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An inhibitor of ornithine decarboxylase in the thymus and spleen of dexamethasone-treated rats Bishop, Paul Burton 1984

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AN INHIBITOR OF ORNITHINE DECARBOXYLASE IN THE THYMUS AND SPLEEN OF DEXAMETHASONE-TREATED RATS by PAUL BURTON BISHOP B-.Sc, THE UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, B.C., 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming _io, the required standard THE UNIVERSITY OF BRITISH COLUMBIA . JULY 1984 c ) PAUL BURTON BISHOP, 1984 i n I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Biochemistry  T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 August 7, 1984 i i ABSTRACT The a c t i v i t y of ornithine decarboxylase, the rate-determining enzyme i n polyamine biosynthesis, decreases markedly i n rat thymus and spleen soon a f t e r the i n j e c t i o n of rats with dexamethasone. This study shows that as early as 2 1/2 hours a f t e r hormone treatment, when enzyme a c t i v i t y i s very low, an i n h i b i t o r of ODC i s present. I n h i b i t i o n of ODC can be r e a d i l y detected at 5 and 12 hours, but not at 24 hours a f t e r g l u c o c o r t i c o i d i n j e c t i o n . The i n h i b i t o r appears to be a protein since i t s a c t i v i t y was destroyed by heat or t r y p s i n . I t retains i t s a c t i v i t y a f t e r d i a l y s i s which d i f f e r e n t i a t e s i t from other ODC i n h i b i t o r s that require small molecular weight substances for a c t i v i t y . The apparent molecular weight of a p a r t i a l l y p u r i f i e d extract was 54,000, which d i f f e r e n t i a t e s t h i s i n h i b i t o r from antizyme, an i n h i b i t o r of ODC found i n several other c e l l types. The i n h i b i t o r appears to act by a non-competitive and non-catalytic mechanism since the amount of i n h i b i t i o n does not change with time and i t s i n t e r a c t i o n with ODC does not a f f e c t the enzyme's a f f i n i t y for or n i t h i n e . The formation of t h i s i n h i b i t o r i s an early event i n lymphoid tissues i n response to dexamethasone and may be important i n causing the i n h i b i t i o n of c e l l d i v i s i o n which precedes the destruction of lymphocytes. i i i TABLE OF CONTENTS Page Abstract L i s t of Tables i v L i s t of Figures v L i s t of Abbreviations v i Acknowledgements v i 1 Introduction 1. General 1-Polyamine Biosynthesis 1. The Regulation of ODC A c t i v i t y 4. Polyamine Metabolism i n Thymus and Other Lymphatic Tissue . . . 6. The E f f e c t of Glucocorticoids on Polyamine Metabolism i n Lymphatic Tissue 8. The Present Investigation 9. Materials and Methods 11. Results 19. Conditions f o r Measurement of ODC A c t i v i t y 19. The Occurrence of an I n h i b i t o r of ODC i n Lymphatic Tissue from Dexamethasone-Treated Rats 25. Characterization of ODC I n h i b i t i n g Substance i n Thymus of Dexamethasone-Treated Rats 35. P u r i f i c a t i o n of ODC I n h i b i t o r 37. The Role of Pyridoxal Phosphate i n Thymus ODC I n h i b i t i o n . . . . 45. Discussion 48. Bibliography 52. i v LIST OF TABLES TABLE Page 1. ODC A c t i v i t y i n Thymus Extracts Prepared Using D i f f e r e n t Buffers 20. 2. ODC A c t i v i t y i n Thymus Extracts of Control Rats Determined Using D i f f e r e n t D i t h i o t h r e i t o l Concentrations 21. 3. I n h i b i t i o n of ODC A c t i v i t y by Aqueous Extracts of Thymus and Spleen from Dexamethasone-Treated Rats 26. 4. Occurrence of an I n h i b i t o r of Thymus and Spleen ODC i n Lubrol Extracted Lymphoid Tissue from Dexamethasone-Treated Rats . 28. 5. I n h i b i t i o n of ODC i n Thymus and Spleen of Dexamethasone-Treated Rats as a Function of Incubation Time 31. 6. A c t i v i t y of Thymus ODC I n h i b i t o r at D i f f e r e n t Substrate Concentrations 32. 7. A c t i v i t y of ODC and ODC I n h i b i t o r i n Thymus Extracts Prepared from Rats at D i f f e r e n t Times a f t e r Dexamethasone Treatment . . . 34. 8. E f f e c t s of Varying the Ratio of I n h i b i t o r to Enzyme on ODC A c t i v i t y 36. 9. E f f e c t of Small Molecular Weight Substances on Thymus ODC In h i b i t o r y A c t i v i t y . . . . . . . . . . . 39. 10. P u r i f i c a t i o n of Thymus ODC I n h i b i t o r using DEAE Sepharose CL-6B Column Chromatography 42. 11. P u r i f i c a t i o n of Thymus ODC I n h i b i t o r using Heparin Sepharose CL-6B Column Chromatography 44. LIST OF FIGURES FIGURE Page 1. L-Ornithine K i n e t i c s of ODC i n Thymus of Control Rats 22. 2. E f f e c t of Incubation Time on Thymus ODC A c t i v i t y 23. 3. E f f e c t of Protein Concentration on Thymus ODC A c t i v i t y 24. 4. The E f f e c t of Lubrol on ODC A c t i v i t y i n Rat Thymus and Spleen . . 29. 5. L-Ornithine K i n e t i c s of Thymus ODC i n a Mixing Assay 33. 6. E f f e c t of Trypsin on A c t i v i t y of Thymus ODC In h i b i t o r 38. 7. E f f e c t of Sodium Chloride on Thymus ODC A c t i v i t y 41. 8. Molecular Weight Determination of Thymus ODC Inh i b i t o r . . . . . 46. v i LIST OF ABBREVIATIONS A280 Absorbance measured at 280 nm. Hepes N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid Hepps N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid Km Michaelis-Menten constant mCi m i l l i C u r i e mM mi l l i m o l a r Mops Morpholinopropanesulfonic acid Mr Molecular weight nmole nanomole ODC ornithine decarboxylase ODIF ornithine decarboxylase i n a c t i v a t i n g factor pmole picomole rpm revolutions per minute SAMD S-adenosyl-L-methionine decarboxylase SDS Sodium dodecyl s u l f a t e UM micromolar PLP pyridoxal phosphate BSA Bovine Serum Albumin v i i ACKNOWLEDGEMENTS The work i n t h i s thesis was performed under the supervision of Dr. J.F. Richards. His valuable assistance and advice were very much appreciated. I would also l i k e to thank Drs. D. Vance and M. Mauk for thei r suggestions and Mr. Ted Peng f o r h i s te c h n i c a l assistance. The f i n a n c i a l support of the Canadian Memorial Chi r o p r a c t i c College i s g r a t e f u l l y acknowledged. 1. INTRODUCTION General Polyamines are natural constituents of most l i v i n g organisms, and c e l l s which are incapable of synthesizing them exhibit marked defects i n protein, l i p i d and carbohydrate metabolism, as well as i n polynucleotide synthesis and function (1). The i n t e r c e l l u l a r concentrations of these compounds i s known to a l t e r before a c e l l d i v i d e s , d i f f e r e n t i a t e s or changes i t s metabolic function (2), and the a c t i v i t y of one or more of the polyamine biosynthetic enzymes has been shown to increase markedly i n a wide v a r i e t y of systems well before a p r o l i f e r a t i v e response occurs (3-7). I f , as i s widely held, polyamine metabolism i s i n e x t r i c a b l y involved with the c e l l u l a r growth process, then decreased polyamine biosynthesis should be associated with a c y t o l y t i c response and the mechanism of producing t h i s response would be fundamentally important i n regulating c e l l d i v i s i o n . A negative growth response preceded by a marked decrease i n the a c t i v i t y of one polyamine biosynthetic enzyme has been demonstrated i n r a t thymus. The enzyme a c t i v i t y was reduced by a factor of 400 at l e a s t 12 hours p r i o r to thymus i n v o l u t i o n and eventual lymphocyte c y t o l y s i s (8). The mechanism involved i n mediating t h i s response i s unknown. It may involve a decrease i n enzyme synthesis, an increase i n degradation, a p o s t - t r a n s l a t i o n a l modification or the synthesis of an i n h i b i t o r that acts s p e c i f i c a l l y on the enzyme. Polyamine Biosynthesis Polyamines are metabolic d e r i v a t i v e s of the amino acids L-methionine and L-ornithine. The three most common products of the pathway are the 2. diamine putrescine and the polyamines spermidine and spermine. Ornithine decarboxylase (ODC), which catalyzes the formation of putrescine from ornithine, i s considered to be the rate l i m i t i n g enzyme i n the biosynthesis of polyamines (9). In r e s t i n g c e l l s , t h i s enzyme has the lowest a c t i v i t y of a l l the biosynthetic enzymes i n the pathway. I t i s well characterized i n several tissues (10-14) and has been p u r i f i e d to electrophoretic homogeneity from a v a r i e t y of sources (15-17). ODC shows a strong dependence on t h i o l s f o r a c t i v i t y and i s often found i n dimers (18,19). Estimates of molecular weight f o r the monomer range from 50,000 to 55,000 i n mammalian systems (20-23) and as high as 80,000 i n other systems (24,25). The enzyme requires pyridoxal phosphate as a cofactor (26). M u l t i p l e species of ODC have been demonstrated which d i f f e r i n t h e i r a f f i n i t i e s f o r pyridoxal phosphate (27). These may represent p o s t - t r a n s l a t i o n a l modifications rather than true isozymes since there i s no evidence to support the existence of d i f f e r e n t ODC genes (28). ODC i s an a c i d i c p rotein having an i s o e l e c t r i c point i n the range of pH 4.1 to 4.8 (29). A f t e r p u r i f i c a t i o n , mammalian ODC has an optimum pH of approximately 7.4 (30). In eukaryotes, the enzyme has a reported range of Km values f o r ornithine of 0.03 to 0.2 mM (31). ODC i s mainly a c y t o s o l i c enzyme and has two important c h a r a c t e r i s t i c s that d i s t i n g u i s h i t from other known eukaryotic enzymes (32). One i s i t s i n d u c i b i l i t y . The ODC a c t i v i t y i n r e s t i n g adult mammalian t i s s u e s , with few exceptions, i s extremely low (33-35). Increases i n a c t i v i t y of up to 1000 f o l d within a few hours have been reported i n response to a p o s i t i v e growth stimulus (36). A second unique c h a r a c t e r i s t i c of ODC i s i t s very short h a l f - l i f e . Depending upon the type of c e l l and stage of d i v i s i o n , the h a l f - l i f e of ODC can be l e s s than 20 minutes, which Is much less than that of any other known eukaryotic enzyme. Another decarboxylase, S-adenosyl-L-methionine decarboxylase (SAMD o AdoMet DC) i s necessary f o r the synthesis of the higher polyamines spermidine and spermine. SAMD converts S-adenosyl-L-methionine to . S-adenosyl-methyl-thiopropylamine which acts as an aminopropyl donor f o r th i s transfer reaction with putrescine, that occurs l a t e r i n the pathway. While SAMD also has a short h a l f - l i f e , i t s properties are fundamentally d i f f e r e n t from those of ODC (37). I t does not require pyridoxal phosphat as a cofactor, but instead appears to belong to a small class of amino acid decarboxylases that uses covalently l i n k e d pyruvate as a prosthetic group. I t s presence i s required f o r enzyme a c t i v i t y . The two remaining polyamine biosynthetic enzymes are propylamine transferases. Their properties contrast with the decarboxylases i n that both are stable enzymes with long h a l f - l i v e s (38). The transferases ( a l s c a l l e d spermidine and spermine synthase) do not require cofactors and hav considerably higher a c t i v i t i e s than ODC and SAMD i n the res t i n g c e l l . Their a c t i o n i s to transfe r an aminopropyl group to putrescine and spermidine which r e s u l t s i n the synthesis of spermidine and spermine re s p e c t i v e l y . While the a c t i v i t i e s of the l a t t e r three biosynthetic enzymes are often linked to ODC a c t i v i t y , i t appears that they can also b regulated independently. Large increases i n ODC i n many stimulated tissues can occur independently of changes i n SAMD a c t i v i t y (39). Again, l i t t l e i s known of the mechanisms f o r c o n t r o l l i n g the a c t i v i t i e s of spermidine and spermine synthase. Putrescine i s a competitive i n h i b i t o r of spermine synthase and therefore high c e l l u l a r l e v e l s of putrescine 4. r e s u l t i n g from a c t i v e growth favor spermidine production. The Regulation of ODC A c t i v i t y Since ODC i s the rate determining enzyme i n the biosynthesis of polyamines, the regulation of polyamine metabolism i s centered around modulating the a c t i v i t y of t h i s enzyme. When looked at i n combination, the two unique c h a r a c t e r i s t i c s of ODC, i t s i n d u c i b i l i t y with a short h a l f - l i f e , mean that the i n t e r c e l l u l a r concentrations of ODC, or the enzyme's a c t i v i t y , can r i s e sharply, but only for an extremely short period of time. This implies that a subsequent rapid deactivation of the enzyme must take place. The regulation of ODC a c t i v i t y appears to take place through a v a r i e t y of d i f f e r e n t mechanisms. In HeLa c e l l s , ODC regulation has been shown to involve changes i n the h a l f - l i f e of the enzyme (40). C e l l s induced with glutamine show a four f o l d increase i n ODC h a l f - l i f e when compared with uninduced c e l l s (40). Regulation through increased ODC protein synthesis has been demonstrated i n mouse f i b r o b l a s t s (41). A 20 f o l d increase i n ODC a c t i v i t y i n transformed f i b r o b l a s t s has been correlated with increases i n the amount of enzyme protein (42). A s i m i l a r e f f e c t has been demonstrated i n androgen stimulated mouse kidney, where large increases i n ODC a c t i v i t y were shown to be associated with decreased rates of degradation and increased rates of ODC synthesis. The rapid deactivation of ODC a c t i v i t y has also been shown to r e s u l t from elevated concentrations of the polyamines themselves. This action i s often probably i n d i r e c t however, since a macromolecular ODC i n h i b i t o r induced by micromolar concentrations of polyamines has been demonstrated (43). I t has been c a l l e d 'antizyme' as i t i s a protein i n h i b i t o r of an 5. enzyme that i s synthesized i n response to the immediate or remote product of the reaction i t catalyzes (44). Antizyme i s a small protein of Mr 26,000 and was f i r s t i d e n t i f i e d i n L1210, neuroblastoma and H35 c e l l s as well as i n rat l i v e r (45). The induction of t h i s i n h i b i t o r i s dependent upon continued protein synthesis and i t s l e v e l s within the c e l l have been shown to decrease r a p i d l y i n the presence of cycloheximide (46). In rat l i v e r i t i s located i n the c e l l nucleus and could be dissociated from ODC i n v i t r o by high s a l t concentrations r e s u l t i n g i n reactivated ODC (47). K i n e t i c studies have revealed that i t i n h i b i t s ODC non-competitively (48). Substances having c h a r a c t e r i s t i c s s i m i l a r to those of antizyme have been found i n other tissues and as a r e s u l t of d i f f e r e n t s t i m u l i . A pyridoxal phosphate s e n s i t i v e ODC regulatory protein of Mr 19,500 was induced by putrescine i n chicken l i v e r (49). The growth stimulus r e s u l t i n g from plucking h a i r from the dorsal skin of rats resulted i n the production of an ODC i n h i b i t o r y substance of apparent Mr greater than 30,000 (50). This protein has some antizyme-like properties and i s thought to be involved i n the c y c l i c a l r e gulation of ha i r growth i n r a t s . ODC i s a l s o known to be regulated through p o s t - t r a n s l a t i o n a l modification. A nucleolar transglutaminase has been i s o l a t e d from guinea pig l i v e r which reduces ODC a c t i v i t y by covalently l i n k i n g a putrescine molecule to each of the enzyme's four glutamyl side chains (51). Furthermore, i t has been shown that the addi t i o n of the ODC-putrescine complex w i l l increase s t o i c h i o m e t r i c a l l y , the a c t i v i t y of RNA polymerase I (52). There i s evidence to suggest that t h i s complex may be the 65,000 Mr subunit of RNA polymerase I. Further evidence of ODC i n h i b i t i o n r e s u l t i n g from p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n has been found i n the slime mold, 6 . Physarum polycephalum (53). I n h i b i t i o n i s achieved through phosphorylation by a protein kinase of Mr 26,000 of a protein of Mr 70,000 having ODC a c t i v i t y (54). Similar protein kinases have been found i n bovine spermatozoa and i n E h r l i c h a s c i t e s c e l l s (55). The l a t t e r phosphorylation i s stimulated by i n t e r f e r o n (56). Others have reported the presence of an ODC i n a c t i v a t i n g f a c t o r (0DIF) i n extracts of adult rat prostate glands (57). The mechanism of a c t i o n of ODIF i s unclear although i t shows a time dependent a c t i v i t y and may p o s s i b l y represent a new class of highly s p e c i f i c proteases (58). Polyamine Metabolism i n Thymus and Other Lymphatic Tissue R e l a t i v e l y l i t t l e i s known about polyamine metabolism i n thymus and spleen. The enzymes have not been characterized and the mechanisms involved i n a l t e r i n g t h e i r a c t i v i t i e s have not been studied i n d e t a i l . There i s evidence to suggest that the thymus, i n p a r t i c u l a r , may o f f e r valuable i n s i g h t i n t o the c h a r a c t e r i s t i c s and regulation of lymphatic ODC. High concentrations of putrescine, spermidine and spermine have been found i n rat thymus (59). E x c e p t i o n a l l y high ODC l e v e l s have also been detected i n t h i s t i s s u e and are known to be associated with the thymocytes and not the stroma (60). The thymus i s an a c t i v e producer of lymphocytes which have a turnover rate f i v e to ten times higher than that of lymphocytes elsewhere. In the human, much of the thymic substance undergoes atrophy by the time of puberty (61), while i n r a t s , i n v o l u t i o n i s known to have taken place at the adult stage (62). Given the close r e l a t i o n s h i p that e x i s t s between polyamine biosynthesis and changes i n the p r o l i f e r a t i v e state of c e l l s , i t would seem p l a u s i b l e that thymus ODC could undergo exceptionally large changes i n a c t i v i t y , more so than i n 7. most other tissues, p r i o r to a p o s i t i v e or negative growth response. The mechanism for regulating the enzyme's a c t i v i t y must be able to accommodate the production of high l e v e l s of ODC before c e l l growth and perhaps sharp decreases to low l e v e l s p r i o r to i n h i b i t i o n of c e l l d i v i s i o n . While the mechanism of regulating ODC i n rat thymus has not been studied, preliminary findings on the c o n t r o l of t h i s enzyme's a c t i v i t y have been reported i n other lymphatic t i s s u e s . Studies c a r r i e d out on S49 lymphoma c e l l s implicate c y c l i c AMP as a mediator of f l u c t u a t i o n s i n ODC l e v e l s , since S49 variants having l e s i o n s i n the pathway of cAMP generation and action w i l l not respond to any of the s t i m u l i that produce an i n h i b i t i o n of ODC i n wild type S49 c e l l s (63). Others have shown that polyamine depletion mediated by an i r r e v e r s i b l e and s p e c i f i c i n h i b i t o r of ODC arrests the growth of mouse lymphoma c e l l s , but that t h i s arrest i s d i s s o c i a b l e from a cAMP mediated a r r e s t (64). There i s also evidence to suggest that ODC l e v e l s i n mouse lymphoma c e l l s may be regulated independently of e i t h e r previously mentioned mechanism since a small percentage of the c e l l s treated with t o x i c l e v e l s of the i r r e v e r s i b l e i n h i b i t o r of ODC are able to survive and p r o l i f e r a t e due to an overproduction of" ODC (65). The synthesis of a regulatory protein may also be required for ODC control i n lymphocytes. Experiments performed using human lymphocytes demonstrate that ODC a c t i v i t y can be g r e a t l y reduced by the addition of low concentrations of i t s product, putrescine, or i t s i n d i r e c t product, spermidine, to the culture medium (66). This e f f e c t appears to involve protein synthesis, since the putrescine induced i n h i b i t i o n decreased from 47% to 5% when cycloheximide was added (67). To date, an i n h i b i t o r of ODC 8. has never been reported i n lymphatic t i s s u e . The E f f e c t of Glucocorticoids on Polyamine Metabolism i n Lymphatic Tissue As a general r u l e , whenever a hormone induces an anabolic response i n i t s target t i s s u e , an early stimulation of ODC i s seen. The time course of t h i s response t y p i c a l l y shows a peak of a c t i v i t y 4 to 6 hours a f t e r the hormone i s administered (68). In kidney and l i v e r , glucocorticoids stimulate a generalized net increase i n p r o t e i n synthesis associated with gluconeogenesis (69). A pharmacological dose of dexamethasone has been shown to produce a rapid and s i g n i f i c a n t decrease i n ornithine and SAM decarboxylases i n rat thymus, whereas the i n j e c t i o n of mineralocorticoids had no e f f e c t (70). In other studies, the s i g n i f i c a n t decrease i n ODC a c t i v i t y detected i n thymus and spleen of rats following the i n vivo administration of g l u c o c o r t i c o i d s was shown to be detectable as early as 2 1/2 hours a f t e r the hormone was given (71). This i s well i n advance of thymus and spleen c e l l l y s i s reported to occur approximately 16 hours a f t e r hormone treatment (72), Other evidence of g l u c o c o r t i c o i d control of lymphatic t i s s u e growth i s shown by the increased thymus weight and lymphocyte m i t o t i c a c t i v i t y found i n mice (73) and i n rats (74) two days and eight days r e s p e c t i v e l y , a f t e r adrenalectomy. A negative growth response r e s u l t i n g from g l u c o c o r t i c o i d treatment has also been demonstrated i n t i s s u e cultures of T-lymphoblast c e l l l i n e s from patients with acute lymphoblastic leukemia (75). To better understand the f i r s t stages of t h i s type of growth response, two studies have t r i e d to quantify the e a r l i e s t changes that thymus c e l l s undergo as a r e s u l t of treatment with g l u c o c o r t i c o i d s . Two-dimensional ge l electrophoresis has i d e n t i f i e d 18 consistent differences when the proteins of hormone r e s i s t a n t c e l l s 9 . were compared with the gels obtained from s e n s i t i v e c e l l s (76). I t i s also reported that one of the major proteins present i n the r e s i s t a n t rat thymus c e l l s , but not i n the s e n s i t i v e c e l l s , migrated to exactly the same po s i t i o n on the 2D g e l as did the l a r g e s t protein from r e s i s t a n t lymphosarcoma c e l l s (76). Another s e r i e s of experiments has demonstrated the absence of a chromatographically separable species of ODC i n the thymus of dexamethasone treated r a t s . When ODC was p a r t i a l l y p u r i f i e d from a crude extract of thymus from co n t r o l rats using ion exchange chromatography, multiple peaks .of a c t i v i t y were observed. This procedure was then repeated using a crude extract of thymus from dexamethasone treated rats and the major peak of enzyme a c t i v i t y was no longer present (71). These r e s u l t s , although preliminary i n nature, suggest that g l u c o c o r t i c o i d s s i g n i f i c a n t l y a l t e r the constituent proteins of rat thymus and i n one instance, markedly a f f e c t one species of ODC well before c e l l l y s i s occurs. The Present Investigation This study was undertaken to determine the reason for the rapid and large decrease i n ODC a c t i v i t y observed i n the thymus and spleen of dexamethasone-treated rats and to i n v e s t i g a t e the r e l a t i o n s h i p of t h i s decrease to the growth i n h i b i t i o n and l y t i c response of the c e l l s . As has been previously discussed, there are several possible reasons for t h i s decrease and we chose, as a s t a r t i n g point, to look f o r the presence of an ODC i n h i b i t o r . I n i t i a l l y , the conditions for assaying ODC i n thymus and spleen were established. These conditions include an e f f e c t i v e method of t i s s u e 10. preparation and c e l l d i s r u p t i o n , the optimum concentrations of sul f h y d r y l reducing agents f o r maximum ODC a c t i v i t y and s u f f i c i e n t s e n s i t i v i t y to detect the low l e v e l s of enzyme a c t i v i t y when necessary. With these c r i t e r i a i n place, the influence of various factors can be assessed. Secondly, a reproducible assay f o r the presence of an ODC i n h i b i t o r was developed. The method chosen i s a mixing assay i n which ODC a c t i v i t y i s measured separately i n al i q u o t s of ti s s u e extract from control rats and from hormone-treated r a t s , and i n mixtures of the two types of extracts. The presence of an i n h i b i t o r i s indicated when the a c t i v i t y observed i n the mixtures i s l e s s than the sum of a c t i v i t i e s i n extracts assayed separately. Procedures f o r i s o l a t i n g the i n h i b i t i n g substance and studying i t s c h a r a c t e r i s t i c s and properties were then developed. F i n a l l y , the properties of t h i s i n h i b i t o r were compared with others described i n the l i t e r a t u r e . 11. MATERIALS AND METHODS Treatment of Animals Intact immature female Wistar rats (UBC s t r a i n ) , age 4-8 weeks, were used i n a l l experiments. The animals were allowed to adjust to t h e i r surroundings f o r at l e a s t 48 hours p r i o r to use i n experiments. They were fed with standard laboratory chow and water ad l i b i t u m and kept under controlled 12 hour l i g h t and 12 hour dark l i g h t i n g conditions. Dexamethasone (Hexadiol phosphate) was dissolved i n ethanol and d i l u t e d to a f i n e suspension i n s a l i n e immediately p r i o r to use. This was administered at a dose of 200 ug by i n t r a p e r i t o n e a l i n j e c t i o n , 5 hours p r i o r to s a c r i f i c e unless otherwise i n d i c a t e d . Rats used for control purposes were injec t e d with 0.9% s a l i n e or were not in j e c t e d . Sample Preparation The animals were k i l l e d by c e r v i c a l d i s l o c a t i o n . The thymus and spleen were removed immediately and placed i n i c e cold Hepes buffer (50 mM, pH 7.4, 10 mM d i t h i o t h r e i t o l and 100 MM EDTA). Tissues from 3 or more rats were pooled and homogenized i n 2 ml of buffer per gram of tissue for tissues from dexamethasone-treated ra t s and 4 ml/g tissue f o r tissues from control r a t s . Homogenization was performed using a Potter-Elvejhem homogenizer with the pestle being driven at approximately 600 rpm during the 8-9 up and down movements. The homogenizer was kept i n i c e throughout t h i s procedure. The r e s u l t i n g homogenate was centrifuged at 20,000 xg at 4°C f o r 20 minutes and the supernatant portions f i l t e r e d through gauze to remove fat and suspended t i s s u e fragments. For tis s u e removed from dexamethasone treated r a t s , the p e l l e t was then resuspended i n a volume of 12. buffer, equal to the f i r s t e x t r a c t i o n , which contained 1% Lubrol. Homogenization was then repeated using 4-5 up and down strokes so as to avoid excessive foaming. The r e s u l t i n g s l u r r y was centrifuged and f i l t e r e d as before with the supernatant being combined with that obtained from the f i r s t e x traction. P e l l e t s from the extracts of tissue of control rats were not extracted a second time, except where indicated. ODC and I n h i b i t o r Assays Al i q u o t s of the supernatant f r a c t i o n s were used f o r assay of ODC and SAMD. The ODC a c t i v i t y was measured by a s l i g h t modification of the previous method (77). The rea c t i o n mixture contained, i n a f i n a l volume of 0.4 ml, 0.2 mM pyridoxal phosphate and L-ornithine at a concentration of 0.136 mM containing 0.125 uCi L - l - 1 " C o r n i t h i n e HC1 (58 mCi/mmole). A l l assay tubes were kept on i c e u n t i l s t a r t i n g the reaction with the addi t i o n of substrate. In the assay [1'*C]C02 was co l l e c t e d i n 0.2 ml hyamine hydroxide following a c i d i f i c a t i o n . Under these assay conditions, a 100 ul a l i q u o t of thymus homogenate gave approximately 1200 cpm. The method used to detect the presence of an ODC i n h i b i t o r took the form of a mixing assay. Aliquots of thymus extracts from control and dexamethasone-treated r a t s were assayed separately f o r ODC and together i n a 1:1 mixture. The predicted a c t i v i t y of the mixing assay would therefore be the sum of the i n d i v i d u a l ODC a c t i v i t i e s . The degree of i n h i b i t i o n could then be determined by comparing the observed ODC a c t i v i t y from the mixing assay with the predicted a c t i v i t y . One unit of i n h i b i t i o n was defined as the di f f e r e n c e i n pmoles/100 ul/30 minutes, between the ODC 13. a c t i v i t y i n an aliqu o t of thymus ODC from co n t r o l rats and the ODC a c t i v i t y measured i n a mixing assay. The SAMD was assayed by a s l i g h t modification of a published method (78) containing, i n 250 ml, 0.6 mM S-adenosyl-L-[carboxy 1 1*C]-methionine (60 mCi/mmole), 2.5 mM putrescine, 0.05 mM pyridoxal phosphate and 0.1 M phosphate buffer, pH 7.1. A l l assays were c a r r i e d out i n duplicate or t r i p l i c a t e and an average value used i n c a l c u l a t i o n s . DEAE-Sepharose CL-6B Column Chromatography A s e t t l e d volume of 20.0.ml of DEAE-Sepharose CL-6B suspended i n standard Hepes buffer was degassed f o r 2 hours. A f t e r decanting the fines the r e s i n was packed i n a 1.5 x 10 cm. Biorad column to a bed volume of 15.0 ml and washed with 3 volumes of buffer. The column was equili b r a t e d i n a cold room at (4°C) before use. The thymus extract from 6 dexamethasone-treated r a t s was prepared as previously described and loaded onto the column. The column was eluted with increasing concentrations of NaCl with 10, 2.5 ml f r a c t i o n c o l l e c t e d at each step. The concentrations of s a l t used were 0, 100 mM, 300 mM and 500 mM NaCl. Each f r a c t i o n was assayed f o r protein content using the method of Bradford (79). The fr a c t i o n s at each s a l t concentration were then pooled and concentrated to approximately 1.0 ml using u l t r a f i l t r a t i o n . This s o l u t i o n was then dialyzed f o r 20-24 hours and then assayed f o r ODC a c t i v i t y and a b i l i t y to i n h i b i t an a l i q u o t of thymus ODC from co n t r o l r a t s . The recovery was determined by comparing the number of i n h i b i t o r units loaded onto the 14. column with the t o t a l number of i n h i b i t o r units from each e l u t i o n . The p u r i f i c a t i o n was calculated by comparing s p e c i f i c a c t i v i t i e s of loaded i n h i b i t o r units with that obtained from each e l u t i o n . Heparin Sepharose Column Chromatography The Heparin Sepharose CL-6B gel was swollen and washed with standard Hepes buffer for 15 minutes on a sintered glass f i l t e r . The gel was then resuspended i n 25 ml of buffer and degassed f o r 2 hours. The beads were packed i n a 0.9 x 15 cm Pharmacia column to a bed volume of 2.5 ml and washed with 10-12 bed volumes of standard buffer. The column was eq u i l i b r a t e d i n a cold room (4°C) before use. With the flow rate adjusted to a minimum l e v e l , a volume of p a r t i a l l y p u r i f i e d thymus ODC I n h i b i t o r (from the DEAE Sepharose step) not exceeding the t o t a l column volume, was applied to the r e s i n . The column was eluted i n the same manner as the DEAE column with increasing s a l t concentrations of 0, 100, 300 and 500 mM NaCl. The r e s u l t i n g f r a c t i o n s were pooled, concentrated, dialyzed and assayed f o r i n h i b i t o r y a c t i v i t y as before. Sephacryl S-200 Gel F i l t r a t i o n A s e t t l e d volume of 180 ml of Sephacryl S-200 was suspended i n standard Hepes buffer and degassed f o r 2 hours. A f t e r decanting the fines the r e s i n was packed i n a 1.5 x 9.0 cm Pharmacia column to a t o t a l bed volume of 100 ml. The packed column was washed with 1 l i t r e of standard buffer and e q u i l i b r a t e d i n a cold room (4°C) before use. The column was c a l i b r a t e d f o r molecular weight determination purposes with commercialy prepared molecular weight standards. These included Blue dextran (2 x 10 6), F e r r i t i n (440,000), Catalase (332,000), Aldolase (158,000), BSA (67,000) and pyridoxal phosphate (247).. The sample size was 1.0 ml and each was prepared at a concentration of approximately 15 mg/ml. The flow rate f or t h i s column system was 10.0 ml/hr as determined by the Pharmacia FRAC 200 f r a c t i o n c o l l e c t o r and the p e r i s t a l t i c pump. The protein content of each 1.0 ml f r a c t i o n was determined by recording the ^ e e l u t i o n volume of each standard was obtained i n t h i s manner and a c a l i b r a t i o n curve was constructed. The e l u t i o n of each standard was ca r r i e d out using standard Hepes buffer. The p a r t i a l l y p u r i f i e d thymus ODC i n h i b i t o r present i n the 100 mM NaCl eluted pool from the Heparin Sepharose column was applied to the Sephacryl S-200 column. The column was eluted as before and each f r a c t i o n was assayed f o r i n h i b i t o r y a c t i v i t y using the mixing assay. The fractions showing high i n h i b i t o r y a c t i v i t y were pooled and concentrated. The r e s u l t i n g volume was then re-assayed to confirm i n h i b i t o r y a c t i v i t y and i t s e l u t i o n p o s i t i o n used to estimate the molecular weight. Miscellaneous Methods a) Trypsin Digestion; The t r y p s i n used i n these experiments was TPCK treated which renders i t free of chymotrypsin a c t i v i t y . I t has been lyophylized from Bovine pancreas and dialyzed free of s a l t . Each assay used 1000 units of a c t i v i t y or 0.11 mg t r y p s i n (9000 BAEE units per mg. protein). A n t i t r y p s i n was prepared i n the same manner as t r y p s i n and used at a 2:1 r a t i o of s p e c i f i c a c t i v i t y to stop the a c t i o n of t r y p s i n p r i o r to ODC assays. A 1.0 ml stock s o l u t i o n (120 ml Hepes pH 7.6 containing 1.7 mg trypsin) of t r y p s i n was prepared and incubated at 37°C i n a shaker water bath. At time zero 1.5 ml of thymus extract from dexamethasone 16. treated rats was added to the incubation mixture. At time zero, 30 and 60 minutes, 500 pi ali q u o t s of the t r y p s i n and thymus extract mixture was removed and incubated with 500 u l of a n t i t r y p s i n stock so l u t i o n (3 mg antitrypsin/1.0 ml Hepes @ pH 7.6) for 5 minutes. ODC and mixing assays were then c a r r i e d out on 100 u l a l i q u o t s of the po s t - a n t i t r y p s i n s o l u t i o n . Control experiments were done to show that the tr y p s i n and a n t i t r y p s i n were a c t i v e . Aliquots of enzyme extract were combined with aliquots of the t r y p s i n preparation and assayed f o r ODC a c t i v i t y at various time i n t e r v a l s i n the absence of a n t i t r y p s i n . This procedure was repeated with a n t i t r y p s i n added to the assay immediately a f t e r the addition of t r y p s i n . Due to the presence of excess a n t i t r y p s i n , i t s i n t e r a c t i o n with the enzyme was also assessed. Aliquots of the a n t i t r y p s i n preparation were combined with a l i q u o t s of the ODC i n h i b i t o r and incubated f o r various time i n t e r v a l s . Mixing assays were then c a r r i e d out to v e r i f y that ODC i n h i b i t i o n was unaffected. b) Polyacrylamide Gel Electrop h o r e s i s . Stock solutions of sodium phosphate buffer (0.5 M, pH 7.2), SDS (10%), ammonium persulfate (1.5%) and acrylamide-bisacrylamide (30:0.8 per 100 ml) were prepared i n advance of the sample preparation. The gel mixture used contained 10% acrylamide. For each g e l , 42 ml of mixture was prepared. This contained 14.0 ml of acrylamide-bisacrylamide, 8.4 ml of sodium phosphate buffer, 0.42 ml of SDS, 2.1 ml of ammonium pe r s u l f a t e and 17.1 ml of d i s t i l l e d water. This mixture was then degassed f o r 10 min at which time 21 u l of TEMED was added. The continuous buffer system used 200 ml sodium phosphate buffer (0.5 M sodium phosphate pH 7.2 per l i t r e ) 10 ml SDS (10%) and 790 ml of d i s t i l l e d water (per l i t r e ) as an electrode buffer. 17. For a 50-100 ug protein sample, a s o l u t i o n of 10 ul sodium phosphate buffer, 100 PI SDS, 25 Ml 6-mercaptoethanol (5%), 50 y l gl y c e r o l (10%) and 50 ul bromophenol blue (0.2%) was boiled f or 3 minutes. This sample was then centrifuged at 20,000 xg for 5 minutes. Samples of 5 ul and 10 ul were loaded onto alternate lanes of the g e l . Electrophoresis was c a r r i e d out at 140 v o l t s for 6 hours. The gel was stained overnight i n a 5:5:2 mixture of Coomassie blue (0.1%), methanol and g l a c i a l a c e t i c a c i d . The destaining was ca r r i e d out using 20% methanol and 7% a c e t i c a c i d . c) U l t r a f i l t r a t i o n . This procedure was c a r r i e d out using the Amicon D i a f l o YM10, YM30 or XM50 membranes. For molecular weight estimations, a minimum extract volume of 10.0 ml was used. Nitrogen was used as a c a r r i e r gas at a pressure of 80 p s i . The f i n a l retentate volume was approximately 1.0 ml. and the f i l t r a t e was c o l l e c t e d i n a graduated cyl i n d e r to monitor the volume reduction. For concentration of column f r a c t i o n s , the YM10 membrane was used as described above. A l l u l t r a f i l t r a t i o n procedures were c a r r i e d out at 4°C. d) D i a l y s i s . A l l d i a l y s i s tubing was prepared i n advance by b o i l i n g for 1 hour i n a so l u t i o n of d i l u t e a c e t i c a c i d and EDTA. I t was cut to si z e and washed thoroughly with d i s t i l l e d water and then buffer immediately p r i o r to use. The d i a l y s i s buffer used was Hepes (50 mM pH 7.4). The d i a l y s i s buffer volume used was at l e a s t 500 x greater than the sample volume and a minimum of three buffer changes were used over a 20-24 period. S t i r r i n g was constantly maintained and a l l d i a l y s i s took place at 18. 8. Chemicals D i t h i o t r e i t o l , pyridoxal phosphate, L-ornithine, putrescine and tryps i n (TPCK-treated) were purchased from Sigma Chemical Company, St. Louis, MO, USA. S-adenosylmethionine was obtained from Calbiochem-Behring, San Diego, CA, USA. L - t l - 1 " C ] O r n i t h i n e was obtained from Amersham Corporation, IL, USA. 19. RESULTS Conditions f o r Measurement of ODC A c t i v i t y Experiments were f i r s t directed toward e s t a b l i s h i n g assay conditions suitable f o r measuring ODC a c t i v i t y i n ti s s u e s of control animals. Of primary importance was optimizing CO2 production and therefore measurable r a d i o a c t i v i t y . The data presented i n Table 1 demonstrates the ODC a c t i v i t i e s obtained from homogenizing thymus from control rats and assaying enzyme a c t i v i t y with d i f f e r e n t buffer systems. Hepes was chosen as the standard buffer because i t c o n s i s t e n t l y gave the greatest a c t i v i t y . ODC i n most tissues requires reduced s u l f h y d r y l groups f o r maximum a c t i v i t y . The r e s u l t s i l l u s t r a t e d i n Table 2 show that 10 mM concentrations of d i t h i o t h r e i t o l were optimum f o r the amount of thymus enzyme, although lower concentrations s t i l l allowed measurable enzyme a c t i v i t y . I t i s also necessary to determine a substrate concentration that w i l l avoid problems associated with substrate l i m i t a t i o n . The L-ornithine k i n e t i c s of crude thymus ODC are i l l u s t r a t e d i n Figure 1. The double r e c i p r o c a l p l o t gave an apparent Km f o r ornithine of 80 pM which i s within the range reported f o r ODC from other sources. The d e t a i l s of the assay conditions are described i n the methods section. The r e s u l t s In Figures 2 and 3 show that thymus and spleen ODC a c t i v i t y i n crude extracts i s l i n e a r with respect to time and protein concentration under optimum assay conditions. This was also shown to be true f o r ODC a c t i v i t y i n extracts of tissues taken from dexamethasone treated r a t s . TABLE 1 ODC ACTIVITY IN THYMUS EXTRACTS PREPARED USING DIFFERENT BUFFERS The r a t s used were e i t h e r s a l i n e i n j e c t e d or non-injected. Each buffer was prepared at a concentration of 50 mM and adjusted to pH 7.4. ODC assays were then conducted as described i n the Methods section. A l l assays were c a r r i e d out i n duplicate or t r i p l i c a t e and the r e s u l t s averaged. BUFFER* (mM) ODC ACTIVITY (pmoles ^COp/lOO Ml/30 min.) HEPES 206 HEPPS 185 MOPS 189 * see l i s t of abbreviations f o r IUPAC nomenclature of buffers 21. TABLE 2 ODC ACTIVITY IN THYMUS EXTRACTS OF CONTROL RATS DETERMINED  USING DIFFERENT DITHIOTHREITOL CONCENTRATIONS ODC assays were c a r r i e d out as described i n the Methods section. The d i t h i o t h r e i t o l concentration was adjusted to the desired l e v e l i n the reaction volume of the assay. DITHIOTHREITOL ODC CONCENTRATION ACTIVITY (mM) (pmoles/100 jil/30 min) 3 285 5 269 10 471 15 317 FIGURE 1 L-ORNITHINE KINETICS OF ODC IN THYMUS OF CONTROL RATS ODC a c t i v i t y i n thymus extracts of co n t r o l rats was determined at various substrate concentrations. A l l assays were c a r r i e d out In t r i p l i c a t e and the r e s u l t s averaged. Km = 80 MM Vmax = 1.6 x 10 s pmoles ' " ^ / l O O Ml/30 min. 23. FIGURE 2 EFFECT OF INCUBATION TIME ON THYMUS ODC ACTIVITY Aliquots of thymus extract from (o) c o n t r o l rats and from (o) dexamethasone-treated ra t s were assayed f o r ODC a c t i v i t y . The assays were stopped at the times indicated. A l l assays were c a r r i e d out i n duplicate or t r i p l i c a t e . 23a. 24. FIGURE 3 EFFECT OF PROTEIN CONCENTRATION ON THYMUS ODC ACTIVITY The ODC a c t i v i t y was measured i n thymus extracts of (o) control rats and of (o) dexamethasone-treated rats using d i f f e r e n t volumes of each extract. The d e t a i l s of the assay procedure are described i n the methods section. 25. The Occurrence of an I n h i b i t o r of ODC i n Lymphatic Tissue from  Dexamethasone Treated Rats. The p o s s i b i l i t y that the rapid decrease i n ODC a c t i v i t y i n thymus and spleen i n dexamethasone treated rats resulted from the production of an i n h i b i t o r or i n a c t i v a t o r was f i r s t investigated by measuring the ODC a c t i v i t y obtained from mixing a l i q u o t s of t i s s u e extract from co n t r o l and dexamethasone-treated r a t s . The r e s u l t s i n Table 3a show that the observed enzyme a c t i v i t y was considerably l e s s than the sum of a c t i v i t i e s i n extracts assayed i n d i v i d u a l l y . In a s e r i e s of ten s i m i l a r experiments, the observed a c t i v i t y ranged from 28% to 61% of the predicted a d d i t i v e value i n thymus and 22% to 54% i n spleen. Since, i n most cases, the a c t i v i t y contributed by the extract from dexamethasone-treated animals was less than 10% of the t o t a l , destruction or i n h i b i t i o n of ODC i n extracts from co n t r o l animals must have occurred. Prompted by the wide range of i n h i b i t i o n or i n a c t i v a t i o n of ODC observed i n these mixing assays, the p e l l e t s r e s u l t i n g from the two t i s s u e preparations were assayed f o r ODC a c t i v i t y and a b i l i t y to i n h i b i t an a l i q u o t of enzyme preparation from control animals. The data presented i n Table 2b show that l e s s than 5% of the t o t a l ODC a c t i v i t y remained i n the p e l l e t s of spleen and thymus extracts from non-injected or c o n t r o l animals. However, i t was found that a s u b s t a n t i a l amount of the i n h i b i t o r y substance remained i n the resuspended p e l l e t s of thymus and spleen from dexamethasone-treated r a t s . Experiments were then designed to study the release of i n h i b i t o r from pe l l e t e d material. A second e x t r a c t i o n of the p e l l e t s with Hepes buffer, s t i l l l e f t i n h i b i t o r y a c t i v i t y u n s o l u b i l i z e d . I t was therefore decided to try e x t r a c t i o n with detergent. Lubrol i s known to be an e f f e c t i v e agent f o r s o l u b i l i z i n g a v a r i e t y of proteins (81). I n i t a l l y each p e l l e t was 26. TABLE 3 INHIBITION OF ODC ACTIVITY BY AQUEOUS EXTRACTS OF  THYMUS AND SPLEEN FROM DEXAMETHASONE-TREATED RATS Rats received e i t h e r s a l i n e ( c o n t r o l group) or dexamethasone (200 Mg) 5 hours before s a c r i f i c e . Tissues were pooled from 3-4 rats per group. Enzyme a c t i v i t y was assayed i n duplicate or t r i p l i c a t e using 100 Ml aliquots of each tissue preparation. Tissue extracts were prepared as described i n the methods sec t i o n . a) BUFFER EXTRACTION OF THYMUS AND SPLEEN Source of Tissue i n Assay ODC A c t i v i t y (pmoles ''COp/lOO Ml/30 min.) Treatment of Animals Tissue Observed Predicted % Predicted 1. Control Thymus 237 - -2. Control Spleen 140 - -3. Hormone Treated Thymus 13 - -4. Hormone Treated Spleen 15 - -5. 1 + 3 127 250 51 6. 2 + 4 70 155 46 7. 1 + 4 140 252 56 8. 2 + 3 91 153 60 b) BUFFER EXTRACTION OF PELLETED MATERIAL Source of Tissue i n Assay ODC A c t i v i t y (pmoles " ,C0 2/100 ul/30 min.) Treatment of Animals Tissue Observed Predicted % Predicted 1. Control Thymus 252 — — 2. Control Spleen 189 - -3. Hormone Treated Thymus 13 - -P e l l e t 4. Hormone Treated Spleen 12 - -P e l l e t 5. Control Thymus 4 - -P e l l e t 6. Control Spleen 4 - -P e l l e t 7. 1 + 3 185 265 70 8. 2 + 4 200 264 76 extracted with a volume of Hepes buffer, equal to the f i r s t extraction, containing 1% Lubrol. These two ext r a c t i o n volumes were then combined and assayed f o r i n h i b i t o r y a c t i v i t y . The data l i s t e d i n Table 4 demonstrate the r e s u l t s of mixing experiments c a r r i e d out using aliquots of Lubrol extracts and of buffer extracts from c o n t r o l animals. In the spleen, the percentage of predicted a c t i v i t y recovered ranged from 14-19%, s i g n i f y i n g 81-86% i n h i b i t i o n of ODC. In the thymus, the range was 27-34% s i g n i f y i n g 66-73% ODC i n h i b i t i o n . To determine whether the increased i n h i b i t i o n observed a f t e r Lubrol e x t r a c t i o n was due to the volume of a d d i t i o n a l i n h i b i t o r or to d i r e c t i n h i b i t i o n of ODC by Lubrol i t s e l f , a series of experiments were c a r r i e d out with Lubrol, and thymus and spleen from control animals. I n i t i a l l y , thymus and spleen extracts were prepared from control animals using Hepes buffer only and assayed f o r ODC a c t i v i t y . Then assays were c a r r i e d out on these extracts a f t e r d i f f e r e n t concentrations of Lubrol were added. The r e s u l t s i l l u s t r a t e d i n Figure 4 show that Lubrol added d i r e c t l y to the assay, at concentrations of up to 2%, has l i t t l e or no i n h i b i t o r y e f f e c t on thymus ODC. However, an i n h i b i t o r y or i n a c t i v a t i n g e f f e c t of approximately 30% i s observed i n spleen ODC assays at Lubrol concentrations of greater than 0.1%. A second series of experiments was c a r r i e d out incorporating d i f f e r e n t Lubrol concentrations i n t o the buffer p r i o r to t i s s u e e x t r a c t i o n . ODC assays were then c a r r i e d out with the r e s u l t s being the same as i n the f i r s t Lubrol experiments (data not shown). Therefore, while i t was possible to demonstrate some sort of ODC i n h i b i t i o n or i n a c t i v a t i o n well i n excess of 30% i n spleen as a r e s u l t of dexamethasone treatment, the e f f e c t i v e removal of Lubrol (to concentrations of l e s s than 0.1%) a f t e r tissue extraction with detergent i s necessary f o r further i n v e s t i g a t i o n of the 28. TABLE 4 OCCURRENCE OF AN INHIBITOR OF THYMUS AND SPLEEN ODC IN LUBROL  EXTRACTED LYMPHOID TISSUE FROM DEXAMTHASONE-TREATED RATS Rats received e i t h e r s a l i n e ( c o n t r o l group) or dexamethasone (200 Mg) 5 hours before s a c r i f i c e . Tissues were pooled from 3-4 rats per group. Enzyme a c t i v i t y was assayed i n duplicate or t r i p l i c a t e using 100 Ml aliquots of each t i s s u e preparation. Tissue extracts were prepared as described i n the Methods section with the second extraction buffer containing 1% Lubrol. Source of Tissue i n Assay ODC A c t i v i t y (pmoles 1 "CO^/lOO M.l/30 min.) Treatment of Animals Tissue Observed Predicted % Predicted 1. Control Thymus 235 - -2. Control Spleen 122 - -3. Hormone Treated Thymus 12 - -4. Hormone Treated Spleen 11 - -5. - 1 + 3 82 247 30 6. - 2 + 4 24 133 18 7. - 1 + 4 86 246 35 FIGURE A THE EFFECT OF LUBROL ON ODC ACTIVITY IN RAT THYMUS AND SPLEEN t Lubrol was incorporated i n t o the assay mixture either d i r e c t l y or at the time of t i s s u e homogenization. The d e t a i l s of t h i s procedure are discussed i n the Methods sect i o n . A l l assays were performed i n duplicate or t r i p l i c a t e , a) (o) ODC a c t i v i t y i n thymus of c o n t r o l r a t s , b) (o) ODC a c t i v i t y i n spleen of con t r o l r a t s . 8 800 1.5 2.0 0.5 1.0 Lubrol Concentration (%) 30. spleen system. For t h i s reason, the majority of experiments directed toward characterizing the i n h i b i t o r y substance were ca r r i e d out using thymus extracts from co n t r o l animals as a source of enzyme. I n h i b i t i o n was shown to be independent of time i n both thymus and spleen. The experimental r e s u l t s described i n Table 5 show that at the incubation times investigated, maximum i n h i b i t i o n was reached at the e a r l i e s t time i n t e r v a l i r r e s p e c t i v e of the r e l a t i v e proportions of ODC from c o n t r o l tissues and extracts prepared from dexamethasone-treated rats . When mixing assays using thymus extracts were ca r r i e d out at d i f f e r e n t substrate concentrations, the degree of i n h i b i t i o n observed remained constant. The r e s u l t s l i s t e d i n Table 6 show that increasing the substrate concentration by a fa c t o r of 50 did not change the i n h i b i t i o n observed. The a f f i n i t y f o r ornit h i n e demonstrated by the active enzyme molecules i n the mixing assay was nearly i d e n t i c a l to that demonstrated by ODC molecules i n thymus extracts from c o n t r o l r a t s . The double r e c i p r o c a l plot i n Figure 5 also shows that the Vmax f o r the 1:1 mixtures of tiss u e aliquots i s approximately one eighth of that measured for enzyme from con t r o l r a t s . As shown i n Table 7, the i n h i b i t o r y substance was detectable i n thymus as ea r l y as 2.5 hours a f t e r treatment, but could not be detected i n rats treated 24 hours p r i o r to s a c r i f i c e . The l e v e l s of i n h i b i t o r y substance appeared to be highest at 5 and 12 hours a f t e r hormone i n j e c t i o n . The degrees, of thymus ODC i n h i b i t i o n was shown to vary i n d i r e c t proportion to the volume of thymus extract from dexamethasone-treated rats 31. TABLE 5 INHIBITION OF ODC IN THYMUS AND SPLEEN OF  DEXAMETHASONE-TREATED RATS AS A FUNCTION OF INCUBATION TIME Extracts from thymus and spleen were prepared as described i n the Methods sec t i o n . The proportions of enzyme and i n h i b i t o r extracts were varied i n the r a t i o s described. A l l assays were done i n t r i p l i c a t e and the r e s u l t s averaged. a) RATIO OF ENZYME TO INHIBITOR OF 1:1 Time of Incubation (minutes) % I n h i b i t i o n of Thymus ODC % I n h i b i t i o n of Spleen ODC 15 30 45 60 70 66 73 70 80 81 86 83 b) RATIO OF ENZYME TO INHIBITOR OF 2.5:1 Time of Incubation (minutes) % I n h i b i t i o n of Thymus ODC % I n h i b i t i o n of Spleen ODC 15 30 45 60 27 28 29 32 56 52 57 56 TABLE 6 ACTIVITY OF THYMUS ODC INHIBITOR AT DIFFERENT SUBSTRATE CONCENTRATIONS Extracts of thymus from s a l i n e (control) and hormone-treated (200 ug dexamethasone) animals were prepared 5 hours a f t e r i n j e c t i o n as described i n the Methods sec t i o n . Tissues were pooled from 3-5 animals per group. ODC i n h i b i t o r y a c t i v i t y was measured as described i n the Methods section at the d i f f e r e n t ornithine concentrations l i s t e d , using duplicate or t r i p l i c a t e measurements for each sample. Ornithine ODC A c t i v i t y , pmoles Co2/30 min i n extract of thymus from concentration i n assay (mM) Controls Hormone-Treated Mixture % I n h i b i t i o n 10 101 2 32 31 50 236 7 74 27 100 292 8 86 29 250 373 13. 127 32 500 426 21 136 31 33. FIGUEE 5 L-ORNITHINE KINETICS OF THYMUS ODC IN A MIXING ASSAY (o) The ODC a c t i v i t y i n a 1:1 mixture of thymus extract from co n t r o l rats and from dexamethasone-treated rats was determined at various substrate concentrations and shown to have a near i d e n t i c a l Km for ornithine to ODC i n thymus extracts from c o n t r o l rats (o). Km = 80 uM Vmax = 1.6 x 10 3 pmoles l"C0 2/100 pl/30 min Vmaxj = 0.2 x 10 3 pmoles 1"C0 2/100 nl/30 min TABLE 7 ACTIVITY OF ODC AND ODC INHIBITOR IN THYMUS EXTRACTS PREPARED  FROM RATS AT DIFFERENT TIMES AFTER DEXAMETHASONE TREATMENT Thymus extracts were prepared from ra t s (3-4 per group) at times indicated a f t e r treatment with dexamethasone. ODC a c t i v i t y was measured i n duplicate i n 100 ul of each extract and ODC i n h i b i t o r y a c t i v i t y was determined i n a mixing assay as described i n the Methods section. Time Aft e r Treatment S p e c i f i c A c t i v i t y % I n h i b i t i o n of ODC (hours) of ODC % of Control i n Mixing Assay 2.5 32 12.3 5.0 4 71.0 12.0 5 48.0 24.0 15 0 added to the mixing assay. The r e s u l t s described by Table 8 also show that when the volume of i n h i b i t o r - c o n t a i n i n g extract was kept constant and the volume of enzyme-containing extract varied, a near l i n e a r decrease i n i n h i b i t i o n was observed. Characterization of ODC I n h i b i t i n g Substance i n Thymus of  Dexamethasone-Treated Rats. Experimental r e s u l t s suggest that the i n h i b i t o r y e f f e c t of the crude thymus extract from dexamethasone-treated rats i s at least . p a r t i a l l y s p e c i f i c f o r ODC. Assays of SAMD i n crude thymus extracts of control and dexamethasone-treated ra t s showed 86% of p r e d i c t i v e additive value i n a mixing experiment. Experimental r e s u l t s show how t h i s r e s u l t d i f f e r s from the recovery of ODC using the same tiss u e preparations (data not shown). In a mixing experiment, only 39% of the predicted ODC a c t i v i t y was observed. When combined with other sources of ODC, the i n h i b i t i n g substance contained i n thymus extracts from dexamethasone-treated rats displayed a s p e c i f i c i t y f o r enzyme from lymphatic ti s s u e . An aliquot of thymus extract from dexamethasone treated r a t s w i l l i n h i b i t thymus ODC from con t r o l rats by 70%, while i t w i l l i n h i b i t ODC i n an aliquot of spleen extract from c o n t r o l r a t s by 60% and ODC from rat kidney by only 30% (data not shown). When the thymus extract from dexamethasone-treated animals was heated at 55°C for 2 minutes, allowed to cool and then mixed with an aliquot of thymus extract from c o n t r o l animals, there was no i n h i b i t i o n observed (data not shown). The crude thymus extracts from dexamethasone-treated rat s were incubated with various enzymes which degrade proteins and/or 36. TABLE 8 EFFECTS OF VARYING THE RATIO OF INHIBITOR TO ENZYME ON ODC ACTIVITY Extracts of thymus were prepared from c o n t r o l rats (as source of enzyme) and from dexamethasone-treated rats (source of i n h i b i t o r ) . Tissues were pooled from 4-6 animals per group. ODC i n h i b i t i o n was measured as described i n the text using d i f f e r e n t amounts of the two extracts. Assays were done i n duplicate or t r i p l i c a t e . Amount of Enzyme Amount of I n h i b i t o r % I n h i b i t i o n of u l u l ODC 100 0 . 0 100 50 37 100 100 56 100 250 72 50 100 " 73 100 100 56 250 100 34 37. nuc l e i c acids and then re-examined for t h e i r a b i l i t y to i n h i b i t ODC a c t i v i t y . Experimental r e s u l t s show that the i n h i b i t o r y substance appeared to be unaffected by nucleases. Incubation with ei t h e r RNAse or DNase f o r 60 minutes did not r e s u l t i n a s i g n i f i c a n t change i n a b i l i t y to i n h i b i t thymus ODC from c o n t r o l rats (data not shown). The s e n s i t i v i t y of the i n h i b i t o r y substance to t r y p s i n was assessed at various times a f t e r incubation. The extract containing the i n h i b i t i n g substance was checked f o r a b i l i t y to i n h i b i t thymus ODC from c o n t r o l r a t s before and a f t e r incubation with t r y p s i n . The r e s u l t s i n Figure 6 show that the a b i l i t y to i n h i b i t ODC decreased markedly with continuing exposure to t r y p s i n . The i n h i b i t i n g substance has been shown to be non-dialyzable and not dependent on a small molecular weight substance f o r a c t i v i t y . The data i n Table 9 show that when thymus extracts from both co n t r o l rats and dexamethasone-treated r a t s were dialyzed separately before a mixing experiment, the degree of i n h i b i t i o n detected i n a mixing assay was the same as that observed using undialyzed e x t r a c t s . Experiments using u l t r a f i l t r a t i o n were conducted to approximate the molecular weight of the i n h i b i t o r y substance. Membranes with solute retention l e v e l s of 10,000, 30,000 and 50,000 daltons, a l l retained the i n h i b i t i n g substance. The r e s u l t s of mixing experiments conducted on the f i l t r a t e and retentate are l i s t e d i n Table 9. In a l l cases, only the retentate demonstrated an a b i l i t y to i n h i b i t ODC from the thymus of c o n t r o l r a t s . P u r i f i c a t i o n of ODC I n h i b i t o r The i n i t i a l attempts to p u r i f y the ODC i n h i b i t o r involved chromatography on ion exchange columns. These included DEAE-Sepharose and 38. FIGURE 6 EFFECT OF TRYPSIN ON ACTIVITY OF THYMUS ODC INHIBITOR I n h i b i t i o n of ODC was determined by mixing assays. A l l assays were done i n t r i p l i c a t e and the r e s u l t s averaged. The d e t a i l s of the tr y p s i n procedure are described i n the Methods sect i o n . 33a. TABLE 9 EFFECT.OF SMALL MOLECULAR WEIGHT SUBSTANCES  ON THYMUS ODC INHIBITORY ACTIVITY The d i a l y s i s and u l t r a f i l t r a t i o n procedures used are described i n the Methods sec t i o n . A l l assays were c a r r i e d out i n t r i p l i c a t e and r e s u l t s averaged. a) DIALYSIS Treatment of Extract 1. Pre D i a l y s i s 2. Pre D i a l y s i s 3. 4. Post D i a l y s i s 5. Post D i a l y s i s 6. Source of Tissue Extract Control Hormone-treated 1 + 2 Control Hormone-treated 4 + 5 ODC A c t i v i t y % I n h i b i t i o n (pmoles '"COp/lOO pl/30 min) of Thymus ODC 173 7 66 174 10 74 37 40 b) ULTRAFILTRATION Treatment of Extract Source of Tissue Extract 1. 2. YM-30 Retentate 3. YM-30 F i l t r a t e 4. 5. 6. XM-50 Retentate 7. XM-50 F i l t r a t e 8. 9. Control Hormone-treated Hormone-treated 1 + 2 1 + 3 Hormone-treated Hormone-treated 1 + 6 1 + 7 ODC A c t i v i t y (pmoles 1 "CO^/lOO U.l/30 min) 152 9 89 157 12 % I n h i b i t i o n of Thymus ODC A c t i v i t y 49 166 44 N i l 70 N i l 40. phosphocellulose. Preliminary experiments indicated that the i n h i b i t i n g substance contained i n detergent extracts of the thymus homogenate from dexamethasone-treated rats bound only marginally to phosphocellulose, but nearly completely to DEAE Sepharose CL-6B. E l u t i o n of the DEAE column was f i r s t attempted by step-wise increases of i o n i c strength. In carrying out the c o n t r o l experiments p r i o r to beginning the DEAE procedure, i t was found that thymus ODC was i n h i b i t e d by s a l t . Figure 7 shows that at NaCl concentrations of 50 mM, thymus ODC a c t i v i t y i s i n h i b i t e d by 20% and at 150 mM NaCl, i n h i b i t i o n i s approximately 56%. This fi n d i n g made the assaying of i n d i v i d u a l column f r a c t i o n s f o r i n h i b i t o r y a c t i v i t y i m p r a ctical. For t h i s reason, immediately a f t e r each step of NaCl el u t i o n , the f r a c t i o n s were pooled and concentrated to a f i n a l volume of approximately 1.0 ml. This volume was then dialyzed overnight and mixing assays were c a r r i e d out the following day to determine i n h i b i t o r y a c t i v i t y . The s a l t concentrations used to elute the protein were 0, 100 mM, 300 mM and 500 mM NaCl. P r o t e i n assays were ca r r i e d out on i n d i v i d u a l f r a c t i o n s throughout the e n t i r e s a l t gradient. The functional d i s t r i b u t i o n of p r o t e i n eluted from the DEAE column i s shown i n Table 10. The large majority of ODC i n h i b i t o r y a c t i v i t y was found i n the 300 mM NaCl e l u t i o n volume. The r e s u l t s i n Table 10 o u t l i n e the d i s t r i b u t i o n of i n h i b i t o r y a c t i v i t y found i n the various concentrated volumes from each e l u t i o n step. Polyacrylamide g e l electrophoresis was then used to estimate the number of proteins contained i n each pool of eluted f r a c t i o n s . The 300 mM NaCl pool showed s i x bands. The darkest staining were a si n g l e band that migrated to a p o s i t i o n corresponding to 120,000 daltons and a p a i r of bands at approximately 60,000 daltons. There was FIGURE 7 EFFECT OF SODIUM CHLORIDE ON THYMUS ODC ACTIVITY ODC a c t i v i t y was assessed at various s a l t concentrations. These a c t i v i t i e s were then compared with the ODC a c t i v i t y measured i n the absence of s a l t . A l l assays were c a r r i e d out i n duplicate or t r i p l i c a t e . Percent Inhibition of ODC Activity TABLE 10 PURIFICATION OF THYMUS ODC INHIBITOR USING DEAE SEPHAROSE CL-6B COLUMN CHROMATOGRAPHY The column conditions used i n t h i s procedure are discussed i n the Methods section. The f r a c t i o n s eluted at each NaCl concentration were pooled and concentrated to a . f i n a l volume of approximately 1.0 ml. Each sample was then dialyzed f o r 24 hours and assayed f o r t o t a l protein content and units of ODC i n h i b i t o r y a c t i v i t y * . A l l assays were done i n duplicate or t r i p l i c a t e . E l u t i n g S a l t Concentration (mM) To t a l Protein (mg) Units of Thymus ODC I n h i b i t i o n * 0 2.38 120 100 1.06 70 300 0.34 1440 500 0.14 0 * one unit of i n h i b i t i o n corresponds to a reduction of enzyme a c t i v i t y from c o n t r o l animals by 1 pmole as measured mixing i n a 30 minute assay (see Methods s e c t i o n ) . also a strong band at 232,000 daltons. DEAE Sepharose CL-6B chromatography was c a r r i e d out ro u t i n e l y p r i o r to any other p u r i f i c a t i o n experiments. I t res u l t e d i n a p u r i f i c a t i o n of approximately 6 f o l d and enabled the concentration and long term storage of the i n h i b i t i n g substance at 0°C. In ad d i t i o n , i t has been demonstrated elsewhere that DEAE Sepharose i s an e f f e c t i v e procedure for near complete removal of the detergent Lubrol (81). Another method for removing Lubrol from the extract containing the ODC i n h i b i t i n g substance i s ammonium sulf a t e p r e c i p i t a t i o n . This procedure was c a r r i e d out on the crude thymus extract, p r i o r to i t s a p p l i c a t i o n onto the DEAE Sepharose column. While a small p u r i f i c a t i o n was obtained, the extensive d i a l y s i s required to remove the s a l t resulted i n only a 30% recovery of i n h i b i t o r y a c t i v i t y , making i t unsuitable f or p u r i f i c a t i o n purposes. Several d i f f e r e n t column chromatography, procedures were investigated for use as a d d i t i o n a l steps of p u r i f i c a t i o n . Neither gel f i l t r a t i o n , chromatofocussing or a second ion exchange column e q u i l i b r a t e d to a higher pH, resulted i n a workable system f o r further p u r i f y i n g the i n h i b i t o r . The use of Heparin Sepharose CL-6B did prove p a r t i a l l y successful, since under c a r e f u l l y c o n t r o l l e d conditions, the p a r t i a l l y p u r i f i e d ODC i n h i b i t i n g polypeptide did bind to the r e s i n . The same procedure was used to elute t h i s column as was developed f o r the i n i t i a l p u r i f i c a t i o n step using DEAE Sepharose. The large majority of i n h i b i t o r y a c t i v i t y eluted from the Heparin Sepharose column i n the 100 mM NaCl f r a c t i o n s . The r e s u l t s l i s t e d i n Table 11 show that a small amount of the I n h i b i t i o n did not bind to the column. Protein assays were again c a r r i e d out on 44. TABLE 11 PURIFICATION OF THYMUS ODC INHIBITOR USING HEPARIN SEPHAROSE CL-6B COLUMN CHROMATOGRAPHY This procedure was c a r r i e d out using the p a r t i a l l y p u r i f i e d i n h i b i t o r obtained from DEAE Sepharose CL-6B chromatography. The column conditions are described i n the Methods section. The f r a c t i o n s eluted at each NaCl concentration were pooled and concentrated to a f i n a l volume of approximately 1.0 ml. Each sample was then dialyzed for 24 hours and assayed f o r t o t a l p rotein content and uni t s of ODC i n h i b i t o r y a c t i v i t y * . A l l assays were done i n duplicate or t r i p l i c a t e . E l u t i n g S a l t T o t a l Protein Units of Thymus ODC I n h i b i t i o n * Concentration (mM) (mg) 0 2.1 1180 100 0.7 3890 300 0.5 630 500 0.4 670 * one unit of inhibition.corresponds to a reduction of enzyme a c t i v i t y from c o n t r o l animals by 1 pmole i n a 30 minute assay (see Methods section ) . i n d i v i d u a l f r a c t i o n s and the r e s u l t s are i l l u s t r a t e d i n Table 11. This step resulted i n an average 4 f o l d a d d i t i o n a l p u r i f i c a t i o n . These two steps of p u r i f i c a t i o n were repeated several times to confirm the r e s u l t and to accumulate s u f f i c i e n t quantities of the i n h i b i t i n g polypeptide f or a molecular weight estimation using gel f i l t r a t i o n . A Sephacryl S-200 column (90 cm x 1.4 cm) was equili b r a t e d with standard buffer and c a l i b r a t e d with f i v e molecular weight standards. The p a r t i a l l y p u r i f i e d i n h i b i t i n g polypeptide was applied to the column and the eluted f r a c t i o n s were checked f o r i n h i b i t o r y a c t i v i t y using the mixing assay. The r e s u l t s i n Figure 8 assign an approximate molecular weight to the ODC i n h i b i t i n g polypeptide of 54,000 daltons. The Role of Pyridoxal Phosphate i n Thymus ODC I n h i b i t i o n Preliminary r e s u l t s suggest that pyridoxal phosphate may be involved i n the mechanism of ODC i n h i b i t i o n i n thymus of dexamethasone-treated r a t s . PAMP A f f i g e l column chromatography has proven e f f e c t i v e i n pur i f y i n g thymus ODC from c o n t r o l r a t s (data not shown). When a crude thymus extract from dexamethasone-treated rats i s passed through t h i s system, an apparent regeneration of ODC a c t i v i t y i s detected at the void volume. When the f r a c t i o n s are assayed f o r i n h i b i t o r y a c t i v i t y , a small peak i s detected near the same p o s i t i o n i n the column p r o f i l e at which ODC eluted when p u r i f i e d from c o n t r o l animals. Mixing assays using extracts from rat thymus and thymus of dexamethasone-treated rats were conducted at various PLP concentrations. These r e s u l t s are inconclusive since the a c t i v i t y of thymus ODC i n c o n t r o l r a t s i s markedly reduced i n the absence of PLP. However, taking that i n t o account, the i n h i b i t i o n measured i n 46. FIGURE 8 MOLECULAR WEIGHT DETERMINATION OF THYMUS ODC INHIBITOR Molecular weight standards were used to c a l i b r a t e a Sephacryl S-200 g e l f i l t r a t i o n column. A p a r t i a l l y p u r i f i e d thymus ODC i n h i b i t o r extract was applied to the column and i t was eluted from the column at a point corresponding to a molecular weight of 54,000 daltons. This procedure i s discussed i n d e t a i l i n the Methods sect i o n . The standards used were: Blue dextran (2 x 10 6 ) , F e r r i t i n (440,000), Catalase (332,000), Aldolase (158,000) and B.S.A. (67,000). (J\A/) * H B ! 9 M JD | nDa | o y y ^ 47. the absence of PLP i s s t i l l higher than that with PLP present. Perhaps a cle a r e r r e s u l t would be obtained i f the mixing assays were car r i e d out using p a r t i a l l y p u r i f i e d i n h i b i t o r and enzyme. 48. DISCUSSION The r e s u l t s reported here suggest that the decrease i n ODC a c t i v i t y i n thymus and spleen of dexamethasone-treated rats i s related to the presence of an i n h i b i t o r of ODC produced i n response to the hormone treatment. The extraction of the i n h i b i t o r from thymus and spleen of dexamethasone-treated rats was s i g n i f i c a n t l y enhanced by Lubrol. A wide v a r i a t i o n i n i n h i b i t o r y a c t i v i t y was observed when the tissues were extracted with Hepes buffer only. Consistently high l e v e l s of i n h i b i t i o n were observed when a second extr a c t i o n , c a r r i e d out using 1% Lubrol, was combined with the aqueous extract. This e f f e c t does not appear to r e s u l t from the release of a d d i t i o n a l p r o t e i n as t o t a l protein concentrations i n thymus and spleen extracts did not r i s e s i g n i f i c a n t l y a f t e r a second Lubrol extraction as compared with a second buffer extraction. I t i s possible that the detergent helped break up the c e l l s i n a more e f f i c i e n t manner to produce a consistent homogenate which could not be accomplished with the motor driven homogenizer alone. The a c t i v i t y of ODC i n crude extracts of spleen from c o n t r o l rats was markedly reduced by concentrations of Lubrol as low as 0.1%. This may r e s u l t from the detergent bringing about the release of p r o t e o l y t i c enzymes, not present i n the thymus, which degrade ODC. The observed i n h i b i t i o n of ODC i s not an a r t i f a c t of the assay conditions or the extraction procedure. No i n h i b i t o r y a c t i v i t y was detected when the thymus of control rats was extracted f o r a second time using 1% Lubrol and then assayed f o r a b i l i t y to i n h i b i t enzyme from c o n t r o l r a t s . Also, the degree of i n h i b i t i o n was shown to remain constant when the substrate concentration was varied, i n d i c a t i n g that i n h i b i t i o n of ODC a c t i v i t y was not due to l i m i t i n g 49. concentrations of substrate. The i n h i b i t o r appears to be a polypeptide, since i t s a c t i v i t y was destroyed by treatment with heat or with t r y p s i n . Some s p e c i f i c i t y of the i n h i b i t o r i s suggested, since experiments showed that i n h i b i t i o n of SAMD did not occur i n the mixing assays. Since i t retains i t s i n h i b i t o r y function a f t e r d i a l y s i s and a f t e r u l t r a f i l t r a t i o n with exclusion l i m i t s of up to 50,000 daltons, the ODC i n h i b i t o r induced i n rat thymus by dexamethasone i s u n l i k e l y to require a small molecule for a c t i v i t y . This would seem to d i f f e r e n t i a t e i t s action from the group of ODC i n h i b i t o r s that function through p o s t - t r a n s l a t i o n a l modification. Both the transglutaminase (51) which requires putrescine, and the protein kinase 2+ (53) which requires Mg /ATP and a polyamine, would be inactivated by extensive d i a l y s i s . Low l e v e l s of i n h i b i t i o n are detected at 2.5 hours a f t e r dexamethasone i s administered, while high i n h i b i t o r y a c t i v i t y i s seen at 5 and 12 hours a f t e r the rats received the hormone. No i n h i b i t i o n i s measurable 24 hours a f t e r dexamethasone treatment. These re s u l t s suggest that i n the e a r l i e s t stages of the hormone induced l y t i c response, most of the i n h i b i t o r may be complexed with enzyme and at a l a t e r time, excess i n h i b i t o r i s present and therefore detectable by a mixing assay. The absence of i n h i b i t o r y a c t i v i t y at the l a t e r stages of the tissue response in d i c a t e s that the large majority of the lymphocytes had been destroyed. I t has been established that the ODC i n h i b i t o r i n thymus of dexamethasone-treated rats i n t e r a c t s with the enzyme i n such a way as to a l t e r i t s chromatographic properties (77). The mechanism of i n h i b i t i o n 50. appears to be non-competitive and non - c a t a l y t i c . I d e n t i c a l Km values for ornithine were determined f o r ODC i n thymus of control rats and ac t i v e ODC molecules i n mixing asssays of co n t r o l enzyme and i n h i b i t o r . Also, while the degree of i n h i b i t i o n v a r i e s s t o i c h i o m e t r i c a l l y with the r a t i o of volumes of i n h i b i t o r extract and enzyme extract, i t does not vary with time. Thus, i f the i n h i b i t o r i n t e r a c t s with the enzyme d i r e c t l y , i t does so i n a non-catalytic manner, possibly to form an i n a c t i v e enzyme-inhibitor complex. The procedure used to p a r t i a l l y p u r i f y the ODC i n h i b i t o r i n thymus extracts of dexamethasone-treated rats involved two column chromatography steps. While the Sephacryl S-200 gel f i l t r a t i o n procedure gave an approximate molecular weight determination, i t did not r e s u l t i n any further p u r i f i c a t i o n . The o v e r a l l p u r i f i c a t i o n of 24 f o l d would be s u b s t a n t i a l l y higher i f the recovery rates associated with both the DEAE Sepharose and Heparin Sepharose column chromatography procedures had been above 35%. I t was found that approximately 20% of the i n h i b i t o r y a c t i v i t y did not bind to the Heparin Sepharose r e s i n . In addition, the method of elut i n g both columns did not optimize recovery, since s i g n i f i c a n t d i l u t i o n of the i n h i b i t o r occurred. A gradient of i o n i c strength for column e l u t i o n would have been preferable, but t h i s was not p r a c t i c a l since i n d i v i d u a l f r a c t i o n s would then have to undergo d i a l y s i s to remove s a l t p r i o r to assay for ODC and i n h i b i t o r a c t i v i t y . The molecular weight of the p a r t i a l l y p u r i f i e d i n h i b i t o r was determined to'be approximately 54,000 at pH 7.4 i n 50 mM Hepes buffer, 10 mM d i t h i o t h r e i t o l and 100 mM EDTA. This f i n d i n g again d i f e r e n t i a t e s the 51. thymus ODC i n h i b i t o r from other reported ODC i n h i b i t o r s . The ODC i n a c t i v a t i n g substance found i n rat prostate (57) and the ODC antizyme found i n many tissues (44) have molecular weights of 26,000 or l e s s . The ODC i n h i b i t o r found i n r a t thymus seems to resemble a substance found i n slow growing rat p r o s t a t i c tumor (80). This i n h i b i t o r had a Mr of 68,000, but has not yet been f u l l y characterized. While i n h i b i t o r s of ODC have been reported by other laboratories, using other b i o l o g i c a l systems, t h i s i s the f i r s t instance i n which the occurrence of an i n h i b i t o r has been observed i n response to a ph y s i o l o g i c a l stimulus with a defined long range e f f e c t . Similar e f f e c t s of d i b u t y r y l cAMP and dexamethasone on the decarboxylases have been found i n cultured S49 lymphoma c e l l s (63). However, an i n h i b i t o r of ODC was not found i n an extract of d i b u t y r y l cAMP i n h i b i t e d c e l l s on mixing with extracts of co n t r o l c e l l s (81). 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