n t o the g l y c o l y t i c pathway w i t h only a n e g l i g i b l e d e p o s i t i o n of glycogen from e i t h e r ex-t r a c e l l u l a r glucose or other substrates (19). This r e s u l t s i n l a r g e amounts of energy, l a c t a t e , and fragments being formed. Presumably t h i s energy and the b u i l d i n g u n i t s so formed are then a v a i l a b l e f o r f u r t h e r tumor development. Acc o r d i n g l y , one of the c h i e f d i f f e r e n c e s between nor-mal l i v e r and tumor l i v e r , i s the r e r o u t i n g of glucose i n the tumor i n t o the g l y c o l y t i c pathway r a t h e r than i n t o the g l y c o -genetic pathway as i n normal l i v e r . Thus the f i n d i n g of l a r g e amounts of energy, l a c t a t e and b u i l d i n g u n i t s due to a h i g h g l y c o l y t i c r a t e i n the hepatoma i s i n agreement w i t h the f i n d i n g s of Greenstein (20) and Le Page (21). Olson (22) and Zamecnik (23) have suggested th a t t h i s departure from the normal metabolic p a t t e r n represents an adjustment to meet the p a r t i c u l a r needs f o r energy and f o r b u i l d i n g u n i t s f o r the r a p i d growth of the l i v e r tumor. Orr, P r i c e and S t i c k l a n d (2b.) have added f u r t h e r s i g n -i f i c a n c e to t h i s abnormality i n t h e i r r e p o r t t h a t , while normal l i v e r u t i l i z e s glycogen p r e f e r e n t i a l l y f o r g l y c o l y s i s , the tumor draws on glucose as the c h i e f i f not the s o l e substrate f o r t h i s process. Olson has attempted to e x p l a i n t h i s abnormality i n the anaerobic phase of carbohydrate metabolism w i t h i n the tumor on the b a s i s of increa s e d a c t i v i t y i n the enzymes, (hexokinase; and phosphohexokinase) which d i v e r t glucose d i r e c t l y i n t o the g l y c o l y t i c pathway. This e x p l a n a t i o n , however, may not com-p l e t e l y e x p l a i n the marked Impairment i n the a b i l i t y of mice bearing Sarcoma 180 to deposit glycogen f o l l o w i n g i n t r a p e r i -t o n e a l glucose i n j e c t i o n s as reported by Young (2f?). Since phosphorylase i s d i r e c t l y concerned w i t h the synthesis of glycogen, and sin c e i t has been w e l l e s t a b l i s h e d that tumors i n general l a c k the a b i l i t y t o form glycogen, i t seemed p o s s i b l e that one of the changes occuring i n tumor-bearing animals might be an a l t e r a t i o n i n the phosphorylase system; and t h i s formed the problem to be i n v e s t i g a t e d . Phosphorylase occupies a paramount p o s i t i o n i n the synthesis and breakdown of glycogen. I t s p o s i t i o n i n the metabolic scheme i n l i v e r i s depicted i n P i g . 2. /=Ws mo/* *s*r (a r- 0-Y G L U C O S E - \- PHOS.PHAT*' |^ ^  ,m7m w 6 C o r i and Green (26) reported that phosphorylase e x i s t s i n muscle i n two forms: phosphorylase a, which i s a c t i v e without the a d d i t i o n of adenylic a c i d , and phosphorylase b, which i s i n a c t i v e without the a d d i t i o n o f , a d e n y l i c a c i d . The proportions of the a and b forms are v a r i a b l e and dependant on a l a b i l e enzyme which C o r i (27) has designated the PR ( p r o s t h e t i c group removing) enzyme. This enzyme con-v e r t s the a form to the b form by the removal of the p r o s t h e t i c group. The nature of t h i s p r o s t h e t i c group has not as yet been e s t a b l i s h e d although i t s i d e n t i t y w i t h a d e n y l i c a c i d has been suggested (28). C o r i (29) however, was unable to demonstrate f r e e adenylic a c i d among the products r e l e a s e d from c r y s t a l -l i n e phosphorylase a by the a c t i o n of the PR enzyme. Adenylic a c i d has been shown by C o r i (30) to a c t i v a t e both a and b forms of the enzyme. Phosphorylase b i n a c t i v e without the a d d i t i o n of ade n y l i c a c i d , i s r e s t o r e d to 90$> of' the maximum a c t i v i t y by the a d d i t i o n of adenylic a c i d . C r y s t a l -l i n e phosphorylase a without added adenylic a c i d e x h i b i t s 6$% of the maximum a c t i v i t y . Phosphorylase was assayed i n the d i r e c t i o n of p o l y -saccharide s y n t h e s i s , u s i n g the Inorganic phosphate l i b e r a t e d from glucose - 1 - phosphate as a q u a n i t a t i v e measurement of phosphorylase a c t i v i t y . 7 METHODS: A. Animals The experimental animals were obtained from the Department of Animal Nu t r i t i o n , University of B r i t i s h Golumbi These were Wistar r a t s , of both sexes, weighing between 120 -270 grams and Cc^Bl mice, of both sexes, weighing between 11}.-234. grams. The animals were fed Purina Pox Chow^ (No. £ Check ers) and water ad libitum. Pasted animals xvere deprived of food twenty-four hours before use. B. Transplantation of Tumors The transplantable Novikoff hepatoma'""'*' of the rat, although growing to one tenth of the body weight or approx-imately 20 grams i n eight to ten days, i s very d i f f i c u l t to detect by palpation u n t i l about the f i f t h day following implantation. On exposure, the tumors are poorly outlined, very nodular and lack any d i s t i n c t capsule. They grow at a rapid progressive rate often reaching a weight of 30 grams i f allowed to continue to grow. Additional tumors, which -"- Purina Pox Chow consists of approximately 22$ protein, 60$ carbohydrate, f a t and 13$ moisture. The rat Novikoff hepatoma was obtained from Professor A. Ham, Department of Anatomy, University of Toronto, through Professor M. Darrach, Department of Biochemistry, University of B r i t i s h Columbia. 8 9 appear i n the l a t t e r stages of tumor development, are u s u a l l y l o c a t e d i n the i n t e s t i n a l mesentery where severe haemorrhaging may be an important f a c t o r i n causing the death of the animal. The tumors o f t e n formed adhesions w i t h surrounding t i s s u e s . On s e c t i o n i n g , the tumors are s o f t , grayish-white i n c o l o u r , and h i g h l y v a s c u l a r i z e d both i n the p e r i p h e r a l and c e n t r a l regions of the tumor. C e n t r a l p o r t i o n s of the tumors are f r e q u e n t l y n e c r o t i c a f t e r the te n t h day of tumor development. The tumors are used f o r t r a n s p l a n t i n g on the ei g h t h day f o l l o w i n g implant-a t i o n and are tr a n s p l a n t e d i n t e r p e r i t o n e a l l y i n t o the abdominal r e g i o n . Dunn (31) has described the t r a n s p l a n t a b l e mammary carcinoma"" of the mouse used i n these experiments as f o l l o w s : "Extremely small tumors are r e a d i l y detected by p a l p a t i o n . The loose a r e o l a r t i s s u e permits them to be f r e e l y movable; they are f i r m i n consistence, and w e l l - o u t l i n e d . The tumors grow pro-g r e s s i v e l y and may reach enormous s i z e ; o f t e n a d d i t i o n a l tumors appear at other s i t e s . The s k i n and other t i s s u e s do not be-come adherent u n t i l the tumor has reached a considerable s i z e , when e x t e r n a l u l c e r a t i o n , b l e e d i n g , and e x t r u s i o n of n e c r o t i c contents may take p l a c e . Death u s u a l l y i s a t t r i b u t a b l e to the l a r g e s i z e of the l o c a l tumor which i n t e r f e r e s w i t h the move-ments and fee d i n g of the animal, and to secondary i n f e c t i o n and n e c r o s i s . The mammary carcinoma was obtained from the Jackson B. Memorial Laboratory, Ann Arbor, Michigan. 10 On s e c t i o n , the small tumors are u s u a l l y s o f t and grayish-white i n c o l o u r . They have an i n d i s t i n c t capsule and u s u a l l y can be s h e l l e d out r e a d i l y from the surrounding t i s s u e . Larger tumors o f t e n c o n t a i n c y s t i c and haemorrhagic areas that are v i s i b l e to the naked eye. N e c r o t i c areas are frequent, and the tumor may be adherent to surrounding s t r u c t u r e s because of inflammatory r e a c t i o n and i n f i l t r a t i v e growth. I n the l i v i n g mouse the margins of the tumor are extremely v a s c u l a r , but the v e s s e l s q u i c k l y c o l l a p s e w i t h the death of the animal. Gross metastases may be detected i n the lungs and r a r e l y i n other i n t e r n a l organs." Before removal of the tumor f o r t r a n s p l a n t i n g , the f u r i n the r e g i o n of the tumor was swabbed w i t h 95% ethanol. An I n c i s i o n was made through the s k i n from a p o i n t below the tumor to a p o i n t above the tumor. The s k i n and f u r were d i s s e c t e d away from a wide area surrounding the tumor s i t e . The exposed t i s s u e s were then swabbed w i t h c o t t o n b a t t e n dipped i n 95% ethanol. The mammary tumors growing subcutaneously and hepatoma implanted w i t h i n the abdomen were removed a s e p t i c a l l y and t r a n s f e r r e d to s t e r i l e 10 ml. syringe a f f i x e d to No. 18 gauge needle. P r e p a r a t i o n of the two tumors f o r t r a n s p l a n t a t i o n f o l l o w e d s i m i l a r procedures. 11 The tumor was then passed through a s e r i e s of s t e r i l i z e d 10 ml. syringes by f o r c e u s i n g No. 19> No. 20, and No. 21 gauge needles s u c c e s s i v e l y to macerate the tumor. To the mammary tumor m a t e r i a l an equal volume of s t e r i l e 0.9$ s a l i n e was added and mixed i n a 5 ml. syringe provided w i t h a No. 21 gauge needle f o r i n j e c t i n g the homogen-i z e d t i s s u e . The r a t hepatoma t i s s u e was d i l u t e d w i t h three volumes of the s a l i n e mixed i n a 10 ml. syringe f o r i m p l a n t a t i o n . The w i s t a r r a t and the C^yBl mice were i n j e c t e d w i t h a .50 ml. and .25 ml. of the described tumor homogenates r e -s p e c t i v e l y . The animals r e c e i v i n g the tumors were swabbed w i t h 95$ ethanol over the area where the i n j e c t i o n was to be made. G. Assay of Phosphorylase The method o u t l i n e d by C o r i (32) f o r assay of phos-phorylase a c t i v i t y i n muscle t i s s u e was f o l l o w e d . The animal was anaesthetized w i t h ether, an i n c i s i o n made i n the abdominal vra.ll and the animal b l e d by s e c t i o n i n g of the a o r t a . Approx-imately two grams of t i s s u e were removed and placed i n an i c e c o l d mortar. A few grains of sand and Ij. ml. of d i s t i l l e d water were added to the t i s s u e . The t i s s u e was then thoroughly ground w i t h the sand. The mixture was f i l t e r e d through No. 2 Whatman f i l t e r i n a Buchner f u n n e l , u s i n g s l i g h t s u c t i o n , w i t h the r e c e i v i n g tube immersed i n i c e . The f i l t r a t e was c a u t i o u s l y adjusted to 12 exactly pH 6.1 with d i l u t e HC1 and then dialyzed i n cellophane tubing against running tap water (5 - 10°C.) f o r three hours. D i a l y s i s was c a r r i e d out to remove Mg ions from the f i l t r a t e , thus preventing the breakdoim of glucose - 1 - phosphate to glucose - 6 - phosphate. The dialyzed extract was then d i l u t e d with an equal volume of 0.015M. cysteine - HC1 (pH 6.8) and kept at 0° C. u n t i l added to the reaction mixture. Green and Cori (33) demonstrated that the reducing agent cysteine not only enhanced the a c t i v i t y of phosphorylase to a small extent, but also greatly increased i t s s o l u a b i l i t y i n weak s a l t solutions. The enzyme was assayed i n the d i r e c t i o n of polysac-charide synthesis using the inorganic phosphate l i b e r a t e d from glucose - 1 - Phosphate as a quanitative measurement of the enzyme a c t i v i t y . The reaction tubes were made up with two tubes f o r each tissue phosphorylase determination. Each tube contained the following reagents: -; 1.0 ml., .015M. cysteine-HCl pH 6.5 1,8 ml., 2% glycogen 1.0 ml., .061}M. glucose - 1 - P0. 13 To one tube 0.2 ml. of a d e n y l i c a c i d (0.025M.) was a l s o added; to the other 0.2 ml. of 0.1N. HC1.* Gori and Green (3l+) have demonstrated t h a t f o r the synthesis of p o l y s a c c h a r i d e s , by phosphorylase preparations f o r mammalian t i s s u e s , i n v i t r o , a branched pol y s a c c h a r i d e such as glycogen or n a t u r a l s t a r c h much be present. Furthermore the polysaccharide synthesized by phosphorylase, In v i t r o , c o n s i s t s i n a lengthening of the s i d e chains of the added poly s a c c h a r i d e by the a d d i t i o n of glucose u n i t s i n 1:1+ glucoside l i n k a g e s . The r e a c t i o n tubes together w i t h the tubes c o n t a i n i n g the enzyme p r e p a r a t i o n were e q u i l i b r a t e d f o r ten minutes vrith a g i t a t i o n at 30°C. Then 2 ml. p o r t i o n s of the enzyme prepar-a t i o n were p i p e t t e d i n t o each of the r e a c t i o n tubes. A 1 ml. a l i g u o t was taken from the r e a c t i o n tubes at the end of one minute f o r the f i r s t sample of Inorganic phosphate content. This served f o r the i n o r g a n i c phosphate content at zero time. A second 1 ml. a l i q u o t was taken f o r i n o r g a n i c phosphate determ-i n a t i o n at the end of t h i r t y minutes of i n c u b a t i o n . The 1 ml. a l i q u o t s were t r a n s f e r r e d to tubes c o n t a i n -i n g 1+ ml. of i c e c o l d 10% t r i c h l o r o a c e t i c a c i d . The p r e c i p i t a t e d p r o t e i n was f i l t e r e d o f f i n an i c e bath u s i n g Whatman Wo. 1 f i l t e r paper. The i n o r g a n i c phosphate was determined by the method of F i s k e and Subbarow. (35>) • *• The glucose-l-POj^ and a d e n y l i c a c i d were obtained from the N u t r i t i o n a l Biochemicals Corporation. The glycogen was obtained from the P f a n s t i e h l Chemical Co. L-cysteine-HCl was obtained from the B r i t i s h Drug Houses. Ik To 2 ml. a l i q u o t s of the f i l t r a t e was added 6.6 ml. of d i s t i l l e d water, 1 . 0 ml. of molybdate I and O.lj. ml. o f l:2:k -amino-naptholsulfonic a c i d . F i v e minutes were allowed f o r the development of c o l o u r . The phosphate i n the t i s s u e samples were read against standards c o n t a i n i n g 20 Y and l^oVof i n o r g a n i c phosphate, 1 ml. of molybdate I, O.lj. ml. of l : 2 : l j . - amino-naptholsulf onic a c i d and d i l u t e d to a f i n a l volume of 1 0 m l . w i t h d i s t i l l e d water. The reading of c o l o u r i n t e n s i t y was c a r r i e d out u s i n g a Beckman B - model Spectrophotometer adjusted to read 1 0 0 $ t r a n s m i s s i o n at 660/wwjC through a blank c o n t a i n i n g the molybdate I and the n a p t h o l s u l f o n i c a c i d reducing agent. D. Determination of Glycogen. A m o d i f i c a t i o n of the method o u t l i n e d by Good, Kramer, and Somogyi ( 3 6 ) was used f o r the assay of glycogen. Centrifuge tubes, each c o n t a i n i n g 3 ml. of 30$ K 0 S , were placed i n a b o i l i n g water bath, brought to the b o i l and weighed. Approximately 0.5 gm. of t i s s u e was p l a c e d i n the hot K 0 H . The c e n t r i f u g e tubes c o n t a i n i n g hot K 0 H together w i t h t i s s u e were then reweighed and returned to the b o i l i n g water bath. The c e n t r i f u g e tubes remained i n the b o i l i n g water bath w i t h o c c a s i o n a l shaking u n t i l the t i s s u e was completely d i s -s o lved. The tubes were then removed and allowed to c o o l i n the a i r . 15 Approximately 5 ml. of 95% ethanol was added to each of the c e n t r i f u g e tubes and the contents of the tube mixed by s t i r r i n g . The c e n t r i f u g e tubes were then placed back i n the b o i l i n g water b a t h . t o • a c c e l e r a t e p r e c i p i t a t i o n of glycogen. As soon as the a l c o h o l began to b o i l , the c e n t r i f u g e tubes were again removed from the b o i l i n g water bath and allowed to c o o l i n the a i r . The tubes were then c e n t r i f u g e d u n t i l the glycogen was completely sedimented, the supernatant poured o f f , and approximately 5 ml. of 1 normal E^SOj^ added to each c e n t r i f u g e tube. The c e n t r i f u g e tubes were then returned to the b o i l i n g water bath and three hours allowed f o r a c i d h y d r o l y s i s of the glycogen. The tubes were then allowed t o c o o l . Each tube was n e u t r a l i z e d w i t h 1 normal NaOH, usi n g phenol red as i n d i c a t o r . A few drops of 1 normal R^SOl}. were added immediately to make the s o l u t i o n s l i g h t l y a c i d . The s o l -u t i o n was then made up to 25 ml. volume i n a 25 ml. volumetric f l a s k w i t h d i s t i l l e d water. Into a narrow bore b o i l i n g tube were p i p e t t e d 2 ml. of the d i l u t e d t e s t s o l u t i o n . Two, 2 ml. p o r t i o n s of the standard sugar solution*"* ( c o n t a i n i n g 50 and 100 micrograms per ml.) and 2 ml. of d i s t i l l e d water to serve as a blank were a l s o set up. Added to each tube was 2 ml. of Copper Phosphate-T a r t r a t e , reducing agent. 1 •>:- Merck reagent grade dextrose. 1 6 The s o l u t i o n s were mixed by shaking and then heated i n a b o i l i n g water bath f o r 1 0 minutes f o r r e d u c t i o n of the copper. The tubes were then cooled and 2 ml. of arsenomoly-bdate colour reagent added to each tube. Each mixture was then d i l u t e d up to a 1 0 ml. volume w i t h d i s t i l l e d water. A l i q u o t s were taken from each tube and read i n the Beckman B. Spectrophotometer at $20m^L . The spectrophotometer was adjusted so as t o read 1 0 0 $ t r a n s m i s s i o n through the blank. E. Determination of P r o t e i n Nitrogen. Fol l o w i n g the method o u t l i n e d by K j e l d a h l ( 3 7 ) f o r assay of p r o t e i n n i t r o g e n , 1 ml. of the d i a l y z e d t i s s u e ex-t r a c t was p i p e t t e d i n t o a d i g e s t i o n tube. I n the same manner 1 ml. of urea standard (0.5 mg. N/ml.), and 1 ml. of d i s t i l l e d water to serve as a standard and a blank were a l s o set up. To each d i g e s t i o n tube was added 1 ml. of concentrated s u l p h u r i c a c i d together w i t h a few g r a i n s of m e t a l l i c selenium. The d i g e s t i o n tubes were placed on the K j e l d a h l rack and heated .• over a low flame (approximately twenty-five minutes) u n t i l a l l the n i t r o g e n had been converted to ammonia. The chamber of the d i s t i l l a t i o n apparatus (micro K j e l d a h l ) was c l e a r e d of any contamination by a blank d i s t i l -l a t i o n . The d i g e s t e d sample xriLth r i n s i n g s was t r a n s f e r r e d to the chamber of the d i g e s t i n g apparatus. 17 Several Erlenmeyer f l a s k s were set up, each c o n t a i n i n g 15 ml. of 2% b o r i c a c i d , which contained broitiocresol green I n d i c a t o r . One of these Erlenmeyer f l a s k s was p l a c e d under the condenser o u t l e t so that the t i p of the o u t l e t dipped below the surface of the b o r i c a c i d s o l u t i o n . Concentrated NaOH s o l u t i o n was .added to the d i g e s t i n the chamber i n an amount i n excess of th a t needed to n e u t r a l i z e the amount of a c i d present. The sample was steam d i s t i l l e d u n t i l 10 ml. of the d i s t i l l a t e had been c o l l e c t e d i n the r e -c e i v i n g f l a s k . The sides of the condenser o u t l e t were r i n s e d w i t h d i s t i l l e d water i n t o the r e c e i v i n g f l a s k and the f l a s k removed. The ammonia present i n the b o r i c a c i d s o l u t i o n was then determined by t i t r a t i o n against 0.01i|-3 N. s u l p h u r i c a c i d . T i t r a t i o n of a d i g e s t e d and d i s t i l l e d standard (0.5 mg. N/ ml.) and blank were used as a b a s i s to c o r r e c t f o r ammonia present i n the reagents used. P. Method of C e l l F r a c t i o n a t i o n . In order to compute the d i l u t i o n f a c t o r s necessary to c o r r e l a t e the a c t i v i t y of the vrhole homogenate w i t h that of the sum of the a c t i v i t y of the f r a c t i o n s , a c a r e f u l record was kept of the weight of t i s s u e used, and of the volumes of s a l i n e added during the experiment. The method used f o r c e l l f r a c t i o n a t i o n was a modif-i c a t i o n of that o u t l i n e d by Schneider (38), 18 F o l l o w i n g a twenty-four hour f a s t , the experimental animal was anaesthized x-jith ether and the d o r s a l a o r t a severed. The t i s s u e to be assayed was q u i c k l y removed, weighed and placed i n a c h i l l e d s t e r i l i z e d Potter-Elvenhjem tube. Homogenizing was c a r r i e d out i n the c o l d w i t h c h i l l e d s a l i n e being added to give a f i n a l homogenate volume of II4- ml. The homogenate was then d i v i d e d i n t o two equal p o r t i o n s of 7 ml. each. One p o r t i o n , used as a measurement of the t o t a l phos-phorylase a c t i v i t y I n the whole c a l l was assayed f o r phosphor-y l a s e a c t i v i t y e x a c t l y as o u t l i n e d on page 11. The second p o r t i o n , was p i p e t t e d i n t o i c e c o l d c e n t r i -fuge tubes, and c e n t r i f u g e d at 20,5>00 xg f o r twenty minutes. The supernatant c o n t a i n i n g cytoplasm and s a l i n e was c a r e f u l l y p i p e t t e d i n t o a small graduate. The p a r t i c u l a t e f r a c t i o n con-s i s t i n g of connective t i s s u e , mitochondria, n u c l e i and some unbroken c e l l s was a l s o placed i n a small graduate. Microscopic examinations of both the cytoplasmic and p a r t i c u l a t e f r a c t i o n s i^ere made, the contents of each f r a c t i o n being noted. Both the cytoplasmic and the p a r t i c u l a t e f r a c t i o n s were each d i l u t e d up to a volume of 7 m. w i t h s a l i n e . S t a r t i n g w i t h the adjustment of the f r a c t i o n s t o pH 6.1 the procedure f o l l o w e d to determine the phosphorylase a c t i v i t y i n each f r a c t i o n was c a r r i e d out as o u t l i n e d on page 11. 19 RESULTS o A* Phosphorylase A c t i v i t y i n Tissues From Twenty-Pour  Hour Fasted Normal and Tumor Bearing Rats and Mice. Comparative assay of phosphorylase a c t i v i t y f o r nor-mal l i v e r , tumor-bearing l i v e r and tumor from 2lj.-ho'ur f a s t e d w i s t a r r a t s are presented i n Table I, ( P i g . I ) . The r e s u l t s i n d i c a t e , that i n the 2l4.-h.our f a s t e d wis-t a r r a t s the phosphorylase a a c t i v i t y * of the tumor-bearing l i v e r e x t r a c t showed only a s l i g h t r e d u c t i o n compared to the phosphorylase a a c t i v i t y of the normal l i v e r e x t r a c t . On the other hand the phosphorylase a a c t i v i t y of the tumor gave a s i g n i f i c a n t l y lower value when compared to e i t h e r t h a t of the normal or that of the tumor-bearing l i v e r . T o t a l phosphorylase a c t i v i t y of a l l three e x t r a c t s w i t h added ad e n y l i c a c i d , the normal l i v e r , tumor-bearing l i v e r and tumor f e l l w i t h i n the same range of a c t i v i t y . The r e s u l t s i n d i c a t e that the phosphorylase present i n the tumor was mostly i n the i n e r t form. In the present s t u d i e s phosphorylase was assayed w i t h and without a d d i t i o n of ad e n y l i c a c i d , the r a t i o of a c t i v i t y without added ad e n y l i c a c i d d i v i d e d by a c t i v i t y w i t h added ad-e n y l i c a c i d m u l t i p l i e d by one hundred (^AIAI^ 1 0 0) s e r v i n g as an index of the r e l a t i v e p roportions of the a c t i v e form of the enzyme present i n the t i s s u e s examined. -* Phosphorylase a c t i v i t y without added a d e n y l i c a c i d . TABLE I COMPARATIVE ASSAY OF PHOSPHORYLASE ACTIVITY IN NORMAL LIVER, TUMOR BEARING LIVER AND TUMOR, IN TWENTY-FOUR HOUR FASTED NORMAL AND HEPATOMA BEARING WISTAR RATS. Exper-iment NORMAL LIVER TUMOR BEARING LIVER TUMOR No. -A.A. *A.A. jAr^'xlOO -A.A. +A.A. 7A*A'xlOO A. • A. • +A.A. - A . A . T l 0 0 •A.A. '•A. A. •A.A. 1. 0.112 0.312 35.9 0.034 0.266 12.8 0.007 0.143 4.9 2. 0.112 0.207 54.1 0.076 0.188 40.4 0.000 0.343 0.0 3. 0.178 0.466 38.1 0.160 0.550 29.1 0.035 0.165 21.2 4. 0.118 0.215 54.8 0.088 0.180 48.8 0.000 0.417 0.0 5. 0.157 0.390 40.2 0.175 0.450 38.9 0.023 0.392 5.9 6. 0.219 0.514 42.6 0.190 0.480 39.6 0.000 0.448 0.0 7. 0.157 0.289 54.3 • 0.143 0.268 53.4 0.000 0.216 0.0 8. 0.114 0.229 49.7 0.108 0.259 41.7 0.000 0.381 0.0 . 9. 0.105 0.245 42.9 0.115 0.327 35.1 0.010 0.173 5.7 10. 0.177 0.400 44.2 0.191 0.491 38.9 0.000 0.267 0.0 Mean 0.145 0.327 45.68 0.128 0*346 34.96 0.007 0.295 3.77 i o.'oss ±0.091 + 6.884 10.049 t 0.150 110.863 10.012 10.104 13.435 A Phosphorylase a c t i v i t y expressed i n terms of mg. P. lib e r a t e d per mg. N. tissue extract during 30 minutes incubation and agitation at 30° G. AA - A.A. - Phosphorylase a c t i v i t y i n absence of added adenylic acid. &&& +A.A. - Phosphorylase a c t i v i t y i n presence of added adenylic acid. O- Standard Error of the mean. FIGURE I. PHOSPHORYLASE ACTIVITY IN TISSUES FROM 24-HOUR FASTED NORMAL AND TUMOR-BEARING WISTAR RATS. .370 O .2 .30C-t? 2 °- .225 -z g a. m JSO J075 NORMAL LIVER ooc. MEAN 1 TB. LIVER -A. A. «A. A. 1 TUMOR -A. A. • A. A. J45 327 J28 346 .007 .295 I036 ±091 ± 0 4 9 ±.ISO ±.OI2 ±.104 50 4 0 3 0 Z • ui 2 0 O a ui Q- I O QQ_ ACTIVITY RATIO ( ^ . x too )% N O R M A L T. a L I V E R T U M O R 45.68 ±6.88 34.96 3.77 ±10.86 ±343 •A.A.-ADENYUC AC ID . Comparing phosphorylase a c t i v i t y r a t i o s , of normal l i v e r , tumor-bearing l i v e r and tumor e x t r a c t s i n Table I and P i g . I , i t was noted that the tumor-bearing l i v e r e x t r a c t phosphorylase a c t i v i t y r a t i o , showed an apparent drop compared to that of the normal l i v e r e x t r a c t . The tumor e x t r a c t phos-phorylase a c t i v i t y r a t i o showed a drop vjhich was s t a t i s t i c a l l y s i g n i f i c a n t when compared t o e i t h e r that of the normal l i v e r or that of the tumor-bearing l i v e r e x t r a c t s . A n a l y s i s of the r e s u l t s i n Table I I ( P i g . I I ) i n d i c a t e , that again the phosphorylase a a c t i v i t y of the tumor-bearing l i v e r extract was only s l i g h t l y lower than that of the normal l i v e r . The phosphorylase a a c t i v i t y of the tumor e x t r a c t was s i g n i f i c a n t l y l e s s than e i t h e r that of tumor-bearing l i v e r or that of normal l i v e r e x t r a c t s . In those r e a c t i o n tubes c o n t a i n i n g a d e n y l i c a c i d , l i t t l e change vras noted i n the t o t a l phosphorylase a c t i v i t y between tumor-bearing l i v e r and normal l i v e r e x t r a c t s . The tumor e x t r a c t phosphorylase a c t i v i t y w i t h added a d e n y l i c a c i d f e l l x^ithin the range of that of normal l i v e r e x t r a c t . . I n the phosphorylase a c t i v i t y r a t i o s , an apparent decrease was noted when tumor-bearing l i v e r was compared to that of normal l i v e r e x t r a c t s ; the tumor phosphorylase a c t i v i t y r a t i o was s i g n i f i c a n t l y lower when compared to e i t h e r that of normal l i v e r or tha t of tumor-bearing l i v e r e x t r a c t s . T A B L E I I COMPARATIVE A S S A Y OF PHOSPHORYLASE A C T I V I T Y I N NORMAL L I V E R , TUMOR B E A R I N G L I V E R , AND TUMOR, I N TWENTY-FOUR HOUR FASTED NORMAL AND MAMMARY CARCINOMA B E A R I N G B.L.CKO M I C E . Exper-iment NORMAL LIVER TUMOR BEARING LIVER TUMOR No. L ~A.A.^.T N A -A.A. +A.A. -A.A.^nn -A.A. •A.A. -A.A. TT nn +A.A. X 1 0° •A.A. •A.A. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 0.282 0.235 0.179 0.208 0.120 0.205 0.123 0.105 0.133 0.055 0.348 0.428 0.239 0.348 0.217 0.432 0.223 0.200 0.283 0.145 81.04 54.90 74.90 59.77 55.29 47.45 55.00 52.50 46.99 37.94 0.317 0.157 0.131 0.200 0.100 0.204 0.117 0.113 0.105 0.039 0.499 0.371 0.196 . 0.515 0.231 0.478 0.224 0.253 0.250 0.139 63.53 42.32 66.84 58.83 43.21 42.68 52.23 44.66 42.00 28.06 0.000 0.018 0.019 0.011 0.007 0.000 0.009 0.007 0.000 0.003 0.235 0.424 0.403 0.311 0.227 0.417 0.191 0.415 0.586 0.173 0.000 4.240 4.710 3.530 3.080 0.000 4.710 1.690 0.000 1.730 Mean 0.165 0.286 56.578 0.148 . 0.316 46.436 0.007 0.318 2.369 10.065 tO.093 +12.042 •0.073 i 0.126 i10.909 10.006 10.097 i1.843 & Phosphorylase a c t i v i t y expressed i n terms of mg. P. l i b e r a t e d per mg. N. t i s s u e e x t r a c t during 30 minutes in c u b a t i o n and a g i t a t i o n at 50° C. M. - A.A. - Phosphorylase a c t i v i t y i n absence of added adenylic a c i d . £&& +A.A. ~ Phosphorylase a c t i v i t y i n presence of added ade n y l i c a c i d . c*w Standard E r r o r of the mean • 1+ O 1+ o ft. OD 1 + '8 i+ — Ol in a> O £ o ft-O *• '+ . .- N co CJ LIBERATION IPCMGJMG.ND O ui O to u O O ui PERCENT — KJ o o (tl o ft. o ui O o O in "D X O cn XI I O 3 > cn m 5 H is S n rn on 5 z OD 73 O I X 5 2 O 73 m H m o O > > z o hcj M s 25 B. Glycogen L e v e l i n Tissues Prom Twenty-four Hour  Fasted Normal and Tumor-Bearing Rats and Mice. Olson (39) and Young (ij.0) reported low l i v e r glycogen l e v e l s i n 2ij.-h.our f a s t e d r a t s b e a r i n g hepatomas and i n 2i(.-hour f a s t e d Sarcoma 180 mice, r e s p e c t i v e l y . I n these s t u d i e s the glycogen l e v e l s i n 2ij.-hour f a s t e d normal and hepatoma-bearing w i s t a r r a t s xfere determined, and a l s o i n normal and mammary carcinoma-bearing CcjyBl mice. The l e v e l s of glycogen, expressed i n grams percent wet v/eight showed a h i g h l y s i g n i f i c a n t drop i n the tumor and an apparent drop i n the l i v e r s of the tumor-bearing r a t s when compared to the glycogen l e v e l i n normal l i v e r Table I I I ( P i g . I I I ) . Comparable data was obtained i n experiments on mice bearing the mammary tumor t r a n s p l a n t s , Table IV, ( P i g . I V ) . There was an apparent decrease i n the glycogen l e v e l of the tumor-bearing l i v e r and the drop i n . t h e glycogen l e v e l of the tumor was s t a t i s t i c a l l y s i g n i f i c a n t when compared to that of the normal l i v e r . G« E f f e c t of Administering Glucose, F o l l o w i n g a Twenty-f o u r Hour Past, on Glycogen Levels i n Tissues Prom  Normal and Tumor-Bearing Rats and Mice. Lundbaek and Goranson (ij.1) reported t h a t refeeding w i t h a h i g h carbohydrate d i e t a f t e r a f a s t lowered the muscle phosphorylase a a c t i v i t y i n the r a t . 26 TABLE I I I PASTING GLYCOGEN VALUES IN NORMAL LIVER WISTAR RATS AND IN THE TUMOR AND LIVER OP WISTAR RATS BEARING HEPATOMA TRANSPLANTS. NORMAL LIYER TUMOR BEARING LIVER TUMOR (grams %) (grams %) (grams %\ .110 ,061j. • .068 .630 .190 .080 .183 .080 .o$k .170 .080 .060 .kSo .170 .110 .290 .180 .100 .300 .130 .073 .166 .096 .036 . l l i f .066 .050 .156 .081 Mean — .257 .HI4. .07© +.159 + .Olj.6 + .022 -^-Standard D e v i a t i o n 0 27 FIGURE I I I EFFECT OF GLUCOSE ADMINISTRATION ON GLYCOGEN DEPOSITION IN 24-HOUR FASTED NORMAL AND TUMOR-BEARING WISTAR RATS 2.SO -iu 2.00 -U J a. 2 z U J (J O u > 1.5 O LOO 5 O.SO O.OO NORMAL LIVER FASTEC? FASTED T. B. LIVER FASTED FASJED GLUCOSE TUMOR FASTED FASTED GLUCOSE MEAN 0.2 6 2.27 . O.I I 0.81 0.07 O.I 6 <T" ± O.I 6 ±0.34 ±0.05 ±0.08 ± 0.02 ± 0.02 •2.S0CC OF 2 0 % GLUCOSE SOLUTION, INTR APERITONEALLY. 28 TABLE IV PASTING GLYCOGEN VALUES IN NORMAL LIVER C ^ B l MICE AND IN THE TUMOR AND LIVER OP C ^ B l MICE BEARING MAMMARY CARCINOMA TRANSPLANTS. NORMAL LIVER TUMOR BEARING LIVER TUMOR . (grams %) (grams %) (grams %) .1+2 . 2 0 . 0 3 0 . 2 5 . 2 7 . 0 6 0 . 2 5 . 2 0 . 001+ . 3 7 . 1 9 . 0 5 0 . 3 6 .21+ . 0 0 5 .5-8 . 15 . 0 0 5 .1+2 . 2 2 .001+ . 3 8 . 18 . 0 5 0 . 3 7 .17 . 0 0 0 .1+1+ . 2 0 . 0 3 8 .21+ . 2 5 . 0 6 1 .1+3 , . 11 .01+7 . 3 7 ' . 17 . 0 0 0 Mean — . 3 7 . 2 0 . 0 2 7 — • .071+ +_ .01+1 + . 0 2 3 * Standard D e v i a t i o n o 29 FIGURE IV. EFFECT OF GLUCOSE ADMINISTRATION ON GLYCOGEN DEPOSITION IN 24-HOUR FASTED NORMAL AND TUMOR— BEARING BLC57M1CE. z L l J u a. U i 0-3.00 2.SO 2.00 NORMAL LIVER T.B. LIVER TUMOR F A S T E D F A S T E D G L U C O S E F A S T E D F A S T E D + G L U C O S E F A S T E D F A S T E D G L U C T J S E I '-so | I.OO o u O. 0.50 O.OO MEAN C7~ 0.37 2.93 O.20 0.9 6 003 0.35 ±0.07 i 0.38 ±0.04 ±O.I8 ±Q02 ±0.07 1 0 . 5 0 C C O F 2 0 V . G L U C O S E S O L U T I O N I N T R A P E R I T O N E A L L T - 30 The e f f e c t of I n t r a p e r i t o n e a l glucose i n j e c t i o n s on phosphorylase a c t i v i t y i n normal l i v e r , tumor-bearing l i v e r and tumor of 2L(.-hour f a s t e d w i s t a r r a t s and C ^ B l mice are compared i n Tables V and VI, and (Figs. V and VI) r e s p e c t i v e l y . Our f i n d i n g s , i n the case of the normal l i v e r , are i n agreement w i t h those of Lundbaek and G-oranson, that i s , there i s a s i g n i f i c a n t drop i n the phosphorylase a a c t i v i t y i n the normal l i v e r upon p a r e n t e r a l a d m i n i s t r a t i o n of carbohydrate. The phosphorylase a a c t i v i t y of the tumor-bearing l i v e r f o l -lowing glucose i n j e c t i o n , however, i n d i c a t e d only an apparent drop from the a c t i v i t y of 2lj.-hour f a s t e d v a l u e s . L i k e w i s e , an apparent decrease was noted i n the phosphorylase a a c t i v i t y of the tumor a f t e r the p a r e n t e r a l i n j e c t i o n of glucose. L i t t l e d i f f e r e n c e i n the a c t i v i t y l e v e l s of t o t a l phosphorylase i n normal l i v e r , tumor-bearing l i v e r or tumor were noted i n the r a t s . However the a c t i v i t y r a t i o of the tumor-bearing l i v e r i n mice was considerably lower than the a c t i v i t y r a t i o of the normal l i v e r . 0 The p a r e n t e r a l i n j e c t i o n of glucose f o l l o w i n g a 2I4.-hour f a s t r e s u l t e d i n a r e d u c t i o n i n the phosphorylase a c t i v i t y r a t i o i n both the normal l i v e r and i n the tumor-bearing l i v e r as compared to the 2l}.-hour f a s t e d v a l u e s . This was s i g n i f -i c a n t l y g r eater i n the normal l i v e r . TABLE V-COMPARATIVE ASSAY OF PHOSPHORYLASE ACTIVITY IN NORMAL. LIVER, TUMOR"BEARING LIVER, AND TUMOR IN TWENTY-FOUR HOUR FASTED NORMAL AND HEPATOMA TRANSPLANTED WISTAR RATS" AFTER GLUCOSE ADMINISTRATION. Exper-iment NORMAL LIVER TUMOR BEARING LIVER TUMOR No. -A.A. +A.A. -A.A. • A . A. •xlOO "A.A. ••A.A. .A. • A • • A . A. •xlOO • A . A . -A.A. ••A.A. •xlOO 1. 0.100 0.930 10.75 0.124 0.567 21.77 0.000 0.650 0.000 2. 0.150 0.732 20.55 0.135 0.510 26.47 0.000 0.773 0.000 u> 3. 0.060 0.279 21.59 0.068 0.259 27.18 0.000 0.248 0.000 H 4. 0.057 0.290 • 23.50 0.062 0.252 23.50 0.000 0.267 0.000 5. 0.075 0.337 22.06 0*112" 0.393 28.72 0.000 0.333 0.000 6. 0.115 0.440 26.13 0.096 0.300 32.00 0.000 0.600 0.000 7. 0.047 0.291 16.15 0.129 0.171 27.39 O.ooo 0.750 0.000 8. 0.098 0.498 19.87 0.140 0.480 29.17 0.000 0.540 0.000 9. 0.228 0.888 25.69 0.275 0.768 35.81 0.000 0.731 0.000 10. 0.104 0.508 20.47 0.379 0.916 41.37 0.000 0.625 0.000 Mean 0.1034 0.5193 20.676 0.1520 0.4616 29.338 0.000 0.5517 0.000 <7v. •0.050 t O .224 J5.262 •0.092 •0.225 ±5.481 10.000 10.172 10.000 Phosphorylase a c t i v i t y expressed i n terms of mg. P. liberated per mg. N. t i s s u e e x t r a c t during 30 minutes i n c u b a t i o n and a g i t a t i o n at 30 C. -A.A. - Phosphorylase a c t i v i t y i n absence of added adenylic a c i d . ' -A.A. - Phosphorylase a c t i v i t y i n presence of added adenylic a c i d . ft Standard E r r o r of the mean EFFECT OF GLUCOSE ADMINISTRATION FOLLOWING A 24—HOUR FAST ON PHOSPHORYLASE ACTIVITY IN TISSUES FROM NORMAL AND TUMOR — BEARING WISTAR RATS. z o u> 5 .625 500 -375 Z o t— < a iii m 250 J25 OOO MEAN NORMAL LIVER - A.A? + A .A . T B. LIVER - A .A . « A . A . TUMOR -A. A. J03 519 J52 462 DOO .552 G*~ ± .050 ±,224 ±J092 ±.22S ± .000 ± J 7 2 * A . A . A D E N Y L I C ACID . 40 30 5 20 IO QO ACTIVITY RATIO x i o o ) % NORMAL L I V E R t a L IVER 20.67 29.34 O.OO ±5.26 -5.48 -O.OO to TABLE VI EFFECT OF ADMINISTERING GLUCOSE, FOLLOWING A • .... £ TWENTY-FOUR FAST, ON PHOSPHORYLASE ACTIVITY IN TISSUES FROM NORMAL AND TUMOR BEARING C 5 7 BL MICE. Exper-iment NORMAL LIVER TUMOR BEARING LIVER TUMOR No. -A.A. •A. A. A. A • •A.A. •xlOO -A.A. •A.A. -A.A. •A.A. •xlOO -A.A. •A.A. -A.A. •A.A. •xlOO 1. 0.120 0.493 24.30 0.123 0.-378 35.97 0.000 0.302 0.000 2. 0.056 0.328 17.28 0.150 0.425 34.89 0.000 0.434 0.000 3. 0.108 0.433 25.12 0.155 0.407 37.80 0.000 0.156 0.000 4. 0.100 0.433 23.26 0.171 0.471 36.39 0.000 0.450 0.000 5. 0.093 0.414 22.46 0.135 0.383 35.53 0.000 0.420 0.000 6. 0.021 0.144 16.60 0.038 0.129 29.20 0.019 0.132 14.620 7. 0.079 0.415 18.81 0.190 0.470 40.43 0.000 0.585 0.000 8. 0.062 0.321 19.37 0.109 0.355 30.28 0.000 0.586 0.000 9. 0.071 0.357 18.69 0.132 0.432 30.69 0.000 0.517 0.000 10. 0.031 0.175 19.05 0.050 0.188 26.58 0.000 0.120 0.000 Mean 0.0741 0.3713 21.094 0.1253 0.3638 33.776 0.0019 0.4062 01.4620 t0.022 +0.139 +2.852 ±0.077 ±0.093 14.171 +0.0417 ±0.147 11.166 & Phosphorylase a c t i v i t y expressed i n terms of mg. P. liberated per mg. N. tissue extract during 30 minutes incubation and agit a t i o n at 30°C. 3t& -A.A. - Phosphorylase a c t i v i t y i n absence of added adenylic acid, ft&ft -A.A. " - Phosphorylase a c t i v i t y i n presence of added adenylic acid. Standard Error of the mean FIGURE 71. EFFECT OF GLUCOSE ADMINISTRATION FOLLOWING A 24-HOUR FAST ON PHOSPHORYLASE ACTIVITY IN TISSUES FROM NORMAL AND TUMOR — BEARING B L C 5 7 MICE. .500. N Q S M A - ' . I V E S Z o o" 5 .400 a .300 z o p.200 < a UJ m jJOO o c a 1 T. 8. LIVER -A.A. +A.A. MEAN 074 371 .12 5 <^  ±022 ±J39 ±077 *A . A . A D E N Y L I C A C I D . TUMOR .363 002 406 :Q93 ±041 ±.14 7 40 30 Z 20 L U U a lO _QO_ ACTIVITY RATIO NORMAL L I V E R T. B. LIVER T'JMOR 21.09 33.77 ±2.8 5 ±4.17 O.IS tl.17 35 D. E f f e c t of A d m i n i s t e r i n g Glucose, F o l l o w i n g A Twenty- fo u r Hour Fas t , on Glycogen Levels i n Tissues from  Normal and Tumor-Bearing Rats and Mice. The d e p o s i t i o n of glycogen i n t i s s u e s from normal and tumor-bearing mice are compared i n Tables V I I , V I I I and (S±@s. **"i=E, VJrSM.). Although a l l t i s s u e s assayed i n d i c a t e d s i g n i f i c a n t increases i n glycogen l e v e l s f o l l o w i n g glucose a d m i n i s t r a t i o n over t h e i r corresponding f a s t e d v a l u e s , the tumor-bearing l i v e r and tumor gave glycogen l e v e l s s i g n i f i c a n t l y lower than i n the normal l i v e r f o l l o w i n g glucose a d m i n i s t r a t i o n . These f i n d i n g s are i n agreement w i t h those reported by Young (ij.2), i n which . mice bearing Sarcoma 180 showed a marked impairment i n t h e i r a b i l i t y to deposit glycogen f o l l o w i n g glucose a d m i n i s t r a t i o n . E. E f f e c t of A d e n y l i c , T J r i d y l i c , Cy t i d y l i e and Guanylic  a c i d s , i n vitro,, on Phosphorylase a c t i v i t y i n Tissues  from Normal and Tumor-Bearing Rats and Mice. The r e s u l t s on u r i d y l i c , c y t i d y l i c and gu a n y l i c a c i d s , Table IX, on the phosphorylase a c t i v i t y i n normal l i v e r , tumor-bearing l i v e r and tumor from both r a t s and mice i n d i c a t e , that although these acids belong t o the purines and the p y r i m i d i n e s , of which a d e n y l i c a c i d i s a member, they have no a c t i v a t i n g or depressing e f f e c t on phosphorylase a c t i v i t y when added i n the same molar concentrations as ade n y l i c a c i d . 36 TABLE VII THE EFFECT OF ADMINISTERING GLUCOSE ON GLYCOGEN LEVELS; IN NORMAL LIVER, TUMOR BEARING LIVER, AND TUMOR IN NORMAL AND TUMOR-BEARING TWENTY-FOUR HOUR FASTED WISTAR RATS. NORMAL LIVER (grams %) 2.63 2.1+3 1.98 2.51+ 1.95 2.62 1.75 2.70 1.83 2.2k Mean -- 2.27 TUMOR BEARING LIVER (grams %) o.ik 0.92 0.77 0.68 0.83 0.90 0.92 0.82 0.81+ 0.66 0.81 • .079 TUMOR (grams fo) 0.12 0.26 0.18 0.19 0.15 0.16 0.13 0.12 0.11 0.13 0.16 * .028 / -^-Standard D e v i a t i o n -37 TABLE V I I I THE EFFECT OF ADMINISTERING GLUCOSE ON GLYCOGEN LEVELS; IN NORMAL LIVER, TUMOR-BEARING LIVER, AND TUMOR IN NORMAL AND TUMOR-BEARING TWENTY-FOUR HOUR FASTED C ^ B l MICE. 5 7 NORMAL LIVER TUMOR BEARING LIVER TUMOR, (grams %) (grams %) (grams fo) 2.32 1.03 0.28 2.71 0.96 0.27 2.66 0.99 0.25 2.95 0.62 0.1+3 2.51 0.71 0.1+8 2.80 1.01 0.37 3.58 1.18 0.35 3.02 0.76 0.38 3.01 1.09 0.33 3.75 1.21 0.31+ Mean — 2.93 0.95 0.31+ *CV — + .38 • .18 * .07 s-Standard Deviation •38 TABLE IX EFFECT OF ADENYLIC, URIDYLIC, CYTIDYLIC AND GUANYLIC ACIDS AS PHOSPHORYLASE ACTIVATORS IN NORMAL LIVER, TUMOR BEARING LIVER AND TUMOR; IN NORMAL AND MAMMARY CARCINOMA BEARING C57BL MICE, IN NORMAL AND HEPATOMA BEARING WISTAR RATS. Experiment NORMAL LIVER Animal c" 5. +A.A. +U.A. + C.A. +G.A. RAT o . i 5 o 0 . 2 7 6 0 . 1 3 3 0 . 9 3 0 . 1 0 0 RAT O.ll f 6 0 . 2 8 1 0 . 1 5 1 0 . 0 9 7 0 . 1 0 8 MOUSE 0.0< S8 0 . 1 9 5 0 . 0 5 5 0 . 0 5 3 0 . 0 6 6 MOUSE 0 . 0 6 0 0 . 1 7 2 0.01+8 0.01+7 .0.01+7 Experiment TUMOR-BEARING LIVER Animal C" +A.A. +U.A. +C.A. +G.A. RAT 0.116 0 . 2 7 9 0 .088 0 .088 0.100 MOUSE 0.031+ 0.11+8 0.026 o.ol+o 0.03l| MOUSE 0 . 0 3 8 0.11+0 0 . 0 3 1 0.03l|. 0.029 Experiment TUMOR Animal C* +A.A. -fU.A. +C.A. +G.A. RAT 0 . 0 0 0 0 . 2 0 5 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 MOUSE 0 . 0 0 0 0 . 1 8 7 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 MOUSE 0 . 0 0 0 0 . 1 6 7 0 . 0 0 3 0 . 0 0 0 0 . 0 0 6 C - - C o n t r o l , phosphorylase a c t i v i t y without added a d e n y l i c a c i d . +A.A.— -- •—phosphorylase a c t i v i t y w i t h added a d e n y l i c a c i d . +U.A.— -- --phosphorylase a c t i v i t y w i t h added u r i d y l i c a c i d . +C.A.-- — --phosphorylase a c t i v i t y w i t h added c y t i d y l i c a c i d . +G.A.-- -- --phosphorylase a c t i v i t y w i t h added gu a n y l i c a c i d . i.39 P. Phosphorylase A c t i v i t y i n C e l l P r a c t i o n s of  Normal and Tumorous Tissues. Phosphorylase a c t i v i t y i n the two c e l l f r a c t i o n s , cytoplasmic and p a r t i c u l a t e , as compared i n Table X do not i n d i c a t e any s i g n i f i c a n t s h i f t i n phosphorylase a c t i v i t y from one f r a c t i o n to the other i n tumor-bearing l i v e r or tumor as compared to normal l i v e r , i n e i t h e r r a t s or mice. The pres-ence of phosphorylase a c t i v i t y i n the p a r t i c u l a t e f r a c t i o n and the greater phosphorylase a c t i v i t y i n the sums of the f r a c t i o n s as compared to the whole homogenate are considered i n the D i s c u s s i o n . 40 TABLE X DISTRIBUTION OP PHOSPHORYLASE ACTIVITY 1 FOLLOWING CELL-ULAR FRACTIONATION IN TISSUES FROM TWENTY-FOUR HOUR PASTED NORMAL AND TUMOR BEARING WISTAR RATS AND C^7 BL MICE. Experimental F r a c t i o n NORMAL LIVER Animal -A. A.""""* • A . A . * * * * RAT # 1 Whole""" Cytoplasm P a r t i c u l a t e 0.352 0.850 0.198 0.912+ 2.130 o.65o RAT # 2 Whole Cytoplasm P a r t i c u l a t e O.lj.28 0.k30 0.8624. 0.902 1.126 < 1.982 Experimental F r a c t i o n TUMOR BEARING LIVER Animal -A.A. -MOUSE # 1 Whole Cytoplasm P a r t i c u l a t e 3.857 0.065 0.0567 9.159 0.131 1.1+513 MOUSE # 2 Whole Cytoplasm P a r t i c u l a t e 0.328 0.166 0.097 1.038 0.22+9 0.115 Experimental Animal F r a c t i o n TUMOR -A.A. +A.A. RAT # 1 Whole Cytoplasm P a r t i c u l a t e 0.162 0.000 0.196 1.131 0.383 1.570 MOUSE # 1 x Whole 0.072+ 0.2+35 Cytoplasm 0.158 0.2+38 P a r t i c u l a t e 0.018 0.012 1 Phosphorylase a c t i v i t y expressed as mg. P. l i b e r a t e d per gram wet weight t i s s u e . Phosphorylase a c t i v i t y i n whole homogenate. -"--x- Phosphorylase a c t i v i t y without added a d e n y l i c a c i d . Writ Phosphorylase a c t i v i t y w i t h added a d e n y l i c a c i d . 41 DISCUSSION AND SUMMARY I t has been shown that the phosphorylase a a c t i v i t y as measured by the i n o r g a n i c phosphate l i b e r a t e d from glucose-1-phosphate i s reduced i n the l i v e r of tumor-bearing animals. In the tumors assayed the phosphorylase was l a r g e l y of the i n e r t form. There e x i s t s two p o s s i b l e explanations f o r t h i s de-creased a c t i v i t y . F i r s t , i n the a v a i l a b i l i t y of a d e n y l i c a c i d , the a c t i v a t i n g group or p r o s t h e t i c group of phosphorylase, which may be present i n tumor-bearing l i v e r s and tumors but not a v a i l a b l e to the enzyme due to i t s d i v e r s i o n i n t o other s y n t h e t i c processes such as n u c l e o - p r o t e i n . s y n t h e s i s . This p o s s i b i l i t y i s I n accord w i t h the recent work of Le Fage, Tyner and Heidleberger (i+3) who have reported evidence of r a p i d i n c o r p o r a t i o n of preformed n u c l e o t i d e s i n t o r i b o n u c l e i c a c i d molecules i n the tumor c e l l . Secondly, the PR enzyme which Is r e s p o n s i b l e f o r the conversion of phosphorylase a to phosphorylase b may be d e f i c -i e n t o r l a c k i n g a l t o g e t h e r . Le Page and co-workers (Ijlf.) have reported t h a t during a f i v e - d a y f a s t , r a t s bearing the Flexner-J o b l i n g tumor, l o s t 31$ of t h e i r body weight and 39$ of t h e i r l i v e r p r o t e i n . The tumors of these f a s t e d animals grew almost as f a s t as the tumors i n the fed animals. I t was concluded that the tumor was able to m a i n t a i n a l l i t s requirements from 42 the blood and t o grow r a p i d l y when the host i s f a s t e d and for c e d to ma i n t a i n the blood c o n s t i t u e n t s by catabolism of normal t i s s u e s . Therefore, i t i s . conceivable t h a t the PR enzyme, one of the more l a b i l e enzymes could be depleted or broken down to a considerable extent during the 2l+-hour f a s t preceding the phosphorylase determination. I n comparing the glycogen d e p o s i t i o n values obtained i n 2i+-hour f a s t e d r a t s and mice f o l l o w i n g the p a r e n t e r a l ad-m i n i s t r a t i o n of glucose, a marked impairment was noted i n the a b i l i t y of both the tumors and the tumor-bearing l i v e r s to deposit glycogen. As p r e v i o u s l y mentioned, these f i n d i n g s are i n agreement w i t h those of Young, who reported a.marked im-pairment' of the tumor-bearing l i v e r and tumor i n the a b i l i t y of mice bear i n g Sarcoma 180, to deposit glycogen f o l l o w i n g the i n t r a p e r i t o n e a l i n j e c t i o n of 100 mg. of glucose. Since phosphorylase i s i n t i m a t e l y t i e d up i n the synthesis of glycogen, and si n c e i t s a c t i v i t y was low i n those tumor-bearing animals t e s t e d , i t i s not s u r p r i s i n g t h a t we re c e i v e d , i n these same animals, low glycogen l e v e l s f o l l o w i n g glucose a d m i n i s t r a t i o n . This marked Impairment i n the glycogen d e p o s i t i o n a b i l i t y of tumor.^bearing animals may, however, be due to an a l t e r a t i o n i n the a c t i v i t y or the content of other enzymes as w e l l as phosphorylase concerned w i t h glycogen formation (Ij.6). .43 A l t h o u g h t h e s i g n i f i c a n t d r o p i n t h e a c t i v i t y r a t i o s o f t h e r e f e d n o r m a l l i v e r a s c o m p a r e d t o t h e f a s t e d v a l u e s o f t h e n o r m a l l i v e r a r e i n a c c o r d a n c e w i t h t h e f i n d i n g s o f L u n -b a e k a n d G o r a n s o n , t h e s l i g h t d r o p i n t h e a c t i v i t y r a t i o s o f t h e t u m o r - b e a r i n g l i v e r s a n d t u m o r s f o l l o w i n g p a r e n t e r a l g l u -c o s e a d m i n i s t r a t i o n i s d i f f i c u l t t o e x p l a i n . I n t h e n o r m a l l i v e r t h e r e i s a s i g n i f i c a n t d e c r e a s e i n t h e p h o s p h o r y l a s e a a c t i v i t y f o l l o w i n g g l u c o s e a d m i n i s t r a t i o n a n d o n l y a s l i g h t d r o p i n t h e p h o s p h o r y l a s e a a c t i v i t y o f t h e t u m o r - b e a r i n g l i v e r a n d tumor u n d e r t h e same i d e n t i c a l c o n d i t i o n s . T h e p i c t u r e i n t h e l i v e r o f t h e t u m o r - b e a r i n g a n i m a l t h u s r e s e m b l e s t h a t o f t h e s t a r v e d a n i m a l : a R e l a t i v e l y l o w g l y c o g e n l e v e l a n d a r e l a t i v e l y h i g h p h o s p h o r y l a s e a l e v e l . T h e o n l y r e a s o n a b l e a n s w e r i s t h a t m e n t i o n e d i n t h e b e g i n n i n g o f t h i s d i s c u s s i o n , t h a t i s , a b r e a k d o w n o r i n a c t -i v a t i o n o f t h e P R e n z y m e d u e t o some u n k n o w n n e o p l a s t i c c h a n g e i n t h e g e n e r a l m e t a b o l i s m o f t h e a n i m a l . T h e e x p e r i m e n t c a r r i e d o u t t o s e e i f p u r i n e s a n d p y r i m i d i n e s o t h e r t h a n a d e n y l i c a c i d w o u l d h a v e a n y a c t i v a t i n g e f f e c t o n p h o s p h o r y l a s e , d i d n o t i n d i c a t e e i t h e r a n a c t i v a t i n g o r d e p r e s s i n g e f f e c t o n p h o s p h o r y l a s e . T h e e x p e r i m e n t s o n t h e d e t e r m i n a t i o n o f p h o s p h o r y l a s e a c t i v i t y i n c e l l f r a c t i o n s o f n o r m a l l i v e r , t u m o r - b e a r i n g l i v e r , a n d t u m o r f r o m 2lf . - h o u r f a s t e d r a t s a n d m i c e w e r e c a r r i e d o u t '44 i n an attempt to determine whether any s h i f t occurred i n the l o c a t i o n of the enzyme phosphorylase or a c t i v a t i n g , substance i n the c e l l due to n e o p l a s t i c changes. These experiments represent only the i n i t i a l stages i n t h i s problem. Por these p r e l i m i n a r y experiments crude f r a c t i o n a t i o n s were c a r r i e d out seper a t i n g only the cytoplasmic and s o l i d p a r t i c u l a t e components of the c e l l . Thus and Hers (I4.7) (I[.8) have reported that phosphor-y l a s e i s centered mainly i n the microsomes on the cytoplasm. I t may be s t a t e d , t h a t otir p r e l i m i n a r y r e s u l t s i n d i c a t e t h a t , the phosphorylase d i d seem t o be centered i n the cytoplasmic f r a c t i o n i n a l l the t i s s u e s s t u d i e d . Some of the phosphorylase a c t i v i t y i n the p a r t i c u l a t e f r a c t i o n may be due to whole c e l l s which excaped rupture during homogenization. The higher a c t i v i t y i n the phosphorylase of the sum of the f r a c t i o n s of the c e l l i n comparison to the whole homo-genate may be due to r e l a t i v e l y h i g h temperatures (up to 5>0°C. ) during c e n t r i f u g a t i o n • of the f r a c t i o n s . C o r i (I4.9) has r e -ported a marked increase i n phosphorylase a c t i v i t y at temper-atures i n the range of k$°G, 145 SUMMARY 1 . The data showed a marked r e d u c t i o n i n phosphorylase a c t i v i t y i n the l i v e r s of both r a t s and mice bearing-tumors f o l l o w i n g a twenty-four f a s t . 2. The phosphorylase present i n the hepatoma and i n the mammary carcinoma was found to be almost e n t i r e l y i n the i n e r t form. 3 . The p a r e n t e r a l i n j e c t i o n of glucose i n t o both species of animals r e s u l t e d i n a r e d u c t i o n i n phosphorylase a c t i v i t y i n the normal l i v e r and i n the l i v e r of the tumor-bearing animal. This was s i g n i f i c a n t l y g r e ater i n the normal l i v e r . Ij.. The glycogen l e v e l s i n both tumors of the f a s t e d animals were very low i n comparison to l i v e r and showed l i t t l e change f o l l o w i n g the i n t r a p e r i t o n e a l i n j e c t i o n of glucose. 5. I t was a l s o found that the l i v e r s of tumor-bearing animals showed a d e f i c i e n c y i n the synthesis of glycogen f o l l o w i n g glucose a d m i n i s t r a t i o n . 6 . C y t i d y l i c , u r i d y l i c and Guanylic acids d i d not show any a c t i v a t i n g or depressing e f f e c t on phosphorylase a c t i v i t y i n normal l i v e r , tumor-bearing l i v e r or tumor from normal and tumor-bearing w i s t a r r a t s and C^ 7 B 1 mice. 7. Phosphorylase a c t i v i t y was found both i n the cytoplasmic and i n the p a r t i c u l a t e f r a c t i o n s ; of normal l i v e r , of tumor-bearing l i v e r and tumor, i n normal and tumor-bearing w i s t a r r a t s and mice. 4& BIBLIOGRAPHY Warburg, 0., Metabolism of Tumors, London: Constable & Co., 1930. Zamecnik, P.C.; L o f t f i e l d , R.B.; Stephenson, M.L.; and S t e e l e , J.M., Cancer Research, 11, 592, 195>1« Greenstein, J.P., Biochemistry of Cancer, Academic Press, Inc. Hew York, N. Y., pp. 389, 191+7. Pones, W.S.; and White, J . , J. N a t l . Cancer I n s t . , 12> 1+23, 1951. Weinhouse, S., Cancer Research, 11, 585, 195l. P o t t e r , Van. R., Cancer Research, 11, 565, 195l. 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(22) Olson, P., Cancer Research, 11, 582, 1951. (23) ' Zamecnik, P.C.; L o f t f i e l d , R.B.; Stephenson, M.L.; and Steele, J.M., Cancer Research, 11, 599, 195l. (2IL) , Orr, J.W.; Price, L.W.; and Stickland, L.H., Biochem. J . , 35, IL79» 19*4.1. (25) Young, N.F.; Kensler, C.J.; Seki, L..; and Hamburger, P., Proc. Soc. E x p t l . B i o l . Med., 66, 323, 19*4-7. (26) Green, A.A. and Cori, G.T,, J . B i o l . Cheml, l£l, 23, 19*4-3. (27) Cori, G.T.; and Green, A.A., J . B i o l . Chem., 151, 31, 19*4-3. (28) Cori, G.T.; and Green, A.A., J . B i o l . Chem., l g l , 38, 19*4-3. (29) Cori, G.T.; and Cori, CP., J . Biol.. Chem., 158, 331, 19*4-5. (30) Cori, G.T.; and Green, A.A., J . B i o l . Chem., l | l , 37, 19*4-3. (3D Dunn, T.B. Mammary Tumors i n Mice, The Science Press P r i n t i n g Co., Lancaster, Pennsylvania., pp. 13, 1914-5. .48 (32) Green, A.A., and C o r i , G.T., J . B i o l . Ghem., 151, 21, 191+3. (33) Green, A.A., and C o r i , G.T., J. B i o l . 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A.; P o t t e r , V.R.; Busch, H.; Heidelberger, C ; and H u r l b e r t , R.B., Cancer Research, 12, 155, 1952. (1+5) C o r i , G.T.; and C o r i , C.P., J. B i o l . Chem., l£8, 321, 191+5. (1+6) Ochoa, S.; and St e r n , J.P., Ann. Rev. Biochem., 21, 51+9, 1952. (1+7) DeDuve, C ; Hers, H.G.; and Berthet, L.; Ind. Chim. beige, 17_, 11+3, 1952. (1+8) Hers, H.G..; Berthet, J . ; Berthet, L.; and DeDuve, ^ B u l l . Soc. Chim. B i o l . , 21, 1951. (1+9) C o r i , C.P.j C o r i , G.T.; and Green, A.A. J. Biol.. Chem., l 5 l , 1+6, 191+3. 

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