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Purification and properties of choline acetyltransferase from chicken brain Ma, Kelvin 1978

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PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM CHICKEN BRAIN by KELVIN ^ MA B.Sc, University of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DIVISION OF NEUROLOGICAL SCIENCES THE DEPARTMENT OF PSYCHIATRY FACULTY OF MEDICINE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1978 © K e l v i n Ma, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I ag ree that the 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 and s tudy . I f u r t h e r agree 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 purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g or pub l i ca t ion of 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 not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f Psychiatry The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 November 10, 1978 Date ' ABSTRACT Choline acetyltransferase (ChAc), the enzyme responsible f o r the synthesis of acetylcholine (ACh), has been extensively p u r i f i e d from chicken brains. P u r i f i c a t i o n procedures included ammonium s u l f a t e f r a c t i o n a t i o n , DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150 column chromatography, and a f f i n i t y chromatography on agarose-hexane-Coenzyme A column. ChAc a c t i v i t y was measured radiochemically. Due to the i n s t a b i l i t y of the enzyme i n the course of p u r i f i c a t i o n , the most active f r a c t i o n obtained a f t e r agarose-hexane-Coenzyme A chroma-tography showed a s p e c i f i c a c t i v i t y of only 0.34 umoles ACh formed/ min./mg protein which corresponded to a 773 f o l d p u r i f i c a t i o n from homogenate. However, on non-denaturing polyacrylamide gel electropho-r e s i s at pH 8.8, the highly p u r i f i e d ChAc preparation showed two d i s t i n c t bands, and ChAc a c t i v i t y was recovered by s l i c i n g and assaying the gel. ChAc a c t i v i t y corresponded to the p o s i t i o n of the fast e r moving band. The same preparation showed one major band and two minor bands on SDS gel electrophoresis. The estimated MW of chicken brain ChAc by gel f i l t r a t i o n and SDS gel electrophoresis was 42,500 daltons and no subunit was observed. Two forms of chicken brain ChAc with d i f f e r e n t Km values for the substrates were eluted from agarose-hexane-CoA column. The pH optimum was estimated to be 2+ 2+ between pH 7.6-8.0. NaCl, KC1, Ca and EDTA stimulated, while Cu , N-ethylmaleimide and CoA i n h i b i t e d the enzyme preparation. The appa-rent Km values for acetyl-CoA and choline were, studied and were found to be s i m i l a r to those of other mammalian species. The ChAc prepara-t i o n also showed species s p e c i f i c i t y by the Ouchterlony double immuno d i f f u s i o n t e s t . E f f e c t and mechanisms of s a l t and EDTA a c t i v a t i o n of i i i . ChAc a c t i v i t y are discussed. Dr. S.C. Sung Supervisor i v TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES i x INTRODUCTION 1 MATERIALS AND METHODS 6 I. Materials and chemicals 6 (i) Tissues 6 ( i i ) Chemicals 6 I I . Methods 7 (i) ChAc assay 7 ( i i ) Protein assay 8 ( i i i ) D i a l y s i s and concentration of enzyme solutions . . 8 (iv) Non-SDS polyacrylamide gel electrophoresis . . . 9 (v) Recovery of enzyme a c t i v i t i e s from polyacrylamide gels 10 (vi) P u r i f i c a t i o n procedures 10 (a) Extraction of ChAc a c t i v i t i e s from chicken brains 10 (b) Ammonium s u l f a t e f r a c t i o n a t i o n (35 - 65% saturation) 11 (c) DEAE-Sephadex (A-25) chromatography . . . . 12 (d) Chromatography on hydroxyapatite column . . 12 V Page (e) Gel F i l t r a t i o n on Sephadex G-150 column . . 13 (f) A f f i n i t y chromatography using agarose-hexane-CoA 14 ( v i i ) Molecular Weight determination 15 (a) Sephadex G-150 gel f i l t r a t i o n 15 (b) SDS polyacrylamide gel electrophoresis . . 16 ( v i i i ) Studies on the general properties of chicken ChAc 17 RESULTS 19 I. ChAc assay 19 I I . Regional d i s t r i b u t i o n of ChAc a c t i v i t y i n chicken brains 22 I I I . ChAc p u r i f i c a t i o n 25 (i) Extraction of ChAc a c t i v i t i e s 27 ( i i ) Ammonium s u l f a t e f r a c t i o n a t i o n 29 ( i i i ) DEAE-Sephadex (A-25) chromatography 29 (iv) Hydroxyapatite column chromatography 32 (v) Sephadex G-150 gel f i l t r a t i o n 32 (vi) Agarose-hexane-CoA chromatography 35 IV. P u r i t y of the f i n a l enzyme preparations 35 V. MW determination and subunit composition 43 VI. E f f e c t of pH on chicken brain ChAc 46 VII. K i n e t i c properties and end product i n h i b i t i o n of chicken b r a i n ChAc 48 VIII. E f f e c t s of s a l t s and s u l f h y d r y l reagent on chicken b r a i n ChAc 54 IX. E f f e c t of cupric ions and EDTA on chicken brain ChAc . 58 v i Page X. Immunodiffusion tests of chicken brain ChAc with antibodies against human brain ChAc 63 DISCUSSIONS 65 BIBLIOGRAPHY 83 v i i LIST OF TABLES page Table I Regional d i s t r i b u t i o n of ChAc a c t i v i t y i n chicken brain 26 Table II Ammonium su l f a t e f r a c t i o n a t i o n of chicken brain ChAc 30 Table I I I Summary of enzyme p u r i f i c a t i o n 31 Table IV Carnitine acetyltransferase (CtAc) a c t i v i t y i n chicken brain crude extract, Fractions 6a and 6b 38 Table V ChAc a c t i v i t y of Fractions 6a and 6b i n the presence and absence of eserine s u l f a t e 39 Table VI E f f e c t of ACh on the ChAc a c t i v i t y of Fraction 6a i n the absence of NaCl 55 Table VII E f f e c t of ACh on the ChAc a c t i v i t y of Fraction 6b i n the presence of 150 mM NaCl 55 Table VIII Summary of k i n e t i c parameters of chicken ChAc 56 Table IX E f f e c t of N-ethylmaleimide on chicken ChAc 56 2+ Table X E f f e c t of Ca on chicken ChAc 59 Table XI E f f e c t of Fract i o n 5 on the ChAc a c t i v i t y of Frac t i o n 6b 62 v i i i LIST OF FIGURES Page F i g . 1 E f f e c t of varying acetyl-CoA concentrations on the rate of ACh synthesis of F r a c t i o n 6b. 20 F i g . 2 E f f e c t of varying choline concentrations on the rate of ACh synthesis of Fraction 6b. 21 F i g . 3 Time course of the ChAc reaction up to 60 min. 23 F i g . 4 Dissection diagram showing the major anatomical regions of the chicken brain 24 F i g . 5 Flow chart of volume, ChAc a c t i v i t i e s , and mg proteins during the course of p u r i f i c a t i o n . 28 F i g . 6 E l u t i o n p r o f i l e of Hydroxyapatite column chromato-graphy . 33 F i g . 7 E l u t i o n p r o f i l e of Sephadex G-150. 34 F i g . 8 E l u t i o n p r o f i l e of Agarose-hexane-CoA chromatography. 36 F i g . 9 Non-SDS gel electrophoresis at pH 8.9 of F r a c t i o n 6a. 40 F i g . 10 Non-SDS gel electrophoresis at pH 8.9 of F r a c t i o n 6b. 41 F i g . 11 Non-SDS gel electrophoresis at pH 7.9 of Fraction 6b. 42 F i g . 12 S e l e c t i v i t y curve of F r a c t i o n 6b obtained from gel f i l t r a t i o n on Sephadex G-150. 44 F i g . 13 MW determination of chicken brain ChAc by SDS gel electrophoresis. 45 F i g . 14 E f f e c t of pH on the rate of synthesis of ACh by ChAc from Frac t i o n 6b. 47 F i g . 15 Double-reciprocal p l o t : 1/V vs. 1/[Acetyl-CoA] of F r a c t i o n 6b 49 F i g . 16 Double-reciprocal p l o t : 1/V vs. l/[Choline] of Fraction 6b. 50 F i g . 17 Double-reciprocal p l o t : 1/V vs. 1/[Choline] of Fraction 6a. 51 lx Page F i g . 18 CoA i n h i b i t i o n of rate of ACh synthesis of Fr a c t i o n 6a. 52 F i g . 19 Double-reciprocal p l o t s : 1/V vs. 1/[Acetyl-CoA] of Fracti o n 6a. 53 F i g . 20 E f f e c t of NaCl on chicken brain ChAc a c t i v i t y . 57 Fig . 21 The e f f e c t of cupric ions on chicken brain ChAc a c t i v i t y 60 F i g . 22 E f f e c t of EDTA on chicken brain ChAc of Fract i o n 6b and Frac t i o n 5. 61 F i g . 23. Immunodiffusion tests on Ouchterlony pla t e s . 64 X ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to Dr. S.C. Sung for his precious advices and assistance, i n v a r i a b l e patience, valuable teachings, understanding and kindness, together which made the comple-t i o n of the present project possible. I would also l i k e to express my deepest appreciation to Dr. E.G. McGeer for allowing me the opportunity to take up t h i s project and for her continuing i n t e r e s t and encouragement throughout the course of thi s work. I would also l i k e to thank my laboratory colleagues Dr. J.H. Peng and Mr. Jim Nagy for t h e i r companionship, encouragement, h e l p f u l suggestions and discussions. I also wish to thank Mr. Peter Kempe of the Department of Mineral Engineering, U n i v e r s i t y of B r i t i s h Columbia for kindly and meticulously performing the assays f o r cupric ions i n the incubation mixture. I am also g r a t e f u l to Mr. David Poll e n of United Poultry Ltd., Vancouver, B.C., for providing chicken heads for the p u r i f i c a t i o n . Also g r a t e f u l l y acknowledged i s Mr. David Tsai for his u n f a i l i n g assistance i n preparation of t h i s manuscript at a time when i t was needed most. F i n a l l y , I would l i k e to thank the Medical Research Council of Canada for providing f i n a n c i a l a i d for t h i s work through a studentship, without which my involvement with t h i s work would be d i f f i c u l t to sustain. 1 INTRODUCTION Acetylcholine (ACh) was the f i r s t neurotransmitter to be discovered. I t i s the neurotransmitter at the vertebrate neuromuscular junction and at s p e c i f i c synapses i n the autonomic nervous system. I t i s also an alleged neurotransmitter i n the vertebrate c e n t r a l nervous system. Be-cause of the important r o l e of ACh i n the nervous system, a considerable amount of research has been prompted into the l o c a l i z a t i o n , i s o l a t i o n , and characterization of the properties of choline acetyltransferase (ChAc), the enzyme responsible f o r ACh synthesis. ChAc catalyzes the transfer offamacety.lgroup from a c e t y l coenzyme A (acetyl-CoA) to choline. Despite a long h i s t o r y of in v e s t i g a t i o n s , the knowledge of c h o l i -nergic mechanisms i n the mammalian ce n t r a l nervous system i s s t i l l not developed to the extent known f o r the neurotransmitter function of ACh i n c e r t a i n peripheral synapses. One great problem facing researchers i n t h i s f i e l d has been the l o c a l i z a t i o n of c h o l i n e r g i c neurons i n the centr a l nervous system; hence one of the main objectives i n the i s o l a -tions of ChAc by various workers i s to use t h i s enzyme as. a d e f i n i t i v e marker f o r c h o l i n e r g i c neurons and th e i r processes. In recent years, immunohistochemical techniques Have offered the opportunity to l o c a l i z e various neurotransmitter synthetic enzymes i n the c e n t r a l nervous system. These techniques require antibodies to be produced against the trans-mitter synthetic enzyme and a good reaction between the antibodies and the antigenic enzyme i n the neuronal tissues which had been f i x e d . These techniques proved to be successful f o r the catecholaminergic sys-tems, GABAergic system and the serotonergic system (1, 2, 3, 4). The + + (CH 3) 3-N-CH2-CH2-OH + CH.j-CO-0-CoA ^  (Choline) (Acetyl-CoA) (ACh) 2 development of such an immunohistochemical method for the ch o l i n e r g i c system would depend on the preparation of pure ChAc and the production of monospecific antibodies of s u f f i c i e n t l y high t i t e r . Ever since ChAc was discovered by Nachmansohn and Machado (5) using an extract of rabbit brain, many workers had attempted to p u r i f y t h i s enzyme from various sources and to characterize i t . However, t h i s enzyme has been proven to be d i f f i c u l t to p u r i f y because of i t s very low concentration i n most tissues and i t s tendency to be unstable during p u r i f i c a t i o n . The r i c h e s t source of ChAc a c t i v i t y known i s DrosophiZa heads, with a s p e c i f i c a c t i v i t y i n the crude homogenate of 0.028 P-moles of ACh formed/min./mg. So f a r , p a r t i a l l y p u r i f i e d prepa-rations of ChAc have been obtained from human placenta (6, 7), r a t brain (8, 9), bovine brain caudate n u c l e i (10, 11), squid head ganglia (12), and even b a c t e r i a (13), cockroach and horse shoe crab (14). Enzyme preparations which are homogeneous e l e c t r o p h o r e t i c a l l y have also been claimed by some groups. These preparations include ChAc p u r i f i e d from human brain (15), human placenta (16), bovine brain (17), rat brain (18), squid head ganglia (19), Torpedo odlifomioa (20) and DrosophiZa melanogaster (21). In addition, immunocytochemical l o c a l i -zation of ChAc i n the c e n t r a l nervous system has been reported using antibodies to the e l e c t r o p h o r e t i c a l l y pure human (22, 23) or bovine enzyme (24). Although the enzyme ChAc has been p u r i f i e d from diverse species, many of the c h a r a c t e r i s t i c s of t h i s enzyme remain confusing and obscure. This enzyme also showed considerable species differences and ChAc from chicken brains, which was p u r i f i e d i n the present study, was also found to be d i f f e r e n t from other vertebrate and invertebrate ChAc i n c e r t a i n ways. 3 Despite the fac t that considerable differences were found i n the properties of ChAc i n d i f f e r e n t species, ChAc does bear a close a n t i -genic r e l a t i o n s h i p between some species. Singh and McGeer (25) reported cross-immunity between antibodies to human ChAc and ChAc from mammalian species but not with ChAc from non-mammalian species such as aves, f i s h , amphibians and r e p t i l e s . These workers concluded that ChAc from these non-mammalian species i s d i f f e r e n t i n structure from the ChAc protein of mammalian species. However, c o n f l i c t i n g r e s u l t s were obtained by another group of workers over the cross-immunity between mammalian ChAc antibodies and ChAc from aves (26). They observed c r o s s - r e a c t i -v i t y of rat ChAc antibodies with brain extracts from pigeon. If such r e s u l t s can be confirmed, then i t would be f e a s i b l e to use antibodies produced against chicken ChAc i n the immunocytochemical analysis of chol i n e r g i c systems i n human as well as other mammalian species. A recent report by Malthe-S^renssen (11) showed that ChAc from chicken brains was in a c t i v a t e d 80% by antibodies to bovine brain ChAc. In the present study, r e s u l t s w i l l be presented to attempt to resolved t h i s controversy using both crude and p u r i f i e d chicken ChAc. If ChAc from chicken brains can be p u r i f i e d and monospecific a n t i -bodies against the chicken enzyme can be prepared, then i t would o f f e r an excellent chance to study the c h o l i n e r g i c pathways i n the optic tectum. The avian o p t i c tectum i s a highly laminated and anatomically well described structure (27) and therefore i s an a t t r a c t i v e model for the study of topographical d i s t r i b u t i o n of neurotransmitters. Recent e l e c t r o p h y s i o l o g i c a l (28, 29) and biochemical (30) studies have sugges-ted a number of putative neurotransmitters i n the tectum. Among these are ACh and GABA. Using monospecific antibodies against chicken ChAc 4 to l o c a l i z e c h o l i n e r g i c structures i n the opti c tectum and the n u c l e i of opt i c lobe would c e r t a i n l y give more d e f i n i t i v e information con-cerning c h o l i n e r g i c neurons i n the optic tectum and aid i n better understanding of the v i s u a l system i n aves. Another reason for the p u r i f i c a t i o n of chicken ChAc i s that i t may also o f f e r the opportunity of examining c h o l i n e r g i c mechanisms i n diseased states where abnormalities of c h o l i n e r g i c systems are suspected, e.g., i n avian muscular dystrophy. Sung (31) reported increased ChAc a c t i v i t y i n the muscle of dystrophic mice and d i s s i m i l a r patterns of acetylcholinesterase (AChE) a c t i v i t y from extracts of normal and dys-trophic mouse muscle upon sucrose gradient sedimentation. Wilson et. a l . found that there were abnormalities i n the regulation and l e v e l s of muscle end-plate AChE i n dystrophic chickens (32, 33, 34, 35). These observations supported the hypothesis that there are a l t e r a t i o n s of the c h o l i n e r g i c systems i n murine and avian muscular dystrophies. Thus, i t seems possible that biochemical and anatomical demonstrations of abnormalities i n the c h o l i n e r g i c systems i n avian muscular dys-trophy can be c a r r i e d out i f a pure or highly p u r i f i e d preparation of chicken ChAc and antibodies against chicken ChAc are a v a i l a b l e . Apparently, a l l the suggested studies mentioned above depend on the preparation of a pure or highly p u r i f i e d chicken ChAc; hence, the immediate aim of the present study i s to p u r i f y ChAc from chicken brains extensively and to study i t s biochemical and p h y s i c a l properties. In view that chicken ChAc has never been p u r i f i e d or studied extensively before, and owing to i t s importance i n neuronal function, we are interested i n determining whether or not ChAc from chicken brain i s comparable i n properties to ChAc from other species; i n p a r t i c u l a r , we wish to study the molecular weight (MW) and subunit composition of 5 ChAc, s a l t e f f e c t s on ChAc, k i n e t i c properties of ChAc, i n h i b i t i o n of ChAc by i n h i b i t o r s and pH e f f e c t on the enzyme. I t has been well demons-trated that CoA, a product of the ChAc reaction, i s also a potent compe-t i t i v e i n h i b i t o r with respect to acetyl-CoA (36). A s p e c i f i c use of competitive i n h i b i t o r i s a f f i n i t y chromatography and the present work u t i l i z e d agarose-hexane-CoA a f f i n i t y chromatography for the f i n a l step i n the p u r i f i c a t i o n of ChAc from chicken brains. 6 MATERIALS AND METHODS I. Materials and chemicals (i) Tissues White Leghorns of both sexes, 3-6 months old, were used as the source of chicken brains f or the present p u r i f i c a t i o n . Chicken heads from dead unprocessed White Leghorns were supplied by United Poultry Ltd., Vancouver, B.C.. ( i i ) Chemicals 14 14 [Acetyl-1- C]-acetyl coenzyme A ( C-acetyl-labeled a c e t y l CoA) (54 mCi/nmole) was obtained from New England Corporation (Lachine, Que.). Choline chloride, acetylcholine bromide (AChBr), acetylcholine chloride (AChCl), eserine s u l f a t e , T r i t o n X-100, DL-carnitine-HCl, and coenzyme A were purchased from Sigma Chemical Co. (St. Louis, MO). Toluene was a product of Anachemia Ltd. (Montreal, Que.). l,4-Bis-[2-(5-phenyl-oxazoyl)]-benzene (POPOP) and 2,5-diphenyloxazole (PPO) were from Kent Laboratory (Vancouver, B.C.). Acrylamide, N,N'-methylenebis-acrylamide, N,N,N'N'-tetramethylethylenediamine, and g-mercaptoethanol were obtained from Eastman Kodak Co. (Rochester, N.Y.). Coomassie Blue G-250 was purchased from SERVA Feinbiochemica (Heidelberg). Ammo-nium persulfate, sodium dodecyl s u l f a t e (SDS), and hydroxyapatite (Bio-Gel HTP) were obtained from Bio-Rad Laboratories (Richmond, CA). Bovine serum albumin (BSA), cytochrome c, catalase, and N-ethylmaleimide were purchased from Calbiochem (La J o l l a , CA). Ammonium su l f a t e and Carbo-wax were from Schwarz/Mann Research Laboratories (Orangeburg, N.Y.). DEAE-Sephadex (A-25) and Sephadex G-150 were obtained from Pharmacia Fine Chemicals (Montreal, Que.). Agarose-hexane-CoA was a product of P.L. Biochemicals (Milwaukee, Wis.). Bromophenol Blue (BPB), ovalbumin, and 7 d i a l y s i s tubings were obtained from Fisher S c i e n t i f i c Co.. Unless otherwise s p e c i f i e d , a l l other common laboratory chemicals were "re-agent grade" and were purchased from Fisher S c i e n t i f i c Co.. II. Methods (i) ChAc assay ChAc a c t i v i t y was assayed radiochemically according to the method of Singh and McGeer (37) but with minor modifications. The assay measured the amount of radioactive ACh formed from -^C-acetyl-labeled a c e t y l -CoA and choline. The reaction mixture of a standard routine assay contained 50 mM sodium phosphate buffer, pH 7.4, 12.5 mM choline chlo-r i d e , 1 mM EDTA, 150 mM NaCl, 0.1 mM eserine s u l f a t e , 50yM 1 4 C - a c e t y l -labeled acetyl-CoA (0.01 Ci/assay), and 10 or 20pl of enzyme preparation i n a f i n a l volume of 80yl. When the protein concentration of the enzyme preparation was low, e.g. l e s s than 0.5 mg/ml, 10yl of BSA (1 mg/ml) was added to the reaction mixture ( f i n a l volume of assay mixture s t i l l 80yl) i n order to protect the enzyme from being denatured i n a d i l u t e environment. Unless otherwise s p e c i f i e d , the incubation was c a r r i e d out at 37°C for 30 minutes, and the reaction was terminated by the addi-t i o n of 1 ml of 0.025 N p e r c h l o r i c acid containing 0.011 N a c e t i c acid and 0.15 mM AChBr. To t h i s , 1 ml of 0.5 M T r i s - a c e t a t e , pH 7.0, was added and the contents were poured on top of Amberlite CG-50 columns (approximately 5 x 40 mm i n size) pre-equilibrated with 2 ml of 0.5 M T r i s - a c e t a t e , pH 7.0. The radioactive ACh that was formed was absorbed on the cation exchange r e s i n . Then, a f t e r washing the columns with 30 ml of d i s t i l l e d water, the radioactive ACh was eluted with 3 ml of 4 N a c e t i c acid d i r e c t l y into the counting v i a l s . Ten ml of s c i n t i l l a n t (570 ml of toluene, 430 ml of T r i t o n X-100, 4 g of PP0 and 150 mg of P0P0P) was 8 added to the v i a l and the r a d i o a c t i v i t y was counted with about 80% e f f -ciency using a Nuclear Chicago l i q u i d s c i n t i l l a t i o n counter. 1 unit of ChAc a c t i v i t y i s defined as 1 ymole of ACh formed/30 min. incubation. ( i i ) Protein assay Fractions c o l l e c t e d from d i f f e r e n t chromatographic columns were assayed for protein concentration by measuring the absorbance (at 260 and 280 nanometers) with a Beckman model DU spectrophotometer. A l l other protein determinations were c a r r i e d out according to the method of Lowry et a l . (38). ( i i i ) D i a l y s i s and concentration of enzyme solutions D i a l y s i s tubings were prepared by b o i l i n g them for 5 to 10 minutes and then washing them extensively. Then they were stored i n 10 mM EDTA at 4°C. The tubings were removed from the EDTA so l u t i o n and washed exhaustively with d i s t i l l e d water before usage. During d i a l y s i s , the d i a l y s i s tubing, f i l l e d with the enzyme s o l u t i o n , was swirled i n a fl a s k or beaker with a magnetic s t i r r e r . A r a t i o of 1 volume of enzyme to 50 volumes of d i a l y s i s buffer was used i n a l l d i a l y s e s . A l l d i a -lyses were performed at 4°C. Concentration of large volumes of .enzyme solutions was c a r r i e d out by ammonium s u l f a t e p r e c i p i t a t i o n (0-90% saturation). The required amount of ammonium su l f a t e to produce a 90% saturation was calculated according to the method of Noda et a l . (39). Ammonium s u l f a t e was added slowly to the enzyme solu t i o n so as to allow the ammonium su l f a t e c r y s t a l s to di s s o l v e . Then the cloudy so l u t i o n was s t i r r e d with a mag-n e t i c s t i r r e r at 4°C for 30 to 60 minutes. The mixture was then cen-trif u g e d and the proteins p r e c i p i t a t e d were dissolved i n small volumes of the appropriate buffer. 9 Concentration of small volumes of enzyme solutions was performed by immersing the enzyme so l u t i o n , placed i n d i a l y s i s tubing, into Carbowax f l a k e s . This procedure was used for concentrating enzyme solu-t i o n of less than 20 ml. The Carbowax flakes absorbed water from the enzyme so l u t i o n and the length of time for t h i s concentration proce-dure depends on the volume of the enzyme so l u t i o n . Concentration was c a r r i e d out at 4°C u n t i l the desired volume was reached. Unless otherwise stated, a l l c e n t r i f u g a t i o n procedures employed i n the present work were c a r r i e d out for 20 minutes at 10,000 rpm i n a S o r v a l l RC2. (iv) Non-SDS polyacrylamide gel electrophoresis Disc gel electrophoresis was performed as follows: 5% polyacrylamide gels were prepared by mixing 11.1 ml of 100 mM of e i t h e r T r i s - g l y c i n e buffer, pH 8.9, or Tris-HCl buffer, pH 7.9, 5 ml of water, 5 ml of acrylamide so l u t i o n (22.2g of acrylamide and 0.6 g of methylenebisacrylamide i n a f i n a l volume of 100 ml of water), 1.1 ml of f r e s h l y prepared ammonium persulfate (15 mg/ml), and 30 y l of tetramethylethylenediamine. Twelve clean tubes (0.5 cm x 7.5 cm) were placed v e r t i c a l l y i n a rack on a f l a t surface. The mixture was pipetted into the tubes c a r e f u l l y using a Pasteur pipet. The tubes were then covered with a few mm of water and allowed to polymerize overnight at room temperature. A f t e r the gel had polymerized, the layer of water was removed by tis s u e paper and the gel was ready for use. 100 y l of sample mixed with 10 y l of BPB (0.02% bromophenol blue i n 70% glycerol) was applied onto the top of the gel using a c a p i l l a r y pipet and then placed into the gel electrophoresis c e l l . Cold reser-v o i r buffer (either 50 mM T r i s - g l y c i n e buffer, pH 8.9, or 50 mM T r i s -HC1 buffer, pH 7.9, depending on the pH desired) was poured into the two res e r v o i r s of the c e l l and was also c a r e f u l l y layered on top of the sample. Then the electrodes were connected and a constant current of 2 mA per gel was applied for 2 hours i n the case of pH 8.9, and 3 hours f or pH 7.9. The electrophoresis power supply was a model 400 from Bio-Rad Laboratories. The samples were run at approximately 4°C. After 2 or 3 hours, the current was stopped and the gels were removed from the tubes. The gels were then either stained with Coo-massie Blue f o r 6 or more hours to detect proteins present i n the g e l , or frozen f o r subsequent ChAc assays. To remove the s t a i n from the gels, they were destained i n destaining s o l u t i o n (263 ml of methanol, 175 ml of a c e t i c a c i d , and water up to 3.5 1) using a d i f f u s i o n de-stainer with constant s t i r r i n g for 2 days. The destainer was a model 172A from Bio-Rad Laboratories. (v) Recovery of enzyme a c t i v i t i e s from polyacrylamide gels The gels, a f t e r being frozen for two hours, were s l i c e d into 1 mm s l i c e s using a g e l s l i c e r (model 190, Bio-Rad Laboratories). 3 ad-jacent s l i c e s were grouped together as one f r a c t i o n , crushed into smaller fragments, and placed into the incubation mixture for assaying 14 ChAc a c t i v i t i e s . The incubation mixture contained 50 yM C-acetyl-labeled acetyl-CoA (0.02 yCi/assay), 50 mM sodium phosphate buffer, pH 7.4, 12.5 mM choline chloride, 1 mM EDTA, 0.15 M NaCl, 0.1 mM ese-r i n e s u l f a t e and 40 p i of BSA (1 mg/ml), i n a f i n a l volume of 160 y l . The incubation was ca r r i e d out for 45 minutes at 37°C. (vi) P u r i f i c a t i o n procedures (a) Extraction of ChAc a c t i v i t i e s from chicken brains Chicken heads were obtained from 3-6 months old White Leghorns. 11 Before the c o l l e c t i o n of brain tissues for p u r i f i c a t i o n had started, the regional d i s t r i b u t i o n of ChAc a c t i v i t i e s i n the chicken brain was studied (discussed i n the RESULTS sectio n ) . Cerebellum was found to have very l i t t l e value for the purpose of ChAc p u r i f i c a t i o n . Whole brains were dissected from chicken heads and cerebella were removed and discarded. The di s s e c t i o n procedures were performed at 4°C, either i n cold room or on i c e . The brains were then weighed and homogenized with an Omni Mixer at a speed s e t t i n g of 10 for 3 minutes. The homo-genizing medium used contained 50 mM potassium phosphate buffer, pH 7.4, and 2 mM EDTA. 9 ml of t h i s buffer was used per gram of brain t i s s u e . The homogenate was then centrifuged at 10,000 rpm for 1 hour. The supernatant (Fraction 1) was retained for enzyme p u r i f i c a t i o n and the p e l l e t was discarded. (b) Ammonium s u l f a t e f r a c t i o n a t i o n (35-65% saturation) The procedures for ammonium s u l f a t e p r e c i p i t a t i o n have been des-cribed i n section ( i i i ) under Methods. Ammonium s u l f a t e was added to Fraction 1 to produce a 35% saturation. The mixture was s t i r r e d for 30 minutes at 4°C and then centrifuged. The supernatant was retained and ammonium su l f a t e was added to i t to produce a 65% saturation. A f t e r 30 minutes of s t i r r i n g at 4°C, the mixture was centrifuged. The p e l l e t , containing ChAc a c t i v i t i e s , was dissolved i n TEMG buffer (45 mM T r i s -HC1, pH 7.9: 1 mM EDTA: 2 mM g-mercaptoethanol: 10% v/v glycerol) using very mild hand homogenization. 1 ml of TEMG buffer was used to dissolve the p e l l e t that was p r e c i p i t a t e d from every 30 ml of Fract i o n 1. A f t e r the p e l l e t had been dissolved, the enzyme so l u t i o n was then dialyzed against TEMG buffer with 3 changes. The no n - d i f f u s i b l e mate-r i a l was then centrifuged o f f and the supernatant was c o l l e c t e d and stored frozen u n t i l a l l of Fract i o n 1 had been subjected to ammonium 12 s u l f a t e p r e c i p i t a t i o n . This supernatant was referred to as Fr a c t i o n 2. 710 ml of Fracti o n 2 were pooled but only 530 ml was subjected to DEAE-Sephadex chromatography. (c) DEAE-Sephadex (A-25) chromatography Beads of DEAE-Sephadex (A-25) were allowed to swell i n 45 mM T r i s -HC1 buffer, pH 7.9, at room temperature for 2 days. The swollen r e s i n was e q u i l i b r a t e d with TEMG buffer for a day with several changes of buffer. Then DEAE-Sephadex chromatography was performed batch-wise by mixing F r a c t i o n 2 with DEAE-Sephadex resins (0.7 g of DEAE-Sephadex beads/ml of Fracti o n 2). The mixture was s t i r r e d gently by hand as often as possible at 4°C for 1 hour. The ChAc a c t i v i t i e s i n the mix-ture were then separated from the resins by centrifugation at 17,000 rpm for 2 hours. The supernatant was then concentrated by ammonium su l f a t e p r e c i p i t a t i o n . The proteins p r e c i p i t a t e d were dissolved i n 10 mM PEMG buffer (10 mM potassium phosphate, pH 6.8: 1 mM EDTA: 2 mM 3-mercaptoethanol: 10% v/v g l y c e r o l ) . 1 ml of PEMG buffer was used for every 6 ml of supernatant that was subjected to ammonium s u l f a t e p r e c i p i t a t i o n . A f t e r the p e l l e t had been dissolved, the enzyme solu-t i o n was dialyzed against PEMG buffer with 3 changes of fresh buffer. A f t e r d i a l y s i s , the p r e c i p i t a t e was centrifuged off and the superna-tant (Fraction 3) was retained for the next step of p u r i f i c a t i o n . (d) Chromatography on hydroxyapatite column Bio-Gel HTP powder was suspended i n 10 mM potassium phosphate buffer, pH 6.8 (1 part HTP powder i n 6 parts b u f f e r ) , with gentle s t i r r i n g . A f t e r the s l u r r y had s e t t l e d down, the fin e s were decanted o f f . This procedure was repeated several times. This HTP suspension was then packed into a column to y i e l d a bed size of 2 cm x 30 cm and 1000 ml 13 of PEMG buffer was passed through to e q u i l i b r a t e the column. 10 ml of Fracti o n 3 was applied to the HTP column. Under these conditions, the ChAc a c t i v i t i e s were adsorbed to the Bio-Gel HTP. The non-adsorptive materials were washed away with 200 ml of 10 mM PEMG buffer. The el u t i o n of proteins was c a r r i e d out by f i r s t washing the column with 75 mM PEMG buffer to remove the materials that were bound not too t i g h t l y to HTP. Then a continuous gradient of increasing i o n i c strength was set up using PEMG buffers of two d i f f e r e n t i o n i c strengths. This was done by allowing one re s e r v o i r of 700 mM PEMG buffer to flow con-tinuously and mix with another r e s e r v o i r of 75 mM PEMG buffer. The buffer that flowed from the l a t t e r r e s e r v o i r into the HTP column thus had increasing i o n i c strength. With t h i s gradual increase of i o n i c strength, proteins were eluted gradually according to the degree of tightness of t h e i r binding to HTP. Throughout washing and e l u t i o n of the HTP column, the flow rate was maintained at 6 ml/hr. The washings (4 ml/fraction) and eluates (2 ml/fraction) were c o l l e c t e d i n test tubes using a f r a c t i o n c o l l e c t o r (Gilson Fractionator model SVM1). The tubes containing ChAc a c t i v i t i e s of s p e c i f i c a c t i v i t y greater than 1.4 units/mg protein were pooled and concentrated by ammonium s u l f a t e p r e c i p i t a t i o n . The p r e c i p i t a t e was dissolved i n 4 ml of 10 mM PEMG buffer and dialyzed against the same buffer with 3 changes of fresh buffer. Because a small volume was desired for the next step of p u r i -f i c a t i o n , the enzyme so l u t i o n was further concentrated into 1.3 ml by using Carbowax. This f r a c t i o n was referred to as Fract i o n 4. (e) Gel F i l t r a t i o n on Sephadex G-150 column Beads of Sephadex G-150 were allowed to swell i n 10 mM potassium phosphate buffer, pH 6.8 at room temperature for 3 days with gentle 14 occasional s t i r r i n g . Excess buffer along with the f i n e p a r t i c l e s were decanted o f f and replaced with fresh buffer; t h i s procedure was repeated 3 times during the swelling period. Then the gel s l u r r y was stored i n a cold room to reach the temperature of column operation of 4°C. The swollen Sephadex gel was poured c a r e f u l l y into a column to y i e l d a bed volume of 0.9 cm x 53.5 cm. Care was taken to avoid forma-t i o n of a i r bubbles i n the column. The column was then e q u i l i b r a t e d with 100 ml of 10 mM PEMG buffer. 1.3 ml of Fr a c t i o n 4 was placed on top of the Sephadex gel and was allowed to pass through the column. As soon as a l l the enzyme solu t i o n had entered the g e l , the column was washed with 50 ml of 10 mM PEMG buffer at a flow rate of 5 ml/hr. This chromatographic technique allowed separation of ChAc a c t i v i t i e s from other species of molecules according to t h e i r s i z e s . F r a c t i o n 4, af t e r passing through the column, was co l l e c t e d i n test tubes (1 ml/ tube) using a Gilson f r a c t i o n a t o r . 6 tubes containing ChAc with s p e c i -f i c a c t i v i t y greater than 1.6 units/mg were pooled and concentrated by Carbowax. The enzyme s o l u t i o n was concentrated to 1.1 ml and was centrifuged to remove trace amounts of impurities. The supernatant was col l e c t e d and was referred to as Fract i o n 5. (f) A f f i n i t y chromatography using agarose-hexane-CoA Agarose-hexane-CoA was supplied as a suspension containing gly-c e r o l and sodium azide. CoA was bound to the agarose by a t h i o l ester linkage with a s i x carbon spacer (hexane). Before packing the suspen-sion into a small column, the g l y c e r o l and sodium azide was washed away with d i s t i l l e d water. 2.5 ml of the washed agarose-hexane-CoA was packed into a very small column (0.6 cm x 6 cm) and was e q u i l i b r a -ted with 100 ml of 10 mM PEMG buffer. 1.1 ml of Fract i o n 5 was applied 15 onto the column and the substances which showed no or very weak a f f i -n i t y for CoA were washed away by passing 30 ml of 10 mM PEMG buffer through the column. The e l u t i o n of proteins bound to the column was c a r r i e d out i n a step-wise manner with increasing i o n i c strength. The following sequence of buffers were used: 10 mM PEMG buffers containing 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, 200 mM NaCl, 250 mM NaCl, 300 mM NaCl, and 500 mM NaCl. Throughout washing and e l u t i o n the flow rate was maintained at 15 ml/hr. The eluates were c o l l e c t e d into test tubes (1 ml/fraction) using a Gilson Fractionator. Fractions with high ChAc a c t i v i t i e s were c o l l e c t e d at 150 mM and 200 mM NaCl e l u t i o n s . Those f r a c t i o n s c o l l e c t e d at 150 mM NaCl e l u t i o n were pooled together, concentrated using Carbowax and dialyzed against 10 mM PEMG buffer. This f r a c t i o n was c a l l e d Fraction 6a. Those f r a c t i o n s c o l l e c t e d at 200 mM NaCl e l u t i o n were also pooled, concentrated and dialyzed i n the same manner. This f r a c t i o n was r e f e r r e d to as F r a c t i o n 6b. ( v i i ) Molecular weight determination The estimation of the MW of chicken ChAc was done by two tech-niques: Sephadex G-150 gel f i l t r a t i o n and SDS polyacrylamide gel electrophoresis. (a) Sephadex G-150 gel f i l t r a t i o n Preparation and packing of the Sephadex gel had been described i n d e t a i l under section v i ( e ) of Methods. Fract i o n 6b, Blue Dextran 2000, and d i f f e r e n t protein standards (BSA, MW 68,000; ovalbumin, MW 45,000; cytochrome c, MW 11,700) were applied separately to the column and eluted separately by 10 mM PEMG buffer. The volume of buffer at which these proteins and ChAc were eluted was referred to as the e l u t i o n volume (Ve). The e l u t i o n volume of Blue Dextran i s c a l l e d the void volume of the column. Blue dextran 2000 was assayed by measuring the 16 absorbance at 630 nm, BSA and ovalbumin at 280 nm, and cytochrome c at 410 nm, using a Beckmann model DU spectrophotometer. By measuring the e l u t i o n volumes of these d i f f e r e n t p rotein stan-dards of known molecular weights, the corresponding K values can be av Ve-Vo calculated according to the formula K = — — — , where Vo = e l u t i o n 6 av Vt-Vo' volume of Blue Dextran 2000, and Vt = t o t a l bed volume. The K av value i s a constant for a given compound when chromatographed on a s p e c i f i c Sephadex gel and i s s i m i l a r to a d i s t r i b u t i o n c o e f f i c i e n t as used i n p a r t i t i o n chromatography. A p l o t of K values versus the logarithm of the MW of each substance i s c a l l e d a s e l e c t i v i t y curve and i s l i n e a r within the f r a c t i o n a t i o n range of the Sephadex ge l employed. A s e l e c t i v i t y curve was constructed using the K values of BSA, oval-av bumin, and cytochrome c. A f t e r the K value for chicken ChAc had av been calculated from i t s Ve value, the MW of chicken ChAc was e s t i -mated d i r e c t l y using the s e l e c t i v i t y curve, (b) SDS polyacrylamide gel electrophoresis The procedures for performing SDS polyacrylamide gel e l e c t r o -phoresis were e s s e n t i a l l y the same as those described for non-SDS electrophoresis except for the following changes: — the gels (0.5 cm x 7.5 cm) were made up of 5 ml of 30% acrylamide, 40 mg methylenebisacrylamide, 10 ml 200 mM sodium phosphate buffer, pH 7.2, 2 ml of 1% SDS, 2 ml of water, 30 y l of tetramethylethylene-diamine and 1 ml of f r e s h l y prepared ammonium per s u l f a t e . — i n i t i a l l y , 3 mA per g e l was applied for 30 minutes, then the cur-rent was increased to 6 mA for 4 hours. — the r e s e r v o i r buffer used was 200 mM sodium phosphate buffer, pH 7.2. 17 Preparations of enzyme sample and MW protein standards for SDS gel electrophoresis were as follows: — 100 y l of enzyme sample (Fraction 6a or 6b) was added to 50 y l of 0.2 M sodium phosphate buffer, pH 7.2, containing 5% SDS-5% 3-mercaptoethanol, and was b o i l e d i n a 100°C bath for 10 minutes. Then 10 y l of BPB was added to each sample and applied onto the top of the SDS gels. — Protein standards used were BSA (MW 68,000), catalase (MW 58,000), and cytochrome c (MW 11,700). 5 yg of each protein standard was added to 150 y l of 0.2 M sodium phosphate buffer, pH 7.2, contain-ing 5% SDS-5% g-mercaptoethanol and b o i l e d i n a 100°C bath for 10 minutes. Then 10 y l of BPB was added to each sample and applied onto the top of the SDS gels. Similar to the s e l e c t i v i t y curve of gel f i l t r a t i o n of the protein standards, a curve of log MW versus the values can be constructed for the three protein standards. R^ value i s defined as the r a t i o of the distance t r a v e l l e d by the protein and BPB down the g e l ; R^ = distance migrated by protein ^ r> i c — , , J , — . After c a l c u l a t i n g the R £ value of distance migrated by BPB dye r chicken ChAc, i t s MW can be estimated by using the l i n e a r protein standard curve. ( v i i i ) Studies on the general properties of chicken ChAc A l l studies on the properties of chicken ChAc were conducted on e i t h e r F r a c t i o n 6a, 6b, or F r a c t i o n 5, by measuring i t s ChAc a c t i v i t y , i . e . the rate of ACh synthesis by the chicken enzyme. The a c t i v i t y was measured according to the method described i n section ( i ) under Methods, except that the time of incubation and the incubation medium was d i f f e r e n t i n each type of study. For example, i n the study of the / 18 e f f e c t of cupric ions on the a c t i v i t y of the enzyme, EDTA, which was rou t i n e l y added to the incubation medium i n ChAc assays, was omitted. In each s p e c i f i c study, the incubation medium and the time of incubation w i l l be s p e c i f i e d i n the Results section. 19 RESULTS I. ChAc assay The method for assaying ChAc a c t i v i t y has been described i n d e t a i l i n Materials and Methods section. It was based on the reaction between l a b e l l e d acetyl-CoA and unlabelled choline to give l a b e l l e d ACh, and the rate of ACh synthesis was defined as the a c t i v i t y of the enzyme. This microassay was proven to be s u i t a b l e f o r the purpose of detecting ChAc a c t i v i t y i n the f r a c t i o n s obtained during the present p u r i f i c a -t i o n . It required only y l amount of enzyme solutions and the blanks were equivalent to 0.01 nmole ACh formed/ 30 min. incubation. The re s u l t s were reproducible and showed a 5% deviation, and hence the assay was also used i n the studies of the properties of the enzyme i n which detection of changes or differences of ChAc a c t i v i t i e s i s required. Even though no other substances besides substrates were absolutely required for detection of ChAc a c t i v i t y , NaCl, EDTA, and eserine s u l f a t e were usually added to the incubation medium to increase the s e n s i t i v i t y of the assay. As w i l l be discussed l a t e r on i n the thesis , both NaCl and EDTA activated the a c t i v i t y of the enzyme. Eserine s u l f a t e serves as an anti-cholinesterase to protect the pro-duct ACh from being degraded. Figure 1 and 2 show the e f f e c t s of varying substrate concentra-tions on the rate of ACh synthesis. The concentrations of substrates used i n a routine ChAc assay were 50 yM of radioactive acetyl-CoA and 12.5 mM choline chloride which, according to Figures 1 and 2, were at saturating l e v e l s . However, the incubation time i n these two studies were only 15 minutes. In a routine ChAc assay i n which the incubation time was 30 minutes, radioactive acetyl-CoA (50 yM) may be the l i m i t i n g •20 r-0-2 F i g . 1. E f f e c t of varying acetyl-CoA concentrations on the rate of ACh synthesis, expressed as nmole ACh formed/assay (15 min. incubation), of Fraction 6b. Choline was kept constant at 12.5 mM. The incubation medium was the same as described i n ChAc assay section under MATERIALS AND METHODS. 0.2 [ C h o l i n e ] in m M F i g . 2. E f f e c t of varying choline concentrations on the rate of ACh synthesis of Fraction 6b. The rate of ACh synthesis was expressed as nmole ACh formed/assay (15 min. incubation). The concentration of radioactive acetyl-CoA was kept at a constant value of 50 yM. The incubation medium was the same as that described i n the ChAc assay section of MATERIALS AND METHODS. 22 substrate, and t h i s may explain the fac t that the ChAc reaction, ope-r a t i n g under the routine assay conditions, was l i n e a r with time only up to 15 minutes (see F i g . 3). The same s i t u a t i o n was also found i n the case of varying enzyme concentration i n the routine ChAc assay, i . e . l i n e a r i t y was observed between rate of ACh synthesis and enzyme con-centration only i f the enzyme a c t i v i t y i n each assay did not exceed 1 nmole ACh formed/30 min. incubation. The reason why the incubation time was not changed to 15 minutes instead of 30 minutes was that the standard method of assay which was developed and established i n our laboratory used 30 minute incubation time and many preliminary studies of the present work had employed t h i s method. Since longer incubation time did not a f f e c t the detection and comparison of ChAc during p u r i f i -cation, i t had been retained. However, i n studies i n which l i n e a r time dependance of ChAc a c t i v i t y i s important, e.g. i n k i n e t i c studies, a 15 minute incubation time was used instead. It should be noted that when s p e c i f i c a c t i v i t i e s of chicken ChAc i n the present p u r i f i c a t i o n were compared to values c i t e d by other i n v e s t i g a t o r s , they would appear to be lower because of the longer incubation time used. Moreover, other factors such as differences i n assay method and species hetero-geneity should also be taken into consideration. It should also be mentioned that choline at 12.5 mM did not show substrate i n h i b i t i o n . I I . Regional d i s t r i b u t i o n of ChAc a c t i v i t y i n chicken brains Chicken brains were dissected into the following regions: t e l e n -cephalon (cerebral hemispheres), thalamus, optic lobes, cerebellum, and brain stem (Fig. 4). Although the d i v i s i o n s were not d i s c r e t e , these regions were taken because they constitute the major d i v i s i o n s of the avian brain (40), and also because the purpose of t h i s study was 23 " 20 4 0 6 0 I n c u b a t i o n t i m e i n m i n F i g . 3. Time course of the ChAc reaction up to 60 min. Chicken brain crude extract (Fraction 1) was used as the enzyme preparation. ChAc a c t i v i t y was expressed as cpm/assay under the assaying conditions s p e c i f i e d i n the ChAc assay section under MATERIALS AND METHODS. Approximately 5000 cpm i s equivalent to 1 nmole ACh formed/assay i n 30 min. of incubation time. Fraction 1 used was 10 times d i l u t e d . • 24 F i g . 4. Dissection diagram showing the major anatomical regions of the chicken b r a i n : 1 - telencephalon, 2 -thalamus, 3 - opti c lobe, 4 - cerebellum, 5 - brain stem, 6 - o l f a c t o r y lobe, 7 - II nerve. (a) side view; (b) v e n t r a l view. 25 merely to give a guideline of what regions of the chicken brain should be used f or p u r i f i c a t i o n . As Table I shows, i n terms of both t o t a l ChAc a c t i v i t y and s p e c i -f i c a c t i v i t y , cerebellum was the l e a s t s u i t a b l e region to use for the purpose of ChAc p u r i f i c a t i o n . On the other hand, optic lobes c o n s t i -tuted the r i c h e s t source of ChAc a c t i v i t i e s i n chicken brains, having a s p e c i f i c a c t i v i t y of 0.028 unit/mg protein. Because of these r e s u l t s , c e r e b e l l a were discarded a f t e r dissections and were not included for p u r i f i c a t i o n . The r e s u l t s presented here are i n complete agreement with the r e s u l t s of Aprison et a l . on the d i s t r i b u t i o n of ChAc i n the pigeon brain (41). It i s i n t e r e s t i n g to note that the avian o p t i c lobes contain high ChAc a c t i v i t y as other workers had also reported extremely high ChAc a c t i v i t y i n the optic lobes of squid, which have three times higher s p e c i f i c a c t i v i t y than that of the . e l e c t r i c organ of Torpedo (42). I I I . ChAc p u r i f i c a t i o n 2525 g of chicken brain tissues (without cerebella) were c o l l e c t e d from 1000 chickens over a one month period. The brain tissues were eith e r homogenized immediately, centrifuged and fractionated by ammo-nium s u l f a t e , or stored frozen for up to a few days. There was l i t t l e or no loss of ChAc a c t i v i t y from the brain tissues during storage under frozen temperatures. Although large quantities of brain tissues were c o l l e c t e d , only a small portion of the ChAc a c t i v i t i e s obtained from these tissues were a c t u a l l y used i n t h i s p u r i f i c a t i o n . Other portions had been used f or t r i a l runs f or each p u r i f i c a t i o n step or l o s t i n unsuccessful attempts of p u r i f i c a t i o n , and approximately 25% to 50% of Fracti o n 3 i s s t i l l stored frozen for future p u r i f i c a t i o n s and inves-26 Table I. Regional d i s t r i b u t i o n of ChAc a c t i v i t y i n chicken brain. The brain regions were homogenized i n 50 mM sodium phosphate buffer, pH 7.4, containing 2 mM EDTA, (9 ml/g t i s s u e ) , and assayed for ChAc a c t i v i t y as described i n MATERIALS AND METHODS. Region ChAc a c t i v i t y S p e c i f i c a c t i v i t y (units/region) (units/mg protein) Telencephalon 1.95 + * 0.09 0.011 + 0.001* Thalamus 0.49 + 0.04 0.0169 + 0.0009 Optic lobes 1.58 + 0.04 0.028 + 0.001 Cerebellum 0.27 + 0.01 0.0054 + 0.0003 Brain stem 0.68 + 0.04 0.0202 + 0.0006 A l l these values represent the means ± S.E.M. of 3 chicken brains. 27 t i g a t i o n s . F i g . 5 shows the account of the amount of volume and ChAc a c t i v i t i e s during the course of p u r i f i c a t i o n . A summary of the r e s u l t s of p u r i f i c a t i o n of chicken ChAc i s reported i n Table I I I . (i) E x t raction of ChAc a c t i v i t i e s Homogenization of chicken brains i n 50 mM potassium phosphate buffer, pH 7.4, containing 2 mM EDTA, s o l u b i l i z e d approximately 55-60% of the t o t a l ChAc a c t i v i t i e s i n the ti s s u e s . E f f o r t s were made to t r y to improve on the extraction of ChAc a c t i v i t i e s from tissues by homogenizing them i n the same homogenizing buffer i n the presence of 200 mM NaCl. However, t h i s procedure had no e f f e c t on the extent of extraction- of ChAc a c t i v i t i e s from the ti s s u e s . Another attempt at solving the problem involved rehomogenization of the p e l l e t i n the presence of 200 mM NaCl. This procedure extracted only 4% more ChAc a c t i v i t i e s from the p e l l e t , hence i t was not employed i n the p u r i f i c a -t i o n . A d i f f e r e n t technique was also t r i e d using T r i t o n X-100 as an extraction agent. Extract obtained from homogenization of the tissues i n the presence of 0.25% T r i t o n X-100 did contain 96% of the t o t a l ChAc a c t i v i t i e s ; however, the s p e c i f i c a c t i v i t y of the extract was 50% lower than that of the extract obtained by the f i r s t method. Since the purpose of the present project i s to p u r i f y ChAc, i t i s undesirable to extract ChAc from the tissues using 0.25% T r i t o n X-100 because i t s o l u b i l i z e s many more undesired proteins from the neuronal membranes. The t o t a l ChAc a c t i v i t i e s obtained from the tissues were 4140 units and showed a s p e c i f i c a c t i v i t y of 0.013 unit/mg protein (see F i g . 5 and Table I I I ) . Extraction using the f i r s t method described extracted a t o t a l a c t i v i t y of 2270 units having a s p e c i f i c a c t i v i t y of 0.032 unit/mg protein (see F i g . 5 and Table I I I ) . 2525 g tissues homogenization HOMOGENATE centrifugation 180 ml of Fracti o n 2 not used f o r t h i s p u r i f i c a t i o n 85 ml of Fraction 3 not used f or t h i s p u r i f i c a t i o n 0.5 ml 1.1 unit 0.18 mg proteinj FRACTION 1 24100 ml 4140 units 311540 mg protein "20720 ml 2270 units 70330 mg protein "710 ml 1680 units 25920 mg protein | about 3/4 used for p u r i f i c a t i o n 530 ml FRACTION 2 FRACTION 2 FRACTION 3 1257 units 19349 mg protein 95 ml 725 units 6040 mg protein | only 10 ml used f or p u r i f i c a t i o n Flo ml 76 units L630 mg protein 1.3 ml 25 units |17.8 mg protein 1.1 ml 17.2 units 1_. 3 mg protein 1.7 ml 4.22 units 0.42 mg protein F i g . 5 Flow chart of volume, ChAc a c t i v i t i e s , and mg proteins during the course o f . p u r i f i c a t i o n . Since only 3/4 of Fraction 2 and about 1/10 of Fract i o n 3 was used for t h i s p u r i f i c a t i o n , a c t u a l l y only 7.9% of the t o t a l a c t i v i t i e s present i n homogenate was used f or t h i s preparation. 29 ( i i ) Ammonium s u l f a t e f r a c t i o n a t i o n 10 ml of Fraction 1 was used i n a preliminary study on ammonium su l f a t e f r a c t i o n a t i o n of chicken ChAc extract. As shown i n Table I I , ChAc a c t i v i t i e s were p r e c i p i t a t e d i n the range of 35-70% ammonium su l f a t e saturation. However, the actual range that was used for the present p u r i f i c a t i o n was 35-65% ammonium su l f a t e saturation as decided by the s p e c i f i c a c t i v i t y of each ammonium s u l f a t e f r a c t i o n . This range p r e c i p i t a t e d 74% of the 2270 units of ChAc a c t i v i t i e s present i n Fraction 1 and recovered 1680 units of ChAc a c t i v i t i e s having a s p e c i f i c a c t i v i t y of 0.065 units/mg protein. Ammonium su l f a t e was removed by d i a l y s i s before Fra c t i o n 2 was assayed and subjected to DEAE-Sephadex chromatography. ( i i i ) DEAE-Sephadex (A-25) chromatography Under the conditions described i n the procedures for DEAE-Sephadex chromatography, the enzyme did not bind to the r e s i n and can be recovered i n the supernatant upon ce n t r i f u g a t i o n of the r e s i n -enzyme mixture. Approximately 1260 units of ChAc a c t i v i t i e s were applied to the r e s i n and 725 units (57%) of enzyme were recovered. This batch-wise procedure yielded an enzyme preparation with a spe-c i f i c a c t i v i t y of 0.12 units/mg protein, a two f o l d enrichment of ChAc s p e c i f i c a c t i v i t y over Frac t i o n 2 and a 9 f o l d p u r i f i c a t i o n over i n i t i a l homogenate. The r e s u l t s of t h i s chromatography may be improved i f column chromatography was employed instead of a batch-wise pro-cedure. However, i t was not f e a s i b l e , i n terms of time and equip-ment, to use column for t h i s technique because of the huge amount of material (530 ml) that was needed to be chromatographed. Table I I . Ammonium s u l f a t e f r a c t i o n a t i o n of chicken brain extract. Values of ChAc a c t i v i t i e s were averages of 2 studies i n which 10 ml of Fraction 1 was used i n each study. ChAc a c t i v i t y was assayed as described i n MATERIALS & METHODS. Enzyme preparation Total ChAc a c t i v i t y (units) % Recovery S p e c i f i c a c t i v i t y (units/mg protein) Fraction 1 (extract) 1.375 100 0.044 0-35% saturated ammonium sul f a t e f r a c t i o n 0.151 11 0.015 35-50% saturated ammonium sul f a t e f r a c t i o n 0.448 33 0.097 50-55% saturated ammonium sulf a t e f r a c t i o n 0.336 24 0.135 55-60% saturated ammonium sulf a t e f r a c t i o n 0.235 17 0.076 60-70% saturated ammonium su l f a t e f r a c t i o n 0.063 0.027 70-100% saturated ammonium sul f a t e f r a c t i o n * 0.095 0.014 Total a c t i v i t i e s and % recovered i n a l l ammonium sul f a t e saturated f r a c t i o n s 1.328 97 * This f r a c t i o n was the supernatant obtained a f t e r the 60-70% saturated ammonium su l f a t e f r a c t i o n was centrifuged. Table I I I . Summary of enzyme p u r i f i c a t i o n . Step ChAc a c t i v i t y ( u n i t s ) * S p e c i f i c a c t i v i t y (units/mg protein) P u r i f i c a t i o n f o l d % Recovery Homogenate •k-k 327 0.013 1 100 Enzyme extract (Fraction 1) ** 179 0.032 2.4 55 Ammonium s u l f a t e f r a c -t i o n a t i o n (Fraction 2) ** 133 0.065 5 41 DEAE-Sephadex (Fraction 3) 76 0.12 9.2 23 Hydroxyapatite (Fraction 4) 25 1.4 107 8 Sephadex G-150 (Fraction 5) 17.2 2.35 181 5 Agarose-hexane-CoA (Fraction 6a) 1.1 6.11 470 0.3 (Fraction 6b) 4.22 10.05 773 1.3 1 unit of ChAc a c t i v i t y represents the enzyme required to form 1 pmole ACh/30 min. incubation. These values were obtained by multiplying the o r i g i n a l ChAc a c t i v i t y values (4140, 2270, and 133, see F i g . 5) by 0.079, since only 7.9% of the t o t a l ChAc a c t i v i t i e s present i n the Homogenate were a c t u a l l y used for th i s p u r i f i c a t i o n . 32 (iv) Hydroxyapatite column chromatography 10 ml of Fract i o n 3 containing 630 mg proteins and 76.2 units of ChAc a c t i v i t i e s were applied to hydroxyapatite column. Approxi-mately 80 mg of unbound proteins were removed from the column by washing with 10 mM PEMG buffer, pH 6.8. As seen i n F i g . 6, a major portion of the undesired proteins were eluted with 75 mM PEMG buffer, pH 6.8. F i n a l l y , ChAc a c t i v i t i e s were eluted at approxi-mately 200 mM PEMG buffer concentration using a continuous gradient e l u t i o n . F r a c t i o n 4 obtained a f t e r t h i s step showed a s p e c i f i c a c t i v i t y of 1.4 unit/mg protein. Although hydroxyapatite column chromatography only recovered about 33% of enzyme a c t i v i t i e s , i t yielded a 12 f o l d p u r i f i c a t i o n over the previous step and a 108 f o l d enrichment of enzyme s p e c i f i c a c t i v i t y over i n i t i a l homogenate. Generally speaking, hydroxyapatite column chromatography seemed to be a good procedure to use for p u r i f i c a t i o n of ChAc as many other workers also employed i t to p u r i f y ChAc from diverse sources (8, 12, 15, 17, 18, 19, 20). The mechanism of the ChAc-hydroxyapatite i n t e r -action w i l l be discussed i n DISCUSSIONS. (v) Sephadex G-150 g e l f i l t r a t i o n Upon Sephadex G-150 gel f i l t r a t i o n , Fraction 4 displayed one major broad protein peak and several very minor peaks, as shown i n F i g . 7. Aft e r g e l f i l t r a t i o n , the enzyme f r a c t i o n (Fraction 5) con-tained about 17.2 units of ChAc a c t i v i t i e s and about 7.3 mg proteins, y i e l d i n g a s p e c i f i c a c t i v i t y of 2.35 units/mg protein. The recovery of enzyme for t h i s step was about 70% and the p u r i f i c a t i o n f o l d over i n i t i a l homogenate was 180. The sing l e enzyme peak shown i n F i g . 7 c l e a r l y suggests that ChAc migrated through the Sephadex column as a singl e MW protein. It* 4 ft 1600 1800 ELUTION BUFFER I N M L F i g . 6. E l u t i o n p r o f i l e of Hydroxyapatite column chromatography. 10 y l of each f r a c t i o n ( d i l u t e d 1:30) was used for assaying ChAc a c t i v i t y . ChAc a c t i v i t y was expressed as t o t a l units of a c t i v i t y present i n each f r a c t i o n . • • Enzyme a c t i v i t y A — A A280 ' 1000 F R A C T I O N N U M B E R F i g . 7. E l u t i o n p r o f i l e of Sephadex G-150. 10 y l of each f r a c t i o n ( d i l u t e d 1:200) was used for assaying. • • Enzyme a c t i v i t y ; v v Protein absorbance at A . 2 o U CAT = ChAc. 35 (vi) Agarose-hexane-CoA chromatography Under the conditions described i n the procedures for agarose-hexane-CoA chromatography, chicken brain ChAc did i n t e r a c t with CoA which was linked to the agarose by a t h i o l ester linkage with a s i x carbon spacer (hexane). Washing the agarose-hexane-CoA column with 10 mM PEMG buffer eluted l i t t l e or no ChAc a c t i v i t i e s , i n d i c a t i n g that chicken brain ChAc was bound to the CoA column. F i g . 8 which depicts the e l u t i o n p r o f i l e for t h i s step, shows the majority of ChAc a c t i -v i t i e s were eluted at 200 mM NaCl while only about 1/5 of the t o t a l eluted ChAc a c t i v i t i e s appeared at 150 mM NaCl e l u t i o n . These two populations of chicken ChAc a c t i v i t i e s had s p e c i f i c a c t i v i t i e s of 10.05 units/mg protein and 6.11 units/mg protein r e s p e c t i v e l y . The recovery of t o t a l ChAc a c t i v i t i e s for t h i s step was 30% from the previous step, hence making the f i n a l t o t a l ChAc recovery 3% from extract and 1.6% from homogenate. The t o t a l ChAc a c t i v i t i e s obtained at the end of t h i s p u r i f i c a t i o n was 5.32 u n i t s ; however, the enzyme a c t i v i t y was not stable and the enzyme l o s t more than 50% of i t s a c t i v i t y a f t e r a week's storage. It was i n t e r e s t i n g t o n o t e that chicken brain ChAc a c t i v i t i e s were eluted at two d i f f e r e n t concen-tr a t i o n s of s a l t suggesting the p o s s i b i l i t y of existence of 2 ChAc isozymes. This w i l l be discussed i n more d e t a i l i n the DISCUSSIONS section. IV. P u r i t y of the f i n a l enzyme preparations Two other known enzymes related to ChAc that are .present i n brain tissues are c a r n i t i n e acetyltransferase and acetylcholinesterase. The p o s s i b i l i t y that the f i n a l ChAc preparation may be contaminated with these two enzymes was tested and the r e s u l t s are shown i n Table IV 36 6000 50 0 0 4000 3000 > < & 2000 < 0 -O 1010 I I I 1 5 o m M i o o m M i s o m M 200mM NaCI NaCI N a C l NaCI 4 I 2 5 o m M 300 m M 500mM NaCI NaCI NaCI | 4 1 A -41 20 40 60 80 F R A C T I O N N U M B E R 100 120 F i g . 8. E l u t i o n p r o f i l e of Agarose-hexane-CoA chromatography. 10 y l of each f r a c t i o n (diluted 1:3) was assayed for ChAc a c t i v i t y . ± A Enzyme a c t i v i t y ; O-—O Protein absorbance at A 2 8 Q . 37 and Table V. Table IV shows that both Fractions 6a and 6b contained extremely low c a r n i t i n e acetyltransferase a c t i v i t y whereas i n chicken brain crude extract c a r n i t i n e acetyltransferase (CtAc) was present i n high q u a n t i t i e s . The parameter CtAc activity/ChAc a c t i v i t y for extract was 4.07, 240 times larger than that of Fractions 6a and 6b, which were 0.017 and 0.016 respectively. Since the parameter CtAc activity/ChAc a c t i v i t y indicates the r e l a t i v e amounts of the two enzymes i n the enzyme preparations, the r e s u l t s i n Table IV showed that CtAc a c t i v i t i e s were not p u r i f i e d along with ChAc a c t i v i t i e s but were eliminated by the p u r i f i c a t i o n procedures. The r e s u l t s of Table V indi c a t e that the presence of eserine s u l f a t e i n the reaction mixture had no e f f e c t on the ChAc a c t i v i t y of Fractions 6a and 6b, thus showing that the enzyme preparations were free of acetylcholinesterase. In order to check further the p u r i t y of the f i n a l enzyme prepara-t i o n s , non-denaturing gel electrophoresis was performed on Fractions 6a and 6b. Fraction 6a showed 2 major and 1 minor bands on the gel obtained by running non-denaturing gel electrophoresis at pH 8.9 (Fig. 9). Fr a c t i o n 6b showed only 2 bands on gel electrophoresis at pH 8.9 and 7.9 as shown i n F i g . 10 and F i g . 11. P a r a l l e l gels were run and assayed f or ChAc a c t i v i t i e s i n a l l three cases to locate which band corresponds to ChAc a c t i v i t i e s . For Fract i o n 6a, there was ChAc a c t i -v i t y associated with the f a s t e r moving band at pH 8.9 (Fig. 9). At the same pH, Fract i o n 6b also showed correspondence between the f a s t e r moving protein band and ChAc a c t i v i t i e s (Fig. 10) while at pH 7.9, ChAc a c t i v i t y seemed to migrate only s l i g h t l y and was associated with the slower moving band (Fig. 11). From these r e s u l t s , i t i s clear that both Fractions 6a and 6b are not e l e c t r o p h o r e t i c a l l y pure and Table IV. Carnitine acetyltransferase (CtAc) a c t i v i t y i n chicken b r a i n crude extract, F r a c t i o n 6a and 6b. Preparation CtAc a c t i v i t y * ChAc a c t i v i t y * CtAc a c t i v i t y / (units) (units) ChAc a c t i v i t y Crude extract 0.399 0.098 4.07 Fract i o n 6a 0.005 0.293 0.017 Fr a c t i o n 6b 0.008 0.484 0.016 * CtAc a c t i v i t y and ChAc a c t i v i t y were assayed by e s s e n t i a l l y the same method as that described f o r ChAc assay i n MATERIALS AND METHODS. Dowex 1 (chloride form) columns of s i z e 0.1 cm x 4 cm were used 14 instead of Amberlite columns. C-acetyl-labeled acetyl-CoA was 14 retained i n the column while C-acetyl-labeled a c e t y l c a r n i t i n e 14 and C-acetyl-labeled ACh were excluded into v i a l s for radioactive counting. The reaction mixture was e s s e n t i a l l y the same as that for the regular ChAc assay except for the case of CtAc assay, car-n i t i n e replaced choline as the substrate. The reaction was stopped by addition of 0.7 ml of i c e cold water and the reaction mixture was then poured onto Dowex columns prewashed with water. Then the columns were washed with two 0.7 ml washings of water. The eluates from the columns were counted for r a d i o a c t i v i t y . 1 unit of CtAc a c t i v i t y i s defined as the enzyme required to form 1 umole of acetylcarnitine/30 min. incubation. 39 Table V. ChAc a c t i v i t y of Fract i o n 6a and 6b i n the presence and absence of eserine s u l f a t e . In order to f i n d out i f there was any degradation of ACh by AChE present i n the enzyme preparations, eserine s u l f a t e which was ro u t i n e l y added to the assay mixture of ChAc assays was eliminated and replaced by 10 y l of water. Preparation Eserine s u l f a t e i n ChAc a c t i v i t y assay mixture (units/ml) Fraction 6a + 0.252 Fracti o n 6a - 0.249 Fracti o n 6b + 0.421 Fracti o n 6b - 0.416 1 0,000 5,000 Top - a _ 2 Gel Length I il c m Bottom F i g . 9. Non-SDS gel electrophoresis at pH 8.9 of Fraction 6a. The top diagram represents the ChAc a c t i v i t i e s recovered along the gel. The bottom diagram shows the p o s i t i o n of the protein bands along the g e l . 100 y l of Fract i o n 6a was applied to the gel, representing an estimated 36 yg of protein. 4,000 to g 3,000 a 2,000 u 1,000 i—rrl I \ I bp j L 1 2 3 4 5 Gel Length in cm TOP F i g . 10. Non-SDS g e l e l e c t r o p h o r e s i s a t pH 8.9 o f F r a c t i o n 6b. The top d i a g r a m r e p r e s e n t s t h e d i s t r i b u t i o n o f ChAc a c t i v i t i e s r e c o v e r e d on the g e l and t h e bot t o m i s t h e p i c t u r e o f t h e g e l showing t h e 2 p r o t e i n bands. 100 y l of F r a c t i o n 6b was used (an e s t i m a t e d 25 yg o f p r o t e i n p r e s e n t on t h e g e l ) . 42 5,000 4.000J > < U) 3,000r 2 Q 2,000f i.ooot T O P Gel Length in c m n T B O T T O M F i g . 11. Non-SDS gel- electrophoresis at pH 7.9 of Fracti o n 6b. The top diagram represents the d i s t r i -bution of ChAc a c t i v i t i e s recovered on the gel and the bottom diagram shows the p o s i t i o n of the protein bands on the gel with respect to ChAc a c t i v i t i e s . 100 y l of Fr a c t i o n 6b, approximately 25 yg of protein, was used. 43 contain one other type of protein besides ChAc even though ChAc appears to be the predominant protein present, judging from the i n -t e n s i t i e s of the bands. Frac t i o n 6b may be considered as a purer preparation because Fraction 6a displayed one a d d i t i o n a l band on the gel. It may also be speculated that the i s o e l e c t r i c point of chicken brain ChAc could be i n the neighborhood of 8 because ChAc migrated down the gel f a s t e r at pH 8.9 than at pH 7.9 and the distance migrated at pH 7.9 was very small. The i s o e l e c t r i c point of ChAc from chicken brain has never been studied before but Malthe-S^renssen demonstrated by i s o e l e c t r i c focusing that pigeon brain ChAc has an i s o e l e c t r i c point of 6.6 (43). V. MW determination and subunit composition Since the f i n a l enzyme preparations were not e l e c t r o p h o r e t i c a l l y pure, i n addition to SDS g e l electrophoresis, gel f i l t r a t i o n was em-ployed to estimate the MW of chicken ChAc. F i g . 12 shows the s e l e c t i -v i t y curve and the MW of F r a c t i o n 6b was estimated to be 41,500 d a l -tons. SDS gel electrophoresis was also performed to estimate MW and to obtain information concerning subunit composition of chicken brain ChAc. F i g . 13b shows that Fraction 6b gave 1 major and 2 much f a i n t e r bands on the SDS gel run at pH 7.2. It was assumed from the r e s u l t s of non-SDS gel electrophoresis that the predominant protein of the two proteins present i n F r a c t i o n 6b was ChAc. If the major band (R^ = 0.485) on the SDS gel was used as the marker for ChAc, then according to F i g . 13a (which shows the standard curve of log MW of protein markers versus t h e i r R^ values), chicken brain ChAc would have a MW of 42,500 daltons. This value i s i n good agreement with that obtained by gel f i l t r a t i o n . Since gel f i l t r a t i o n , which B S A (6 8,0 0 0 ) O v a l b v m i n C4 5,0 0 0) C A T C41.5 o o) c y t c C11,7 0 o ) 4 - • 9 K a v F i g . 12. S e l e c t i v i t y curve of Fraction 6b obtained from gel f i l t r a t i o n on Sephadex G-150. ChAc was eluted at 21.5 ml and Kav was ca l c u l a t e d to be 0.423. Vt (bed volume) = 33.8 ml; Vo = void volume = 12.5 ml; protein standards used were: BSA (MW 68,000; Ve = e l u t i o n volume =18.6 ml); ovalbumin (MW 45,000; Ve = 21 ml); and cytochrome c (MW 11,700; Ve = 28.4 ml) Ve - Vo Kav = — — . CAT = ChAc. Vt - Vo 45 2 O) o B S A C6 8,00 0 ) C a t a l a s e C5 8.0 0 0 ) C A T C4 2,5 0 0 ) c y t c C11.7 0 0 ) TD (b) TOP BOTTOM Fi g . 13. MW determination of chicken brain ChAc by SDS g e l electrophoresis. F r a c t i o n 6b was used. (a) Curve of log MW of protein standards Vs. the i r values. From SDS gel electrophoresis, R^ for the protein standards were: BSA (MW 68,000; R f = 0.35); catalase (MW 58,000; R f = 0.39); cytochrome c (MW 11,700; R f = 0.88). T> _ Distance migrated by protein . _ f Distance migrated by dye estimates the MW of undissociated ChAc, gave a value of 41,500 daltons, the major band (42,500 daltons) on the SDS gel probably represents the chicken brain ChAc monomer. The two f a i n t e r protein bands, having smaller values and higher MW, probably represent subunits of the other non-ChAc protein present i n Fraction 6b rather than ChAc sub-u n i t s . Although these experiments did not disprove the existence of ChAc subunits because of the heterogeneity of the enzyme preparation, i t seems possible from these r e s u l t s that chicken ChAc does not contain subunits. VI. E f f e c t of pH on chicken brain ChAc a c t i v i t y ChAc a c t i v i t y of F r a c t i o n 6b was measured i n a ser i e s of buffers with varying pH's ranging from pH 4 to pH 9 and the r e s u l t s are shown in F i g . 14. Below pH 5, the enzyme was p r a c t i c a l l y i n a c t i v e . Above pH 5, the enzyme was not too s e n s i t i v e to pH changes over a broad pH range of 6.5 to 8. D i f f e r e n t buffers were used i n t h i s experiment according to t h e i r b u f f e r i n g ranges. This created a problem since the a c t i v i t i e s obtained by assaying i n sodium acetate buffers were much lower than those obtained by assaying i n sodium phosphate b u f f e r s . At pH 6, ChAc assayed i n sodium phosphate buffers had a value more than 2 times that obtained by assaying i n sodium acetate buffers (see F i g . 14). This fact accounts for the d i s c o n t i n u i t y of the pH depen-dence curve. The same s i t u a t i o n was observed when Tri s - H C l buffer was used instead of sodium phosphate buffer; T r i s - H C l buffer yielded a smaller enzymic a c t i v i t y than sodium phosphate buffer at pH 8, but a greater a c t i v i t y than sodium phosphate buffer at pH 7.6. Despite the d i f f i c u l t y i n i n t e r p r e t i n g these r e s u l t s , i t i s s t i l l possible to see a d e f i n i t e pattern of pH dependence of ChAc over the pH range 47 Fig. 14. Effect of pH on the rate of synthesis of ACh by ChAc from Fraction 6b (1:10 diluted). The following buffers were used: pH 4.5 - 5.7: sodium acetate buffer; pH 5.7 - 7.6: sodium phosphate buffer; pH 8 - 9: Tris-HCl buffer. The assays were performed by the same procedures as described i n METHODS AND MATERIALS txcept that different buffers of different pH's replaced the normal buffer used, A A Tris-HCl as buffer; • • sodium phosphate as buffer; A A sodium acetate as buffer. 48 studied and the pH optimum can be estimated to be around pH 7.6 - 8. VII. K i n e t i c properties and end product i n h i b i t i o n of chicken brain ChAc The e f f e c t of varying substrate concentrations on the rate of ACh synthesis was studied and the r e s u l t s are shown i n Figs. 15, 16, 17 and 19, expressed as Lineweaver-Burke p l o t s . These double-reciprocal p l o t s were l i n e a r over the range of substrate concentrations studied. The apparent Km values f o r the substrates were calculated from the x - i n t e r -cepts of the p l o t s (Km = the negative r e c i p r o c a l of the x- i n t e r c e p t ) . In the presence of 300 mM NaCI, Fract i o n 6a had an apparent Km for acetyl-CoA of 17 yM; i n the absence of NaCI the value decreased to 11 yM (see F i g . 19, graph a and graph b). The apparent Km's for choline of Fractions 6a and 6b i n the presence of 150 mM NaCI were 200 yM and 455 yM re s p e c t i v e l y (Figs. 16 and 17). According to F i g . 15, the apparent Km for acetyl-CoA of Fract i o n 6b i n the presence of NaCI was calculated to be 6 yM. The i n h i b i t i o n of ACh synthesis by CoA i n the presence or absence of NaCI i s shown i n F i g . 18. In the presence of 300 mM NaCI, CoA at a concentration of 100 yM brought about a 50% i n h i b i t i o n of ACh syn-thesis whereas i n the absence of NaCI, i t caused a 80% reduction i n ChAc a c t i v i t y . I t took only 40 yM of CoA to cause a 50% i n h i b i t i o n of rate of ACh synthesis i n the absence of NaCI. Thus for chicken brain ChAc, NaCI seemed to provide protection against CoA i n h i b i t i o n . The e f f e c t of NaCI on the K i of CoA was then studied and the re-su l t s are shown i n F i g . 19. F i r s t , comparing ei t h e r graph a to c or graph b to d, i t can be seen that CoA exhibited competitive i n h i b i t i o n on chicken brain ChAc. Secondly, K i values can be calculated by the formula: x-intercept = , n—, • T n e K i for CoA was calculated r Km(l + 100/Ki) 49 m F i g . 15. Double-reciprocal p l o t : 1/V vs. 1/[Acetyl-CoA] of Fract i o n 6b. ChAc a c t i v i t i e s were assayed as described i n MATERIALS AND METHODS. Incubation time was 15 min. and 150 mM NaCl was present i n the assay mixture. Since the varying substrate was radioactive acetyl-CoA, the amount of r a d i o a c t i v i t y i n each assay was kept between 0.01 - 0.02 uCi by d i l u t i o n with non-radioactive acetyl-CoA. [Choline] was kept cons-tant at 12.5 mM. [Acetyl-CoA] was varied from 2.3 yM to 100 yM. The enzyme was d i l u t e d 1:10. The x-intercept was estimated to be -1.65 (M X 10^) . 1/V was expressed as 1/nmoles of ACh formed per assay. ^[Choline J fm M ) F i g . 16. Double-reciprocal p l o t : 1/V Vs. 1/[Choline] of Fraction 6b. Incubation time was 15 min. [Acetyl-CoA] was kept constant at 50 uM. [Choline] was varied from 0.03 mM to 4 mM. 150 mM NaCI was present i n the assay mixture. The enzyme was d i l u t e d 1:10. The x-intercept was estimated to be -2.2 mM 1/V was expressed as 1/nmoles ACh formed per assay. ' ' ' ' ' ' ' ' ' ' I I I I I I 4 8 12 16 20 24 28 32 [Choline]^"1^1) F i g . 17. Double-reciprocal p l o t : 1/V Vs. 1/[Choline] of Fraction 6a. 15 min. incubation time was used. [Acetyl-CoA] was kept constant at 50 yM. [Choline] was varied from 0.03 mM to 1 mM. 150 mM NaCl was present i n the assay mixture. The enzyme was d i l u t e d 1:5. The x-intercept was estimated to be -5 mM \ -J 100 200 300 400 500 600 700 [CoA] IN jblM F i g . 18. CoA i n h i b i t i o n of rate of ACh synthesis of Fraction 6a. Incubation time was 20 min. [Acetyl-CoA] was 50 pM and [Choline] was 12.5 mM. The enzyme was d i l u t e d 1:5. O O i n the presence of 300 mM NaCI; • • , i n the absence of NaCI. 800 9 00 —9 1 000 53 10 0 (d) o E u l < </> a) o E ' c (c) (b) - 2 . -1 ^ / [ A c e t y I - C o A](M" 1 x 1« 5 ) F i g . 19. Double-reciprocal p l o t s : 1/V vs. 1/[Acetyl-CoA] of Fraction 6a. Incubation time was 20 min. Since the varying substrate was radio-active acetyl-CoA, the amount of r a d i o a c t i v i t y i n each assay was kept between 0.01 - 0.02 yCi by d i l u t i o n with cold acetyl-CoA. [Acetyl-CoA] was varied from 2.3 yM to 50 yM. [Choline] was kept constant at 12.5 mil. The enzyme was d i l u t e d 1:5. (a) v v i n the presence of 300 mM NaCl, x-intercept = -0.6 X l o V 1 ; (b) * A no NaCl, x - i n t e r -5 -1 cept = -0.9 X 10 M ; (c) O O i n the presence of 300 mM NaCl plus 100 yM CoA, x-intercept = -0.15 X 10JM 1 ; (d) CoA, x-intercept = -0.1 X 10 5M - 1. no NaCl, 100 yM 54 to be 34 yM i n the presence of 300 mM NaCl and 12.3 yM i n the absence of NaCl. Hence NaCl increased the K i of CoA by 2.8 times and i t i s probable that t h i s decrease of a f f i n i t y of CoA for ChAc i s responsible for the decrease i n i n h i b i t i o n . This speculation needs further experi-ments to confirm. ACh i n h i b i t i o n of chicken brain ChAc a c t i v i t y was also i n v e s t i -gated and the r e s u l t s are shown i n Tables VI and VII. In the absence of NaCl, ChAc seems to be uninhibited by ACh up to 100 mM. In the presence of 150 mM NaCl, ChAc a c t i v i t y was i n h i b i t e d s l i g h t l y and i t took 200 mM ACh to cause a 28% i n h i b i t i o n . The e f f e c t of NaCl on ACh i n h i b i t i o n cannot be speculated further because F r a c t i o n 6a was used i n the f i r s t case while F r a c t i o n 6b was used i n the second study. Also, the chloride s a l t of ACh was used and t h i s complicated the i n t e r -pretation of r e s u l t s of the study i n which NaCl was excluded. However, i t does seem that ACh exerts very l i t t l e e f f e c t on chicken brain ChAc under conditions of saturating choline concentrations and 150 mM NaCl. Table VIII summarizes the k i n e t i c parameters studied f o r the chicken brain ChAc. VIII. E f f e c t s of s a l t s and s u l f h y d r y l reagent on chicken brain ChAc When the e f f e c t of NaCl on chicken brain ChAc was studied (Fig. 20), a c t i v a t i o n of ChAc was observed up to 0.6 M NaCl. [NaCl] greater than 0.7 M i n h i b i t e d ChAc a c t i v i t y . Maximum a c t i v a t i o n of ChAc a c t i v i t y occurred at 0.3 M NaCl which activated ChAc a c t i v i t y of Fract i o n 6a by 98%. Ef f e c t s of other s a l t s were also studied and at 150 mM KC1 and 50 mM sodium c i t r a t e , ChAc a c t i v i t y of Fract i o n 5 was increased by 93% and 80% res p e c t i v e l y . Ammonium su l f a t e at 75 mM i n h i b i t e d ChAc a c t i v i t y of Fr a c t i o n 5 by 30%. Ammonium chloride at 150 mM also 55 Table VI. E f f e c t of ACh on the ChAc a c t i v i t y of Frac t i o n 6a i n the absence of NaCI. Saturating choline concentration (12.5 mM) was used. [ACh-Cl] (mM) ChAc a c t i v i t y (% control) 0 100 5 87 25 104 50 99 100 96 Table VII . E f f e c t of ACh on the ChAc a c t i v i t y of Frac t i o n 6b i n the presence of 150 mM NaCI. Saturating choline concentra-t i o n (12. 5 mM) was used. [ACh-Cl] (mM) ChAc a c t i v i t y (% control) 0 100 5 93 25 99 50 86 100 79 200 72 56 Table VIII. Summary of k i n e t i c parameters of chicken ChAc. Preparation [NaCI] Apparent Km Apparent Km K i (CoA) (choline) (acetyl-CoA) Frac t i o n 6a 300 mM 17 yM 34 yM Frac t i o n 6a 0 mM 11 yM 12.3 yM F r a c t i o n 6a 150 mM 200 yM Fraction 6b 150 mM 455 yM 6 yM Table IX. E f f e c t of N-ethylmaleimide on chicken ChAc. Fracti o n 5 was used for t h i s study. Incubation time was 20 minutes. N-ethylmaleimide was added to the assay mixture and immediately assayed for ChAc a c t i v i t y . No pre-incubation of the i n h i b i t o r with the enzyme was done. ChAc a c t i v i t y was expressed as % of c o n t r o l . [N-ethylmaleimide] % of control (ChAc a c t i v i t y ) 0 mM 100 0.5 mM 30 1 mM 2.4 2 mM 0.5 Q4 0.3 > < CO W 02 < CO LU _ l o s 0.1 c U / / / u / / / / X 0.1 0.2 03 0.4 0.5 0.6 0J 0.8 0.9 1.0 N a C l I N M F i g . 20. E f f e c t of NaCl on chicken brain ChAc a c t i v i t y . F r a c t i o n 6a was assayed for ChAc a c t i v i t y as described i n MATERIALS'-AND METHODS except that NaCl i n the assay mixture was varied from 0 to 1 M. Incubation time was 15 min. The enzyme f r a c t i o n was d i l u t e d 1:5. ChAc a c t i v i t y was expressed as nmoles/assay, which represents nmoles ACh formed i n 15 min. incubation. 58 i n h i b i t e d ChAc a c t i v i t y of Fraction 5 by 30%. The e f f e c t of a l k y l a t i n g s u l f h y d r y l reagent was studied and the re s u l t s are shown i n Table IX. N-ethylmaleimide was found to be a powerful i n h i b i t o r of chicken brain ChAc a c t i v i t y . 2+ The e f f e c t of Ca was examined and the r e s u l t s are shown i n Table 2+ X. Generally, Ca had an a c t i v a t i n g e f f e c t on chicken ChAc although there was no d e f i n i t e pattern of a c t i v a t i o n observed. IX. E f f e c t of cupric ions and EDTA on chicken brain ChAc Cupric ions are known to i n h i b i t many enzymes and t h e i r e f f e c t on chicken brain ChAc was examined using F r a c t i o n 5. F i g . 21 demonstrates the i n h i b i t i o n by cupric s u l f a t e of the a c t i v i t y of chicken brain ChAc. Cupric ions are potent i n h i b i t o r s of chicken brain ChAc, reducing the enzyme a c t i v i t y by 50% at a concentration of 30 yM. The stimulatory e f f e c t of EDTA on chicken brain ChAc a c t i v i t y was investigated over a wide range of EDTA concentrations and the r e s u l t s are demonstrated i n F i g . 22. Chicken brain ChAc was activated almost maximally at 0.1 mM EDTA. According to the graph, Fraction 5 was stimulated to a much greater extent by EDTA than Fraction 6b and t h i s was investigated further i n another study. The r e s u l t s are shown i n Table XI. In the absence of EDTA, the a c t i v i t y obtained when the two f r a c t i o n s were added together was 16% (2356 cpm/assay compared to 2033 cpm/assay) greater than the sum of the i n d i v i d u a l a c t i v i t i e s of the two f r a c t i o n s . Hence the p o s s i b i l i t y that there may be an i n h i b i t o r y factor present i n Fraction 5 which was removed upon addition of EDTA was considered u n l i k e l y (discussed l a t e r i n DIS-CUSSIONS) . 2+ Table X. E f f e c t of Ca on chicken ChAc. Fracti o n 6b was used f o r t h i s study. Calcium chloride of d i f f e r e n t concentrations were added to the assay mixture and ChAc assay was performed according to the procedures described i n MATERIALS AND METHODS. Incubation time was 30 minutes. EDTA was removed from the assay mixture. 150 mM NaCl was present i n the assay mixture. Fra c t i o n 6b was dialyzed against 10 mM potassium phosphate buffer, pH 6.8, containing 2 mM g-mercaptoethanol and 10% g l y c e r o l to re-move EDTA from the enzyme f r a c t i o n . [Ca2+] ChAc a c t i v i t y (% control) 0 mM 100 0.05 mM 107 0.1 mM 206 0.5 mM 168 1 mM 248 3 mM 190 60 100 O DC o O 50 50 100 [ C U 2 + ] i n / A M F i g . 21. The e f f e c t of cupric ions on chicken brain ChAc a c t i v i t y . ChAc was assayed according to procedures described i n MATERIALS AND METHODS except that NaCI and EDTA were removed from the incubation mixture. Incubation time was 20 min. Fraction 5 was used and i t was dialyzed against 10 mM potassium phosphate buffer, pH 6.8, containing 2 mM 3-mercaptoethanol and 10% g l y c e r o l to remove EDTA from the enzyme f r a c t i o n . Cupric s u l f a t e was used as the source of cupric ions. % c o n t r o l represents % of ChAc a c t i v i t y present i n the f r a c t i o n com-2+ pared to the value of ChAc a c t i v i t y i n the absence of Cu ions. 4,000 -o [EDTA] in m M F i g . 22. E f f e c t of EDTA on chicken brain ChAc of Fraction 6b, (O O ) and of Fraction 5 (•—-#) . Both f r a c t i o n s had been dialyzed against 10 mM potassium phosphate buffer, pH 6.8, containing 2 mM g-mercaptoethanol and 10% g l y c e r o l to remove EDTA present i n the enzyme f r a c t i o n s . [EDTA] used were 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, and 4 mM. ChAc a c t i v i t y was expressed as cpm obtained i n the assay. 5000 cpm represents 1 nmole ACh formed i n 30 min. incubation. F r a c t i o n 5 was d i l u t e d 1:20 and Fraction 6b was dilut e d 1:10. Table XI. E f f e c t of Fract i o n 5 on the ChAc a c t i v i t y of Fract i o n 6b. 20 minute incubation time was used. Frac t i o n 5 was 1:30 d i l u t e d and F r a c t i o n 6b was 1:7 d i l u t e d . Preparation 1 mM EDTA ChAc a c t i v i t y (cpm/assay) Fr a c t i o n 5 - 243 Fraction 6b - 1790 Fracti o n 5 + Fra c t i o n 6b - 2356 Fracti o n 5 + 2044 Fracti o n 6b + 3840 Fracti o n 5 + Fra c t i o n 6b + 5972 In the absence of EDTA, the sum of ChAc a c t i v i t y of Fract i o n 5 and Fracti o n 6b i s 2033 cpm/assay (243 + 1790). In the presence of EDTA, the sum of ChAc a c t i v i t y of Fract i o n 5 and Fract i o n 6b i s 5884 cpm/assay (2044 + 3840). 63 X. Immunodiffusion tests of chicken brain ChAc with antibodies  against human brain ChAc Immunodiffusion tests were set up and performed on Ouchterlony type double d i f f u s i o n plates (Hyland Div. Travenol Laboratories, Inc. CA) as shown i n F i g . 23. The antiserum against human brain ChAc was obtained from Dr. J.H. Peng i n our laboratory, who injec t e d human brain ChAc (which showed 1 band on non-SDS gel electrophoresis) into rabbits (44). The anti-human ChAc antibodies showed a sing l e p r e c i -p i t i n band against human brain ChAc on double d i f f u s i o n Ouchterlony pla t e s . The antiserum also i n h i b i t e d 80% of human ChAc a c t i v i t y . When t h i s antiserum was tested against crude extract or highly p u r i -f i e d chicken brain ChAc, with d i f f e r e n t d i l u t i o n s of both antiserum and antigen, no p r e c i p i t i n band was seen. The normal rabbit antiserum also showed no reaction on double d i f f u s i o n Ouchterlony plates. 64 AS o 1/1 AS o i / i d "Oi/16 Ol/2 l/16o 0 l / 2 'o d" eP d 6B d o O d d d d (a) 1/4 a.) 1 / 8 1/4 F i g . 23. Immunodiffusion tests on Ouchterlony plates. Figs, (a) and (b), i n the center w e l l was placed about 10 y l of the antigen, either F r a c t i o n 6a or 6b. In the outer wells were placed about 10 y l (repeated 2 times) of rabbit anti-ChAc serum (AS), undiluted, 1/2, 1/4, 1/8, and 1/16 d i l u t e d . In Figs, (c) and (d) 10 y l of rabbit anti-ChAc serum was placed i n the center well and 10 y l (repeated 2 times) of antigen (either F r a c t i o n 6a or 6b), undiluted, 1/5, 1/10, and.1/20 d i l u t e d were placed i n the outer wells. 10 y l of chicken brain crude extract (EX) was also placed i n one of the outer wells. In F i g . (e), 10 y l of undiluted and 1/2, 1/4, 1/8, and 1/16 d i l u t e d normal rabbit serum (NS) was tested against 10 y l of chicken crude brain extract (EX) The plates were kept at 4°C for c was observed i n any of the cases. overnight periods. No p r e c i p i t i n band 65 DISCUSSION Using chicken brains as a source of ChAc, a f r a c t i o n that approached homogeneity has been i s o l a t e d using a procedure involving ammonium s u l f a t e f r a c t i o n a t i o n , DEAE-Sephadex, hydroxyapatite, Sepha-dex G-150 column chromatography, and agarose-hexane-CoA a f f i n i t y chromatography. This highly p u r i f i e d ChAc preparation showed 2 bands on non-SDS gel electrophoresis and had a s p e c i f i c a c t i v i t y of 0.34 ymole ACh formed/min./mg protein (10.05 units/mg p r o t e i n ) . Electropho-r e t i c homogeneity of ChAc p u r i f i e d from human brain or placenta has been obtained by Singh and McGeer (15) and Roskoski et a l . (16), and t h e i r preparations had s p e c i f i c a c t i v i t i e s of 0.01 and 0.04 ymole ACh formed/min./mg protein r e s p e c t i v e l y . Also, Chao (17) obtained a pure preparation of ChAc from bovine brain caudate n u c l e i with only a 48.6 p u r i f i c a t i o n \ f o l d J Hence, the s p e c i f i c a c t i v i t y of the ChAc prepara-t i o n obtained from the present p u r i f i c a t i o n does not seem low compared to these values e s p e c i a l l y considering the fact that the preparation i s not e l e c t r o p h o r e t i c a l l y pure. On the other hand, various groups had reported ChAc preparations with s p e c i f i c a c t i v i t i e s as high as 20, 30, and 92 ymole ACh formed/min./mg protein and yet they showed heterogeneity on non-SDS gel electrophoresis (6, 8, 11). It becomes d i f f i c u l t to explain such large discrepancies of r e s u l t s among the various groups of workers. One major factor that can account for the seemingly low s p e c i f i c a c t i v i t y of the present preparation and of some of the e l e c t r o p h o r e t i c a l l y homogeneous preparations obtained by some workers i s enzyme i n s t a b i l i t y . The present p u r i f i c a t i o n , along with p u r i f i c a t i o n s of other workers (6, 8, 15, 16, 18, 21), experienced 66 marked loss of a c t i v i t y during the l a t e r stages of p u r i f i c a t i o n . I, along with these workers, favor the view that the f i n a l ChAc prepara-t i o n contained i n a c t i v e ChAc. It i s well known that many enzymes are unstable at low protein concentrations. This i s the case with ChAc from quite a few species and various workers experienced such d i f f i c u l t y during p u r i f i c a t i o n (6, 8, 16, 18, 21). Rossier (8) found that when the protein concen-t r a t i o n was lower than 0.5 mg/ml, the 100 f o l d p u r i f i e d enzyme l o s t 50% of the a c t i v i t y i n 24 hours at 4°C. If such an enzyme preparation was d i l u t e d to 0.1 mg/ml with b i d i s t i l l e d water or with buffer, the enzyme l o s t a l l of i t s a c t i v i t y immediately a f t e r the d i l u t i o n . In the p u r i f i c a t i o n of ChAc from Drosophila melanogaster (21), loss of a c t i v i t y was minimized by maintaining protein concentrations above 0.1 mg/ml. During the course of the present p u r i f i c a t i o n , i t was not always possible to maintain the enzyme preparation at such a high protein concentration, e s p e c i a l l y i n the l a t e r stages of p u r i f i c a t i o n and when the enzyme preparation was d i l u t e d during passage through the chromatographic columns. Adding 10% v/v g l y c e r o l to the p u r i f i c a t i o n medium helped but did not eliminate the problem of denaturation of ChAc. The p o s s i b i l i t y that considerable ChAc denaturation had occurred during hydroxyapatite and agarose-hexane-CoA chromatography could also account for the presence of denatured chicken brain ChAc i n the f i n a l f r a c t i o n . In both cases, the active s i t e of ChAc was believed to be involved i n the binding to the column. In the course of binding and e l u t i o n , the active s i t e structure may be altered through i n t e r -action with hydroxyapatite, CoA and even agarose. These i n t e r a c t i o n s 67 w i l l be discussed i n more d e t a i l s i n a l a t e r section. Another d i f f i c u l t y encountered i n p u r i f i c a t i o n of ChAc i s low y i e l d . As i s the case with other ChAc p u r i f i c a t i o n s which had y i e l d s of l e s s than 3% (8, 15, 18, 19), the present p u r i f i c a t i o n had only 1.6% recovery of enzyme a c t i v i t y . This low y i e l d r e f l e c t s the number of steps i n the p u r i f i c a t i o n process, the retention of only the f r a c t i o n s of highest a c t i v i t y from each step and the i n s t a b i l i t y of the enzyme i n the f i n a l p u r i f i c a t i o n procedures. It has been w e l l established that ChAc can be recovered both i n the soluble form and p a r t i c u l a t e form from sucrose brain homogenates. In the present p u r i f i c a t i o n , the extraction procedure f o r ChAc also showed that only 55% of t o t a l ChAc a c t i v i t i e s was recovered i n the soluble form, the other 45% was associated with the r e s i d u a l membranes. It was observed that at low pH and low i o n i c strength, a l l the ChAc i n synaptosomes are bound to membranes, and i n increasing pH and i o n i c strength, ChAc l o s t i t s binding properties and became soluble (45). The r e v e r s i b l e binding of rat ChAc to membranes suggested that the enzyme has a p o s i t i v e charge and i s therefore attracted by negatively charged membranes. Fonnum (46) suggested that ChAc binds both to synap-tosome membranes and non-nervous membranes and that the binding of ChAc i s a general property of membranes. It was further proposed that a simple i o n i c exchange reaction takes place between negatively charged membranes and p o s i t i v e l y charged ChAc molecules. The binding reaction of ChAc to membranes may be expressed i n the following way: y MembraneX : Na + + x ChAc y + >• MembraneX : ChAc^ + + (y+x) Na + J x •< y x where x and y denote valencies of membranes and ChAc. The proportion 68 of membrane-bound ChAc i s therefore dependent on the concentration of cations such as Na + because they would force the equilibrium to the l e f t , and on the extent of p o s i t i v e charge on ChAc. The more p o s i t i v e the charge i s on ChAc, the more d i f f i c u l t i t would be for Na + to d i s -place ChAc from the membranes. Both increasing i o n i c strength and increasing pH would enhance release of ChAc from the negatively charged membranes. In agreement with t h i s theory, mammalian ChAc appears to be a basic enzyme usually showing multiple forms on i s o -e l e c t r i c focusing, e.g. p i values reported for the cat enzyme were 7.0, 7.8, and 8.4 (46) and 7.1, 7.5, and 8.4 for the rat enzyme (47). Moreover, ChAc from d i f f e r e n t species also has a strong a f f i n i t y for d i f f e r e n t c a t i o n i c resins such as Amberlite CG-50-11 and Carbbxymethyl-Sephadex. The binding of the enzyme to c a t i o n i c resins mimics i n a l l d e t a i l s the binding of ChAc to synaptosome membranes (48) and the experiments strongly suggested that the enzymes have a p o s i t i v e surface charge. In agreement with t h i s suggestion i s the r e s u l t observed i n t h i s p u r i f i c a t i o n with DEAE-Sephadex chromatography. Most of the ChAc was not absorbed by DEAE-Sephadex at pH 7.9 and i o n i c strength of 75 mM sodium phosphate. It was also observed i n the present work that chicken brain ChAc binds to Carboxymethyl-Sephadex. However, attempts to p u r i f y the enzyme using t h i s cation exchanger were unsuccessful, as the applied enzymatic a c t i v i t y could not be recovered using media (sodium phosphate buffer pH 5.9 containing 1 mM EDTA, 2 mM 3-mercapto-ethanol and 10% v/v glycerol) with increasing NaCl concentration up to 1.0 M. Baker (49) also encountered exactly the same d i f f i c u l t y with Carboxymethyl-Sephadex when p u r i f y i n g the Torpedo enzyme. From the foregoing discussions, i t seems that for extraction of chicken brain ChAc from membranes, r a i s i n g pH and i o n i c strength of the homogenizing buffer should be b e n e f i c i a l since chicken ChAc i s also believed to possess p o s i t i v e surface charge. Higher i o n i c strength was attempted by adding 200 mM NaCI to the homogenizing buffer but no improvement i n ChAc extraction was obtained. Extraction using homoge-n i z i n g buffer with higher pH has not been attempted yet. A further suggestion for ChAc extraction from chicken brain tissues i s to use a homogenizing buffer of high i o n i c strength and of a pH i n the range near the i s o e l e c t r i c point of chicken brain ChAc which would bring about a considerable loss of charge on the enzyme. As shown by the r e s u l t s of hydroxyapatite chromatography, chicken brain ChAc has a high a f f i n i t y f o r hydroxyapatite. Fonnum (48) con-cluded from h i s experiments that the binding mechanism for the enzyme to hydroxyapatite i s d i f f e r e n t from binding to synaptosome membranes, i . e . , not through charge i n t e r a c t i o n s . Rossier further proposed that the binding of the enzyme to hydroxyapatite columns was r e l a t e d to the hydro-phobic nature of the enzyme (50). It was suggested that ChAc contains the nucleotide f o l d and one of the c h a r a c t e r i s t i c s of the nucleotide f o l d i s i t s high hydrophobicity. Most of the nucleotide-metabolizing enzymes were observed to contain t h i s common s t r u c t u r a l domain. Thompson observed that a l l the enzymes containing the so-called 'nucleo-t i d e f o l d ' were i n h i b i t e d by Blue Dextran (51). Rossier found that Blue Dextran i s a potent ChAc competitive i n h i b i t o r with respect to acetyl-CoA (50). Moreover, the 'nucleotide f o l d ' was proposed to be involved i n the binding of ChAc to the nucleotide-containing coenzymes: acetyl-CoA or CoA. Rossier suggested that CoA w i l l bind through i t s adenosine moiety i n a hydrophobic nucleotide-binding s i t e close to the 70 imidazole r i n g . This concept of ChAc containing a dinucleotide f o l d i n the active s i t e was supported by a recent report by Malthe-Scirenssen (11). Hence, i n the present p u r i f i c a t i o n , a f f i n i t y chromato-graphy using agarose-hexane-CoA column was employed. However, the r e s u l t s were not as good as expected, possibly because i n the previous step of hydroxyapatite chromatography, the same sort of hydrophobic i n t e r a c t i o n may be involved. As shown i n F i g . 8, two ChAc peaks appeared upon e l u t i o n with 0.15 M and 0.2 M NaCl. This may be explained by the observation that rat brain ChAc, because of i t s hydrophobic nature, also binds to column of agarose-hexane alone (50). In the case with the chicken enzyme, there may be only one population of ChAc, with some bound to agarose-hexane and some bound to CoA. I n t e r e s t i n g l y , CoA-Sepharose was also employed by D r i s k e l l et a l . (21) to p u r i f y ChAc from Drosophila melanogaster heads, but only one peak of ChAc was eluted at 0.5 M NaCl. However, i n general, the k i n e t i c parameters of ChAc from invertebrates and verte-brates d i f f e r markedly and i t has been shown that squid ChAc does not bind to Blue Dextran or CoA columns (50). Caution should be taken when these r e s u l t s are compared to those from chicken brain ChAc. The two forms of chicken brain ChAc obtained from agarose-hexane-CoA chromatography do show differences i n t h e i r apparent Km values for both acetyl-CoA and choline (see Table V I I I ) . Malthe-S^renssen also obtained two d i f f e r e n t molecular forms of ChAc on Carboxymethyl-Sephadex chromatography (11) d i f f e r i n g i n Km's for acetyl-CoA (8 yM and 15 yM), but having same immunological and ph y s i c a l properties. It i s d i f f i c u l t to decide whether the differences i n Km values i n both cases are s i g n i f i c a n t enough to suggest the existence of isozymes 71 of ChAc p h y s i o l o g i c a l l y , e s p e c i a l l y considering that the k i n e t i c pro-p e r t i e s of ChAc also change i n the presence of s a l t (see Table V I I I ) . With the squid enzyme, Prempeh (52) suggested the existence of more than one d i s c r e t e form of ChAc with d i f f e r e n t s e n s i t i v i t i e s to s a l t s . It should be noted that isozymes of ChAc i n several species have been demonstrated. ChAc from both human brain and squid head ganglia had isozymes 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 c e l l u l o s e phosphate on column chromatography (12, 53). The human ChAc isozymes have s i m i l a r MW's but d i f f e r on the basis of s t a b i l i t y and antibody-producing capa-c i t y , while the squid ChAc isozymes d i f f e r i n heat s t a b i l i t i e s and i n th e i r c a p a c i t i e s to be activated by s a l t . S i m i l a r l y , Husain (19) also reported three forms of ChAc isozymes with d i f f e r e n t MW's, having d i f f e r e n t a f f i n i t i e s to c e l l o l o s e phosphate and sulfopropyl-Sephadex. In the rat and cat, three and two forms of ChAc res p e c t i v e l y , were observed by Malthe-S^renssen (43) to have d i f f e r e n t i s o e l e c t r i c points. From these r e s u l t s , i t i s not s u r p r i s i n g to observe two forms of chicken brain ChAc during t h i s p u r i f i c a t i o n ; however, i t s t i l l has to be stressed that these two forms may not e x i s t p h y s i o l o g i c a l l y but only represent the altered forms of ChAc a f t e r many p u r i f i c a t i o n steps. From the r e s u l t s of Table IV, i t can be concluded that Fractions 6a and 6b had extremely small acetyltransferase a c t i v i t y when c a r n i t i n e was used as a substrate i n place of choline. It i s d i f f i c u l t to decide whether t h i s extremely low c a r n i t i n e acetyltransferase a c t i v i t y indeed shows presence of c a r n i t i n e acetyltransferase contamination or the limi t e d a b i l i t y of ChAc to use c a r n i t i n e as substrate. Another p o s s i -ble contamination i n Fractions 6a and 6b i s acetylcholinesterase, but the fact that the presence of eserine s u l f a t e i n the reaction had no 72 e f f e c t on the enzyme a c t i v i t y of Fractions 6a and 6b (Table V) i n d i -cates that the preparations are free of acetylcholinesterase. ChAc exhibits a broad pH optimum (6.8-9.5) but a majority of species shows a peak at pH 6.8-7.4 (17, 20, 21, 54, 55, 56). The pH optimum of chicken brain ChAc was estimated to be between 7.6-8.0 and i t also exhibits a broad pH optimum (Fig. 14). It has generally been accepted that ChAc contains e s s e n t i a l sulfhy-d r y l groups (57, 58, 59) because of i t s s e n s i t i v i t y to s u l f h y d r y l rea-gents. Fraction 5 was used to test whether N-ethylmaleimide, an a l k y l a t i n g s u l f h y d r y l reagent, would i n h i b i t ChAc a c t i v i t y or not. At 1 mM of N-ethylmaleimide, Fra c t i o n 5 l o s t 98% of i t s ChAc a c t i v i t y (Table IX). This tends to suggest that chicken brain ChAc may also contain e s s e n t i a l s u l f h y d r y l groups. However, i t was recently proposed by Currier and Mautner (60) that the mode of action of the s u l f h y d r y l reagents was i n d i r e c t . The s u l f h y d r y l reagents may f i r s t react with the product of the ChAc reaction, CoA forming a mixed d i s u l f i d e which would be the a c t i v e compound. For example, methylmethanethiolsulfo-nate (CH^-S-SO^-CH^) w i l l react with CoA and form the mixed d i s u l f i d e CoA-S-S-CH^ which i s the most potent ChAc i n h i b i t o r ever synthesized (50, 61). The existence of a free s u l f h y d r y l group i n the active s i t e i s s t i l l a c o n t r o v e r s i a l problem (62). Nevertheless, one observation which favors the hypothesis that ChAc contains e s s e n t i a l s u l f h y d r y l groups i s that a s u l f h y d r y l reagent bound to a column was u s e f u l i n the p u r i f i c a t i o n of ChAc (17, 19). Of a l l the ChAc studied so f a r i n various species, most of the MW values f a l l i n the range of 62,000-69,000 daltons. A MW of 69,000 was reported for ChAc from Drosophila melanogaster (21), 65,000 and 73 69,000 for ChAc from bovine s t r i a t e n u c l e i (10, 11), 67,000 for ChAc from rabbit brain (63), 62,000 and 67,000 for human brain ChAc (64, 37), 60,000-65,000 and 68,000 for rat brain ChAc (18, 65), 67,000 and 68,000 for ChAc from human placenta (16, 6), and 63,000 for ChAc from Torpedo (20). But rather d i f f e r e n t values have been reported for squid head ganglion ChAc isozymes having MW's of 125,000 and 200,000 (19). Other lower MW values c i t e d were 59,000 and 50,000 for the placental (63) and rat brain enzymes (66) res p e c t i v e l y . Chao and Wolfgram (17) found that p u r i f i e d ChAc from bovine caudate nucleus s p l i t s into non-i d e n t i c a l active subunits with MW of 51,000 and 69,000. Their p a r t i a l l y p u r i f i e d enzyme migrated with a MW of 120,000 i n d i c a t i n g that the two subunits combine to form a s i n g l e active dimer. Later, the same authors (67) found that ammonium s u l f a t e p r e c i p i t a t i o n promoted the formation of aggregates of approximately 1,600,000 daltons i n s i z e . More recently, Chao (68) claimed that the MW of bovine ChAc was 84,000 due to s i x subunits of 14,000 daltons. Despite the existence of such a v a r i e t y of r e s u l t s on MW of ChAc, there has been no report on MW of any avian species. The present i n v e s t i g a t i o n of MW of chicken brain ChAc led to a value of 41,500 by gel f i l t r a t i o n and 42,500 by SDS gel electrophoresis. This value of about 42,000 daltons i s a l i t t l e low compared to the range of 62,000-69,000 reported for most other species. Also, no aggregation of chicken b r a i n ChAc was observed and there was no evidence for the existence of active forms or subunits with MW other than 42,000. The agreement between MW determined for the chicken enzyme under denaturing and non-denaturing conditions suggests that the enzyme from t h i s source contains a single polypeptide chain of MW about 42,000 daltons. The apparent Km values f o r acetyl-CoA and choline of chicken ChAc obtained i n t h i s study (see Table VIII) agree quite well with the values reported for other species i n the present l i t e r a t u r e . The Km's for choline of ChAc from most species studied f a l l i n the range of 450 yM to 1000 yM, and the Km's for acetyl-CoA are 8-25 yM (10, 36, 37, 54, 69, 70, 71, 72). Considering the fac t that Km values of ChAc change i n d i f f e r e n t i o n i c environments and that d i f f e r e n t assay methods and conditions were used i n these studies, Km values of ChAc can be said to be r e l a t i v e l y constant between species. However, higher and lower values were reported by some workers: for Km for choline, values of 47 yM for DrosophiZa melanogaster (21), 42 yM (36) and 3120 yM (73) f o r human placenta, and 11.5 mM (49) for Torpedo were obtained; and f o r Km for acetyl-CoA, values of 110 yM for human placenta (73) and 90 yM for DrosophiZa meZanogaster (21) were c i t e d . CoA was found to i n h i b i t a cetylcholine synthesis by competing with acetyl-CoA (see F i g . 19). The K i was calculated to be 34 yM i n the presence of 300 mM NaCI and 12.3 yM i n the absence of NaCI. These values are not too f a r o f f from those reported for ox s t r i a t e n u c l e i of 16 yM (10) and 1.8 yM for rat brain (50). A higher value of 75 yM was obtained by Currier and Mautner (61) for ChAc of squid ganglia. It should be noted that squid ganglia ChAc has quite d i f f e r e n t k i n e t i c properties and i t s Km for acetyl-CoA i s 66 yM. For the chicken enzyme, i t i s i n t e r e s t i n g to note that the K i of CoA has the same magnitude as the Km for acetyl-CoA. This i s i n accord with the suggestion discussed e a r l i e r that ChAc binds with both CoA and acetyl-CoA through a s i m i l a r i n t e r a c t i o n , namely, through a dinucleotide f o l d . White and C a v a l l i t o had suggested the possible r o l e of CoA as a regulating factor i n ACh 75 synthesis (13, 69). The present r e s u l t s with chicken ChAc showed that CoA i s a potent competitive i n h i b i t o r of ChAc and that CoA i s a much stronger i n h i b i t o r of ChAc than ACh (Tables VI and VII). These r e s u l t s tend to f i t i n with the hypothesis that CoA may be involved i n end product regulation of ACh synthesis. The e f f e c t of ACh on the rate of ACh synthesis by chicken brain ChAc at 12.5 mM choline and 50 yM acetyl-CoA was shown i n Tables VI and VII. Both i n the presence and absence of NaCl, chicken ChAc was insen-s i t i v e to ACh. ACh at 200 mM caused only 72% i n h i b i t i o n i n the presence of 150 mM NaCl. Both Glover (10) and Morris (36) reported s i m i l a r r e s u l t s for bovine ChAc and human pl a c e n t a l ChAc, namely, that there was no i n h i b i t i o n by ACh at d i f f e r e n t acetyl-CoA concentrations when choline was present at a saturating concentration. With d e t a i l e d k i n e t i c studies, Morris concluded that ACh i n h i b i t s ACh synthesis competitively with respect to choline and that ChAc has an ordered mechanism of the Theorell-Chance type. One d i s t i n c t i v e feature of the Theorell-Chance mechanism i s that there should be no i n h i b i t i o n by ACh with respect to acetyl-CoA at saturating concentrations of choline. In the present study, chicken ChAc also was not i n h i b i t e d by ACh at a saturating concentration of choline but unfortunately only one concentration of acetyl-CoA was tested (50 yM) for ACh i n h i b i t i o n , hence further experiments are needed i n order to understand the k i n e t i c s of chicken ChAc. Many studies have demonstrated that various s a l t s increase ChAc a c t i v i t y . This e f f e c t has been observed with the enzyme from bovine b r a i n (10, 67), human placenta (72, 74), human brain (37), rat brain (68, 75), rabbit brain (76), squid (77), Torpedo (49), Drosophila 76 melanogaster (21), and s n a i l , cockroach and horseshoe crab (14). A c t i v a t i o n of the enzyme from squid has been found to c o r r e l a t e with an increase i n the Km's for both choline and acetyl-CoA, and also an increase i n the concentration of acetyl-CoA needed to s t a b i l i z e the enzyme towards thermal denaturation (77). Both Morris et a l . (36) and Hersh et a l . (74) using enzyme from human placenta, also observed an increase i n the Km's for choline and acetyl-CoA when the concentration of NaCI was increased and s i m i l a r observations have been made with the enzyme from rat brain (75). Baker et a l . also showed a 39% increase i n the Km for choline i n the presence of 150 mM KC1 with the Torpedo enzyme (49). It thus appears that the stimulating e f f e c t of s a l t on ChAc a c t i v i t y i s rel a t e d to i t s e f f e c t on the k i n e t i c properties of the enzyme. However, recently, Hersh (74) tested various s a l t s on the Vmax and Km (choline) of the pl a c e n t a l enzyme and concluded that the s a l t e f f e c t s are non-specific and are d i r e c t l y related to i o n i c strength and that there was no e f f e c t on the k i n e t i c parameter Vmax/Km (choline). It was proposed that the conformation of the enzyme i s d i f f e r e n t at high i o n i c strength and at low i o n i c strength, and that these d i f f e r e n t conformational states of the enzyme r e s u l t i n d i f f e r e n t rate-determi-ning steps of the reaction. In addition to t h i s , Prempeh (52) suggested the existence of two isozymes of ChAc, one s e n s i t i v e to high s a l t environment and one s e n s i t i v e to low s a l t environment. In the case of chicken ChAc, Km for both substrates were also increased i n the presence of NaCI (Table V I I I ) . The increase of the apparent Km's for both choline and acetyl-CoA shows that the chicken enzyme has les s a f f i n i t y f o r i t s substrates with increasing s a l t con-centrations, an e f f e c t which w i l l l i k e l y decrease the rate of the 77 ChAc reaction. But t h i s i s contrary to the observed e f f e c t of s a l t which i s a c t i v a t i o n of ChAc. To elucidate t h i s paradox, the proposition that the e f f e c t of i o n i c strength on the conformation of the enzyme i s r e f l e c t e d i n the e f f e c t on i t s a c t i v i t y can be used. Hersh (74) has shown that there i s an increase i n the heat l a b i l i t y of the human plac e n t a l ChAc with increasing i o n i c strength which suggests that i o n i c strength induces a change i n the structure of the protein, making i t thermodynamically le s s stable. In turn, t h i s conformational change r e s u l t s i n a change i n the k i n e t i c properties of the enzyme i n which the rate-determining step of the reaction i s changed. It i s possible that the same r a t i o n a l e may be applied to the chicken enzyme. The assumption of the existence of two d i f f e r e n t conformational states of chicken ChAc i n the presence and absence of s a l t would explain the changes of a f f i n i t y of the enzyme for the substrates. Another possible explanation of the change i n enzyme a f f i n i t y f o r the substrates i n the presence of s a l t i s that Na + or CI may compete with the substrates for some anionic or c a t i o n i c s i t e s on the enzyme and hence a l t e r the Km values. In Table VIII, i t was shown that NaCl has an e f f e c t of increasing the K i values of CoA; i n addition, NaCl was observed to o f f e r protection for chicken ChAc against CoA i n h i b i t i o n (Fig. 18). The increase of K i of CoA with increasing s a l t concentration may again be explained by conformational change of the enzyme or competition of Na + or CI for s i t e s on the enzyme. The increase of K i could i n turn be used to explain the s l i g h t protection of s a l t against CoA i n h i b i t i o n since at larger K i values, the enzyme has le s s a f f i n i t y for the i n h i b i t o r . It i s d i f f i c u l t to speculate on the p h y s i o l o g i c a l s i g n i f i c a n c e of such 78 findings. However, Rossier hypothesized that end product i n h i b i t i o n by ACh may be s i g n i f i c a n t p h y s i o l o g i c a l l y i n terms of ACh synthesis regu-l a t i o n . From h i s r e s u l t s on changes of K i of ACh i n the presence of s a l t , he suggested that ChAc may be either completely i n h i b i t e d by ACh i n the absence of CI or f r e e l y a c t i v e i n the presence of CI . However, as discussed e a r l i e r , the present r e s u l t s and the r e s u l t s of other workers had indicated that CoA i s a much more potent ChAc i n h i b i -tor than ACh. A s i m i l a r hypothesis of CoA regulating ACh synthesis through changes of s a l t concentrations has not been proposed yet but such an involvement of s a l t i n ACh synthesis regulation needs much more further i n v e s t i g a t i o n s to delineate. EDTA was found to activa t e chicken brain ChAc a c t i v i t y (Fig. 22). Even though there was no absolute requirement for EDTA for detection of enzyme a c t i v i t y , F r a c t i o n 6b was stimulated 5 f o l d i n enzyme a c t i -v i t y over co n t r o l i n the presence of 0.1 mM EDTA, and a les s p u r i f i e d preparation, Fr a c t i o n 5, showed a 20 f o l d increase i n enzyme a c t i v i t y at 0.1 mM EDTA. This d i f f e r e n c e i n degree of stimulation of ChAc a c t i v i t y by the same concentration of'EDTA i s unexpected but reprodu-c i b l e . ChAc from other sources such as Torpedo and human brain also show stimulation of a c t i v i t y by EDTA (49, 37) while Glover et a l . (10) found no e f f e c t of 0.1 mM EDTA on bovine brain enzyme. EDTA i s commonly used i n many ChAc assay mixtures and has been shown to have a s t a b i l i z i n g e f f e c t on b a c t e r i a l , bovine brain and rat brain ChAc a c t i v i t y (13, 42). Most of the ChAc p u r i f i c a t i o n s reported so f a r had EDTA added i n the enzyme so l u t i o n throughout the p u r i f i c a t i o n procedures because of i t s s t a b i l i z i n g e f f e c t on ChAc. Although EDTA was commonly used i n assaying mixtures, the exact 79 mechanism of stimulation and s t a b i l i z a t i o n i s not known yet. It i s generally believed that EDTA acts as a chelating agent i n ChAc assay mixtures to remove small amounts of metal ion impurities i n the incu-bation mixtures, e s p e c i a l l y cupric ions which are. potent i n h i b i t o r s of ChAc (49, 37, 79). Cupric s u l f a t e i n a c t i v a t e s ChAc from various species completely at very low concentrations: 20 yM for the Torpedo enzyme (49), 1 mM for the DrosophiZa enzyme (21), 10 yM for the rat enzyme (66), 0.1 mM for the bovine and human caudate enzyme (67, 37). Both EDTA and t h i o l reagents l i k e sodium t h i o g l y c o l l a t e or d i t h i o t h r e i t o l are able to circumvent the i n h i b i t i o n of ChAc a c t i v i t y by cupric s u l f a t e , presumably through the metal-chelating properties of the former and the high a f f i n i t y f o r cupric ions of the l a t t e r . However, i t has 2+ been suggested that Cu may be reacting with imidazole groups at the active s i t e , rather than by i n t e r a c t i n g with enzyme s u l f h y d r y l groups (69, 80). The present r e s u l t s of EDTA e f f e c t on chicken ChAc has led to a speculation that the EDTA e f f e c t may have another mechanism other than the metal-chelating action. If EDTA increases ChAc a c t i v i t y by removing metal ions from the assay mixture, one would expect that 0.1 mM EDTA would increase ChAc a c t i v i t y of both Fractions 5 and 6b to the same degree over the c o n t r o l . Instead, i t was observed repeatedly that Fr a c t i o n 6b was stimulated 5 f o l d i n ChAc a c t i v i t y while Fra c t i o n 5 was stimulated 20 f o l d . There are a few possible explanations f o r t h i s observation. F i r s t , a f t e r agarose-hexane-CoA chromatography, the en-zyme (Fraction 6b) may have changed i n some way such that i t became 2+ less susceptible to Cu i n h i b i t i o n . Secondly, EDTA may a c t u a l l y i n t e r a c t with the enzyme i n causing the stimulation of a c t i v i t y . Frac-80 tio n 6b may indeed be d i f f e r e n t from Fraction 5 i n such a way that EDTA does not cause as large a stimulation i n Frac t i o n 6b as i n Fract i o n 5. Another p o s s i b i l i t y i s that the less p u r i f i e d F r a c t i o n 5 may contain an i n h i b i t o r y f a c t o r which can be counteracted by EDTA. However, the r e s u l t s shown i n Table XI discount t h i s p o s s i b i l i t y because when Fractions 5 and 6b were combined and assayed, i t showed a small increase i n ChAc a c t i v i t y rather than a decrease. F i n a l l y , one cannot disregard the p o s s i b i l i t y 2+ that Fraction 6b may contain less bound Cu than Fra c t i o n 5 due to p u r i f i c a t i o n . However, considering that both f r a c t i o n s were dialyzed against PEMG buffers before assaying and that the concentration of EDTA used was quite large (0.1 mM), i t seems u n l i k e l y that t h i s p o s s i b i -l i t y can explain the observation that F r a c t i o n 5 was stimulated more than Fraction 6b. Looking at the EDTA a c t i v a t i o n curve i n F i g . 22, for both Fractions 5 and 6b, maximum a c t i v a t i o n was not reached u n t i l EDTA reached a con-2+ centration of 0.1 mM. I t seems u n l i k e l y that the amounts of Cu and other metal ions present i n the assay mixture are so large that they require 0.1 mM EDTA to remove them. As a matter of f a c t , the concentra-2+ * ti o n of Cu i n the assay mixture was measured to be about 2 ± 0.1 yM. 2+ There are also other ions such as Ca i n the assay mixture that are 2+ chelated by EDTA, but Ca was found to stimulate the a c t i v i t y of chicken brain ChAc rather than to i n h i b i t (see Table X). Hence, i t seems that EDTA i s playing a r o l e other than removal of metal ions i n a c t i v a -ti n g chicken brain ChAc. * 2+ The measurement of Cu i n the assay mixture was done by Mr. Peter Kempe, Department of Mineral Engineering, U.B.C., using the technique of atomic absorption spectroscopy. In t h i s measurement, radioactive acetyl-CoA was replaced by d i s t i l l e d water. 81 Another evidence which supports the foregoing suggestion i s that the extent of a c t i v a t i o n by EDTA (5-20 fold) i s too large to be accoun-2+ 2+ table by Cu i n h i b i t i o n alone. I t i s assumed that Cu are the major ions i n the assay mixture that cause ChAc i n h i b i t i o n . From F i g . 21, 2+ i t can be seen that 5 pM Cu i n h i b i t s F r a c t i o n 5 by only 9%, the actual figures being from 0.217 down to 0.197 pinoles ACh formed/ml/20 min.. 2+ Since only about 2 pM of Cu are present i n the assay mixture, the 2+ i n h i b i t i o n that r e s u l t s from Cu i s not expected to be too large. But EDTA stimulation of ChAc a c t i v i t y was large; hence, presumably the mecha-nism of stimulation i s not j u s t by r e l i e v i n g ChAc from the small i n h i b i -2+ t i o n exerted by 2 pM Cu In addition to these f i n d i n g s , i t should also be noted that Potter et a l . (10) found no e f f e c t on 0.1 mM EDTA on bovine caudate ChAc a c t i v i t y . A l l these observations made i t tempting to speculate that EDTA stimulates chicken brain ChAc, and possibly ChAc of most other species, not only by i t s metal-chelating property but possibly also by binding to the enzyme and changing the property of the enzyme. Singh and McGeer (81) have already suggested the p o s s i b i l i t y that human brain ChAc might aggregate i n the presence of EDTA. In the study of c r o s s - r e a c t i v i t y between human ChAc antibodies and chicken brain ChAc, no reaction was observed between the antibodies and antigens of various d i l u t i o n s (see F i g . 23). In F i g . 23c, no v i s i b l e p r e c i p i t i n band could be observed with approximately 5 pg of antigen. However, i n another t e s t using p u r i f i e d human ChAc, about 2 pg of antigen was s u f f i c i e n t to produce a v i s i b l e p r e c i p i t i n band (personal communication with Dr. J. H. .Peng). The above observations would suggest that the negative r e s u l t with chicken brain ChAc was not due to i n s u f f i c i e n t amount of antigen but rather was due to immunological d i f f e -rences between chicken brain ChAc and human brain ChAc. However, since the chicken antigen was not e l e c t r o p h o r e t i c a l l y homogeneous, the above i n t e r p r e t a t i o n should be taken with caution. Unfortunately, studies on i n h i b i t i o n of chicken brain ChAc by human brain ChAc antibodies were not done to investigate the problem further. From the present studies, r e s u l t s i n d i c a t e that chicken brain ChAc shows s i m i l a r properties to mammalian ChAc, namely, a c t i v a t i o n by NaCI, 2+ KC1 and EDTA, i n h i b i t i o n by N-ethylmaleimide, Cu , and CoA, pH optimum, and Km values for acetyl-CoA and choline. The notable differences that 2+ chicken brain ChAc show are stimulation by Ca and the low MW of 42,500 daltons. 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