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Purification and initial characterization of deacetylase from trout testes (Salmo qairdnerii) Stevenson, Barry W. A. 1972

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c-l P u r i f i c a t i o n and I n i t i a l Characterization of deacetylase from Trout testes (Salmo ga i r d n e r i i ) by BARRY WAYNE A. STEVENSON B.Sc., Simon Fraser University, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Novembe r, 19 72 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make itf r e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada Date NOVEMBER 3 0 , 1 9 7 2 Abstract This investigation describes the i s o l a t i o n and p u r i f i c a t i o n of a deacetylase from trout testes (Salmo gairdnerii) and i t s i n i t i a l characterization, the res u l t s of which are summarized as follows; Histone deacetylase a c t i v i t y was demonstrated i n the 230,000 x g supernatant f r a c t i o n of trout testes by a new and sensi t i v e assay. The deacetylase was p u r i f i e d by s a l t p r e c i p i t a t i o n , molecular seive chromatography and subsequent ion exchange chromatography. Two major fractions of enzyme a c t i v i t y were demonstratable with t h e i r approximate molecular sizes estimated. The s p e c i f i c i t y of the enzyme towards d i f f e r e n t histone fractions appeared to undergo dramatic changes, depending on p r i o r treatments. Preliminary results as these furnished some basis for specu-l a t i o n regarding (a) the possible s i g n i f i c a n c e of deacetylation not only as a histone modification process but also i n the role of gene regulation and (b) the l o g i c a l p r e d i c t i o n as to the e x i s t -ence of enzyme species exhibiting varying substrate s p e c i f i c i t i e s to cope with the d i s t i n c t classes of histones unmasked i n trout testes. i i . Table of Contents Page Abstract L i s t of Figures and Tables i i i . Acknowledgments v. Dedication vi. I. Introduction 1 II. Experimental Procedures 6 (a) Materials 6 (b) Methods 6 (i) Protein Determination 6 ( i i ) C e l l Incubations 6 ( i i i ) I s o l a t i o n and Fractionation of Histones 7 (iv) Preparation of Crude Enzyme 8 (v) Assay of Enzyme A c t i v i t y 9 II I . Results and Discussion 10 (a) Assay Method 10 (b) Crude Enzyme 13 (c) Ammonium Sulphate Fractionation 13 (d) Sephadex G-200 Chromatography 18 (e) DEAE-cellulose Chromatography 23 (f) Degree of P u r i f i c a t i o n 23 (g) I n i t i a l Characterization and S p e c i f i c i t y 26 (i) Molecular Weight Estimation 26 ( i i ) S t a b i l i t y of Enzyme 26 ( i i i ) S p e c i f i c i t y 28 IV. Bibliography 30 L i s t of Figures and Tables Figure 1 - Comparison of the NH 2-terminal region of trout testes histones and the s i t e s of acetylation. Figure 2a - CM-cellulose (CM-52) assay system - enzymatic reaction. Figure 2b '- CM-cellulose (CM-52) assay system - control reaction. Figure 3a - Time vs. a c t i v i t y study of crude enzyme prep-aration . Figure 3b - Enzyme concentration vs. v e l o c i t y study of crude eznyme preparation. Figure 3c - Substrate concentration vs. v e l o c i t y study of crude enzyme preparation. Figure 4 % Ammonoim Sulphate Fractionation of crude enzyme preparation. Figure 5 - Enzyme-protein concentration vs. v e l o c i t y study of 0-30% (NH^)2SO^ p r e c i p i t a t e . Figure 6a - Sephadex G-200 chromatogram of 0-30% ( N H O 2 S O 1 , p r e c i p i t a t e . Figure 6b - Sephadex G-200 chromatography of 0-30% ( N H 1 J 2 S C U p r e c i p i t a t e (overloaded). Figure 7 - Enzyme protein concentration vs. v e l o c i t y study of the pooled and lyophilysed enzyme f r a c t i o n from the Sephadex G-200 chromatogram (Fig. 6a). Figure 8 - DEAE-cellulose (DE-52) chromatogram of the pooled and lyophilysed enzyme f r a c t i o n from the Sephadex G-200 chromatogram (Fig. 6a). Figure 9 - Estimation of the molecular weight of the Sephadex G-200 chromatogram pool A and pool B enzyme fractions (Fig. 6b). Figure 10a - I n i t i a l S p e c i f i c i t y study of 0-30% (NH^^SO.* p r e c i p i t a t e . Figure 10b - I n i t i a l S p e c i f i c i t y study of the Sephadex G-200 enzyme f r a c t i o n pool B (Fig. 6b). Figure 10c - I n i t i a l S p e c i f i c i t y study of the DEAE-cellulose (DE-52) enzyme f r a c t i o n (Fig. 8). Table 1 Summary of P u r i f i c a t i o n V. Acknowledgment I wish to thank Dr. Peter Candido, Dr. Andrew Louie, and Dr. Stewart Gilmour for valuable suggestions and discus-sions and I would es p e c i a l l y l i k e to thank my f r i e n d Dr. Tai Wing Wu for his encouragement and concern. F i n a l l y , I would l i k e to acknowledge my wife, Janice, for her unselfish and loving devotion during the course of t h i s work. Dedication To JANICE 1 . I. Introduction P h i l l i p s (21) f i r s t noted the presence of N-acetyl groups i n c a l f thymus histones. It was l a t e r shown that the NH2-terminal of several histone fractions was blocked by an acetyl group (22). However, subsequent investigations have revealed that the major s i t e s of acetylation of c a l f thymus (1,2,3) pea seed-l i n g (4) and trout testes histones (5,6) are at the e-NH2 group of i n t e r n a l l y s y l residues. DeLange et a l . (4) found that l y s y l residue 16 was approximately 50% acetylated i n c a l f thymus histone IV. Studies on pea seedling histone IV revealed that, i n addition to l y s y l residue 16, at least one of the l y s y l residues 5,8, or 12 was p a r t i a l l y acetylated. Candido and Dixon (5) have reported that a l l four of the l y s y l residues 5,8,12 and 16 i n histone IV from developing trout testes are acetylated i n vivo, and that further evidence presented suggests that a l l four s i t e s can be independently modified leading to a complex mixture of p a r t i a l l y acetylated histone IV molecules. Subsequently, Candido and Dixon (6) have reported that histone I l b i from developing trout testes contains at least one acetylation s i t e , and that histones I I b 2 and III each have at least two acetylation s i t e s (Fig. 1). DeLange et a l . (7) also found two acetylated l y s y l residues i n the p a r t i a l sequence of histone III from c a l f thymus. Thus, not only do histones appear to be acetylated at s p e c i f i c l y s y l residues but also this modification appears mainly i n the N-terminal region of histones I l b i , l i b * , III and IV. His-tone I appears not to undergo e-NH2 acetylation (6). With t h i s 2. Comparison of N-Terminal Region* of Hlstonn of Trout Toslte 7 -. j - — ; Pp. Ac\ I Ao , A»i , Ac' Minor* IV ^to-sir-ofy-Aii-eir-t.r*,— loir-&r-*l* .1' 6 I flfri'l— Cry 10 Hilton* lib, i Ac^-«r->«r^Cff-i ) r,-n>f.«r^/y.Lriy*/«-4f»-^/»-Lri ) V|!« ^ Crjr^f/ - A/« -1/» -J>«»»-«/» -Ar»-Lr»-« IM I tt I I I I I I L n 1 ,Atu-tn-^ir-^t-fArft-Bt— I 1 is |" ( /"" ~| | ' /-——| i • » fo » ao • as!..-.• HMon* I lb. Ac I Ae I Ac 10 IB ao . HMono «v SmpMne* roatong* af 1,ianJ. Hlttomllbf Caff mymm fOhon «f af. Iff?/, Treat t—llt (Bmllmfnd Dtnm.tori.i, Hilton* III Celt MrmwafOtaon ctl^tml.Troml HcUclCcMMc ana? Dtxmn.ltTt).rWMamIIb^ffaaf •<mtJtTO) Tnt nilclCmtMdo mtUmn.tm) Figure 1. * Comparison of the NH 2-terminal regions of trout . t e s t i s histones and the s i t e s of acetylation (From Candido and .Dixon, 6) . • > V 3. degree and s p e c i f i c i t y of acetylation i n histones, has come the concept of histone modification as a l i k e l y area of gene reg-u l a t i o n at the l e v e l of histone-DNA interactions. A l l f r e y et a l . (8) have shown that chemical acetylation of arginine-rich h i s -tones progressively decrease the i n h i b i t o r e f f e c t histones have on DNA-directed RNA synthesis, as catalyzed by UNA polymerases. In agreement with t h i s finding A l l f r e y ' s group (9,10) has also reported that the chromatin of c a l f thymus lymphocytes can be separated i n t o fractions which d i f f e r i n t h e i r morphology and i n RNA synthetic a c t i v i t y . That i s , the 'condensed* chromatin f r a c t i o n , which contains"the bulk of the DNA of the nucleus (about 80%), i s r e l a t i v e l y i n e r t i n RNA synthesis and shows very l i t t l e acetate incorporation into histones; the 'diffuse' chromatin—the f r a c t i o n which i s most active i n RNA s y n t h e s i s — i s also most active i n the acetylation of the arginine-rich histones. However, i t should be pointed out that acetate incor-poration i s only part of the story, and that i t most l i k e l y i s a balance between acetylation and deacetylation which determines the net acetyl content of the arginine-rich histones. In order to discuss t h i s modification, both systems must be considered. Pogo et a l . (11), i n comparing histone acetylation i n normal and sham operated rat l i v e r , with regenerating rat l i v e r , found a decreased rate of deacetylation i n the l a t t e r . This observation may account for the r i s e i n the acetyl lysine content of the histones at p a r t i c u l a r s i t e s i n the chromosome, even i f the acetyl group incorporation rate should remain unmodified. With 4. This evidence of histone modification v i a acetylation and/or deacetylation, has come a need to investigate these areas more close l y with the idea of i s o l a t i n g and p u r i f y i n g the enzyme fractions responsible. Deacetylase (aceto-amido hydrolase), an enzyme which removes the acetyl groups from e-N-acetyl l y s y l residues i n histone, has been studied to date by two other groups of investigators, namely Inoue and Fujimoto (12,13) and Boffa et a l . (14), both i n c a l f thymus extract. Libby (15) has reported a c t i v i t i e s of histone deacetylase i n r a t l i v e r and Novikoff Hepatoma. Although Wigle (16) reported finding a deacetylase a c t i v i t y i n trout t e s t i s high speed supernate which would remove formyl and acetyl groups from formylated and acetylated methionine, no d i r e c t evidence had been reported to show deacetylase a c t i v i t y with respect to acetylated histone fractions from trout t e s t i s p r i o r to t h i s i n v e s t i g a t i o n . Trout testes were selected for t h i s study since much i s known of the acetyl-modification of histones from t h i s species (5,6). With the increasing i n t e r e s t i n defining the role of histones i n the regulation of gene a c t i v i t y , i t appears necessary to investigate the enzymatic factors involved. An attempt was made to demonstrate the existence of deacetyl-ase a c t i v i t y i n trout t e s t i s (Salmo g a i r d n e r i i ) , to i s o l a t e and p a r t i a l l y p u r i f y the enzyme, and to i n i t i a l l y characterize i t . 5. In t h i s work, a reproducible and sen s i t i v e assay was developed. This i n turn f a c i l i t a t e d p u r i f i c a t i o n of the deacetylase. During the p u r i f i c a t i o n process, some int e r e s t i n g results were noted which have not been reported elsewhere and would warrant further study. For example, i t was noted that a change of s p e c i f i c i t y arises as the deacetylase i s p u r i f i e d suggesting, among other p o s s i b i l i t i e s , m u l t i p l i c i t y of deacetylase a c t i v i t y . The l i t e r a t u r e review presented r e f l e c t s the shortage of information presently available i n thi s f i e l d of study. 6 . I I . Experimental Procedures (a) Materials DEAE-cellulose (DE-52) and CM-cellulose (CM-52) were ob-tained from Whatman, Sephadex G-200 from Pharmacia, and Bio-Gel P-10 from Bio-Rad Laboratories. Trout testes were co l l e c t e d from naturally maturing trout (Salmo gair d n e r i i ) from August to December at the Sun Valley trout farm, Mission, B r i t i s h Columbia. The testes were transported to the laboratory on i c e , washed and stored at -80°C u n t i l use or used fresh. Sodium acetate-1- 1*C (61 mCi/mmole) was obtained from Amersham-Searle Corporation, Arlington Heights, I l l i n o i s . A l l other chemicals obtained were of reagent grade. (b) Methods (i) Protein Determination: Enzyme protein determination was made by measuring the absorbance of the unknown solution at 280 my against a blank i d e n t i c a l i n content to the unknown minus protein. An absorb-ance of 1 at 280 my was assumed to be given by 1 mg/ml of protein solution. ( i i ) C e l l Incubations: Fresh trout testes were sc i s s o r minced on ice i n 2 volumes of i c e cold TMKS(TMK plus 0.25 M Sucrose)-1% glucose and a c e l l suspension was prepared by gentle hand homogenization (three strokes up and down) i n a Potter-Elvehjem homogenizer with a Teflon pestle. The suspension was f i l t e r e d through four layers f 7. of cheesecloth, to which cycloheximide was added to a f i n a l con-centration of 10 M^. This suspension was preincubated at 16°C for 10-15 minutes. Then, 10 yCi/ml of sodium acetate-1- 1 h C was added and the incubation continued at 16°C for 90-120 minutes on a gyratory water bath (150 rpm). Afte r incubation, the c e l l s were c o l l e c t e d by centrifugation and washed i n TMK(50 mM T r i s -HC1, pH 7.4; 2 mM magnesium chloride; 25 mM potassium c h l o r i d e ) , pH 7.4. ( i i i ) I s o l a t i o n and Fractionation of Histones: Washed c e l l s were resuspended i n ice cooled TMK; pH 7.4 and homogenized (5000 rpm) i n a Potter-Elvehjem homogenizer with a motor driven pestle (TRI-R Instruments, Inc., Jamaica 35, New York) for 2-3 min. Nuclei were coll e c t e d by centrifugation (5000 x g, 10 mins). This process was repeated once more, and the nuclear p e l l e t was resuspended i n 0.01 M TRIS(Tri-(hydroxy) methylamino methane), pH 7.4 and centrifuged at 10,000 x g for 10 minutes. This process was repeated once again. Basic pro-teins were extracted from the above p e l l e t with at least 4 volumes of 0.4N H2SOi» (0°C, 30 min) (which removed the bulk of the nucleic acids) and p r e c i p i t a t e d with 4 volumes of 95% ethanol (-20°C for 24-36 hrs.). The p r e c i p i t a t e was c o l l e c t e d by cen-t r i f u g a t i o n (10,000 x g, 10 min), washed with cold ethanol (95%), and dried under vacuum. Basic proteins were then converted to t h e i r acetate form by d i a l y s i s against .IN acetic acid and l y o p h i l i z e d . The basic proteins were fractionated at room temp-erature on long columns of Bio-Gel P-10 (30 mm x 3.2 m) e q u i l i -8. brated with .01 N HCl. Protein concentration was determined by absorbance at 230 nm. Histone fractions I l b i , IIb2, III and IV were separately pooled, l y o p h i l i z e d and assayed for r a d i o a c t i v i t y . Aliquots of these fractions ( I l b i , IIb2, III and IV) as well as whole histone, were separated by electrophoresis on urea-aluminum lactate buffered starch gels as described by Sung and Smithies (20) for further i d e n t i f i c a t i o n and estimation of purit y . Electrophoresis was carr i e d out at 6 vol t s per cm. (180 volts across g e l , 35 mA) with water cooled gel trays i n the cold room (6°C). (iv) Preparation of Crude Enzyme: The 230,000 x g supernatant f r a c t i o n , routinely prepared as described by Gilmour and Dixon (18), was used as the source of the crude enzyme i n t h i s study. Fresh trout testes were sci s s o r minced and homogenized i n 2 volumes (w/v) TMKS-1% glucose i n a Potter-Elvehjem homogenizer at 0°C, then passed through 4 layers of cheesecloth. The sus-pension was centrifuged at 5000 x g for 10 minutes. The p e l l e t was resuspended i n 2 volumes TMKS, homogenized with a motor driven pestle for 3 minutes and centrifuged at 10,000 x g for 10 minutes. The supernatant was then placed i n tubes and cen-tr i f u g e d at 30,000 x g for one-half of one hour. The resultant supernatant was then placed i n a Spinco ultracentrifuge (L-65 head) and centrifuged at 60,000 rpm (230,000 x g) for 3 hours. This supernatant was then l y o p h i l i z e d and placed at -20°C for storage. 9. (v) Assay of Enzyme A c t i v i t y ; The enzyme a c t i v i t y was determined by measuring the re-lease of a c e t y l - 1 - 1 g r o u p s i n i t i a l l y bound to histone e-N-l y s y l residues. The standard assay procedure was as follows: Incubation of the enzyme solution with [ 1 4 C ] a c e t y l - l a b e l e d histone (1 mg; 3000 cpm) at 18°C i n a f i n a l volume of 1 ml with 0.001 M phosphate buffer, pH 7.4. After an eight hour incubation, the reaction was stopped by b o i l i n g for 8-10 min and placed on small CM-52 columns ( 1 x 4 cm) i n .001 M phosphate buffer. The CM-52 columns had previously been regenerated with 0.IN HCl and 0.IN NaOH, washed with water and equilibr a t e d to pH 7.0 with phos-phate buffer (.001 M, pH' 7.4). The eluant from the CM-52 columns, containing the released 1'*C-acetate was co l l e c t e d i n 3-5 fr a c -tions each of which was placed i n 10 ml of Bray's s c i n t i l l a t i o n f l u i d (17), and the r a d i o a c t i v i t y measured i n a Unilux l i q u i d s c i n t i l l a t i o n counter. Values were corrected for the e f f i c i e n c y (60%) of counting 1^C i n Bray's s c i n t i l l a t i o n f l u i d . 10. I l l . Results and Discussion (a) Assay Method This method involved the incubation of [ 1kC]-acetylated histone with the enzyme preparation i n .001 M phosphate buffer, pH 7.4 at 18°C. The p r i n c i p l e i s that the deacetylase enzyme cleaves the a c e t y l - 1 - 1 k C groups from the e-N-lysyl residues which have bound acetate and by a se l e c t i v e process these free a c e t y l -l - 1 I f C groups are separated from bound acetate. By employing a cation exchange column, such as CM-52, the [ ^^C]-acetylated histone, being a c a t i o n i c protein, i s bound to the column, and the free [ 1^C]-acetate elutes with the buffer. It i s c o l l e c t e d for measurement of r a d i o a c t i v i t y . Figure 2a, 2b shows the release of [ 1^C]-acetate by the enzymatic reaction as compared to a control reaction with denatured enzyme. [ *^C]-Acetate i s released i n the enzymatic reaction but only s l i g h t l y i n the control 2b. The histone adsorbed to the column was eluted with 10% formic acid, and the r a d i o a c t i v i t y (corrected for quenching) was determined by the method outlined under 'Assay of Enzyme A c t i v i t y 1 . I t can be seen that the r e l a t i v e amount of radio-a c t i v i t y held on the columns i s i n agreement with the amount i n i t i a l l y eluted due to free 1 > fC acetyl groups cleaved by the enzyme. The recovery of r a d i o a c t i v i t y (cpm) from the columns was over 90%. This pattern was representative of the subsequent assays performed and the results reproducible. In r e l a t i o n to other assay methods for measuring release of [XI*C] acetyl groups, (Inoue and Fujimoto (12,13)), i t was found that the CM-52 ca t i o n i c exchange method was more repro-f/?/7CT/oNS coui-ecreo (M.) . i ::X .' . . . ' - • ' > : i 1 " Figure 2a. CM-cellulose (CM-52) assay system: the coljamn (1 x 4 cm) was charged with the enzymatic reaction -solution (20 00 cpm) and eluted as shown with buffer and th^n 10% formic ._. _acid. Fractions 4-6 contain [ 1 ^ C]-acetate *|(1350 cpm) and fractions i3-15"coritailiT~ 1'^ICT-acetylal^dTTTiU^fone ("5"5"0_"cprir)~ I t I Figure '2b. CM-cellulose (CM-52) assay system: the .column was charged with a control reaction (2010 cpm) and eluted as i n Figure 2a.. The majority of the r a d i o a c t i v i t y (1880 cpm) was '~ **" " eruted~as~[~^C]acetylated—fri-stone^ • ——• — — — 13. ducible. Attempts were made at the beginning of thi s project to u t i l i z e e a r l i e r assay methods (12,13,14), which were based on the extraction of [ 1^C]acetate into ethyl acetate, but the results were inconsistent and variable. (b) Crude Enzyme Attempts were made to show that the deacetylase a c t i v i t y mentioned was present i n the crude preparation. Assays were car r i e d out with the res u l t s shown i n figures 3a, 3b, and 3c. As shown, the i n i t i a l v e l o c i t y was l i n e a r with increasing enzyme-protein concentration. Time studies showed that the enzyme was slow acting and l o s t a c t i v i t y gradually af t e r the f i r s t two hours of incubation. V e l o c i t y vs. substrate (whole histone) studies showed a decrease i n slope a f t e r the addition of greater than approximately 0.5 mg of [ l l fC] acetylated histone. Beyond 0.9 mg of histone, the v e l o c i t y declined, suggesting substrate i n h i b i t i o n . The crude enzyme was found to possess a general deacetylating a b i l i t y but the examination of i t at t h i s stage was by no means conclusive, rather i n d i c a t i v e of i t s presence only. (c) Ammonium Sulphate Fractionation Aliquots of thawed high speed supernate (crude enzyme) were mixed with ( N H i J 2 S O i j to saturation levels ranging from 10-70%. The re s u l t i n g p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f -ugation at 2000 rpm and dialysed against 0.001 M phosphate buffer (pH 7.4) for 18-24 hours at 4°C i n the cold room. Each f r a c t i o n was assayed for deacetylase a c t i v i t y as mentioned 14. / Z 3 H 6 £ 7 9 9 , — — — — \ rr— ^ , —1-Figure 3a... Time vs. a c t i v i t y study of crude enzyme prep-a r a t i o n . The r e a c t i o n c o n d i t i o n s were as des-c r i b e d under' 'Assay of Enzyme A c t i v i t y ' , , using: 2 mg of enzyme-protein. i Figure 3b. Enzyme concentration.vs. v e l o c i t y study of crude enzyme p r e p a r a t i o n . The r e a c t i o n c o n d i t i o n s were as described under 'Assay of Enzyme A c t i v i t y 1 . r 16. Figure 3c. Substrate concentration vs. v e l o c i t y study of, crude enzyme preparation. The-reaction con-ditions were as described under 'Assay o f ' Enzyme A c t i v i t y ' using whole histone as a sub^ str a t e . I 17. Figure 4.. Ammonium Sulphate F r a c t i o n a t i o n of crude enzyme pr e p a r a t i o n . The r e a c t i o n c o n d i t i o n s were as . - d e s c r i b e d under 'Assay of Enzyme1 A c t i v i t y 18. e a r l i e r . The findings were as shown i n figure 4. The maximal a c t i v i t y of deacetylation was found i n the 0-30% p r e c i p i t a t e . This p r e c i p i t a t e was used for subsequent p u r i f i c a t i o n steps. A deacetylase a c t i v i t y also appeared at the 70% saturation range but was not used i n the present study. The dialysed p r e c i p i t a t e from the 0-30% ( N H i + ) 2 S O i t f r a c t i o n -ation procedure also showed l i n e a r i t y of i n i t i a l v e l o c i t y with increasing enzyme-protein concentration ( f i g . 5). This pre-c i p i t a t e , when assayed for s p e c i f i c i t y towards histone f r a c -tions I l b i , I I b 2 , H I and IV, gave results as shown i n figure 10a, which w i l l be discussed l a t e r under I n i t i a l Characterization and S p e c i f i c i t y . (d) Sephadex G-200 chromatography The dialysed 0-30% ( N H i f ) 2 S O i f p r e c i p i t a t e was applied to a Sephadex G-200 column (2.75 x 50 cm), equilibrated with .001 M phosphate buffer (pH 7.4). The column was developed using the same buffer. Figure 6a shows the r e s u l t of the separation technique. Most of the protein came o f f i n the f i r s t 150 mis of eluant followed by a le s s e r , broad f r a c t i o n containing the major deacetylase a c t i v i t y . This r e s u l t was reproducible upon subsequent t r i a l s . Again, l i n e a r i t y of assay was demonstrated as shown i n figure 7. However, on some occasions, (Fig. 6b) es p e c i a l l y when the column was overloaded, multiple peaks of a c t i v i t y appeared. There are several possible reasons for t h i s r e s u l t . There may, for example, be more than one form of de-acetylase, each having a s p e c i f i c molecular weight, or there may 19. Figure 5. Enzyme p r o t e i n c o n c e n t r a t i o n vs. v e l o c i t y study of 0-30% (NHi,) 2SOi» p r e c i p i t a t e . . The r e a c t i o n c o n d i t i o n s were as described under 'Assay of Enzyme A c t i v i t y ' . There.was approximately v 7 ; • mg/iril of .enzyme-protein p r e c i p i t a t e . 1 20 Figure 6 a . : Sephadex G - 2 0 0 chromatogram of 0-30% (NHi») 'zSO^  p r e c i p i t a t e . 5 7 . 4 mg of protein was loaded onto the Sephadex G - 2 0 0 column as described i n the. Text. 10 mi-Fractions were coll e c t e d at a flow rate of * about 6 mls/hr. 0 . 3 ml of each f r a c t i o n was assayed for enzyme a c t i v i t y as described e a r l i e r . • 21 1.4 1.3 U I.I I.O-•1 o* 00.7-<M V •3 Pool A [• Poode I 1 1 I /\ / IOO 200 300 +00 ELUTION V O L U M E (mJ) 190 i7» l(,o l5o l«fo IX« MO 100 <?!> 40 7o U io MO 3» io io u z H UJ V/> UJ a. u Figure 6b. Sephadex G-200 chromatogram of 0-30% (NH ^  ) 2 SO 4 p r e c i p i t a t e (overloaded). Conditions are as noted i n Figure 6a, except that a load of 150 mg of protein was 'used. . ' -\3 •• 'O.o/ .' o*9f o-lp o.iif e.zo o.*S Figure 7. Enzyme-protein concentration vs.- Velocity of the .pooled and lyophi II zed •.'enzyme f r a c t i o n from, the Sephadex G-200 chromatogram (Fig. 6a). The reaction conditions were as described under 'Assay of Enzyme A c t i v i t y ' . ; also be varying states of aggregation of a p a r t i c u l a r deacetylase The p o s s i b i l i t i e s of either or both of these factors may e x i s t . However, i n so far as t h i s project was concerned, no further attempt to study t h i s phenomonen was undertaken. I t would be in t e r e s t i n g to pursue this r e s u l t further i n a l a t e r study. The lyophilysed enzyme from the major deacetylase peak was assayed for substrate s p e c i f i c i t y (Figure 10b). The re s u l t s w i l l be discussed l a t e r under I n i t i a l Characterization and S p e c i f i c i t y . (e) DEAE-cellulose Chromatography Fractions from Sephadex G-200 showing the most s i g n i f i c a n t deacetylase a c t i v i t y were pooled, lyophilyzed and placed on a DE-52 column (7.5 x 12 cm) equilibrated with .001 M phosphate buffer (pH 7.4). The wash was free of deacetylase a c t i v i t y as we as protein. The bound protein was then eluted i n a 0-1 M NaCl gradient. The deacetylase a c t i v i t y was observed between 0.05-0.15 N NaCl along the i o n i c gradient (Fig. 8). Fractions between 150-300 ml were pooled, lyophilyzed and assayed f o r s p e c i f i c i t y of deacetylase a c t i v i t y (Fig. 10c). The s p e c i f i c i t y of this deacetylase f r a c t i o n (Fig. 10c) w i l l be discussed l a t e r under I n i t i a l Characterization and S p e c i f i c i t y . (f) Degree of P u r i f i c a t i o n A summary of the p u r i f i c a t i o n procedure described above i s presented i n Table 1. From th i s table, we can see a 161 f o l d p u r i f i c a t i o n of the deacetylase enzyme from i t s i n i t i a l crude form. The f i n a l product however showed a s t r i k i n g l y d i f f e r e n t 24. l o o o \Soo E L U T I O M V O L U M g Cml) 2 0 0 0 Figure 8. DEAE-cellulose (DE-52) chromatogram of the pooled and lyophilyzed enzyme f r a c t i o n from the Sephadex G-200 chromatogram (Fig. 6a). Conditions are as described i n the Text . . .(p. 2 3 ) . ; - ' • - •: < \ TABLE 1. Summary of P u r i f i c a t i o n Fraction Total A c t i v i t y ( u n i t s ) a Protein (mg) Sp e c i f i c A c t i v i t y (units/mg) Y i e l d P u r i f -i c a t i o n (fold) i) Crude Enzyme 12400 2000 6.2 100 1 i i ) 0-30% ( N H i t ) 2 S 0 i f 5300 336 15. 8 43 2.6 i i i ) Sephadex G-200 eluant, lyophilyzed and dialysed 4000 4 1000 32 161 A unit of enzyme a c t i v i t y i s expressed as the cpm released/hr. 26. s p e c i f i c i t y toward histone fractions than did e a r l i e r levels of p u r i f i c a t i o n . This r e s u l t w i l l be discussed under I n i t i a l Characterization and S p e c i f i c i t y . (g) I n i t i a l Characterization and S p e c i f i c i t y (i) Molecular Weight Estimation: Both Y~glokulin (human) and hemoglobin (human) were applied separately to the Sephadex G-200 column used i n the p u r i f i c a t i o n of the deacetylase. Figure 9 shows the log molecular weight vs. e l u t i o n volume (Ve) relat i o n s h i p of the resultant standards and deacetylase enzyme. I t would appear that the deacetylase enzyme from pool B, shown i n F i g . 6b, has an approximate molecular weight of 50,000 ± 500, while pool A has a molecular weight of approximately 100,000 ± 5000. I t i s not conclusive to say that these enzymes possess these molecular weights, due to the lack of s u f f i c i e n t standards. However, they serve to give a crude estimation of molecular weight. I t i s i n t e r e s t i n g to compare these findings with that of Boffa et a l . (14) who reported a molecular' weight of 150 ,000. ( i i ) S t a b i l i t y of Enzyme: The deacetylase enzyme was generally stable at a l l stages of p u r i f i c a t i o n . L y o p h i l i z a t i o n , freezing and thawing, and dialyses did not r e s u l t i n appreciable loss of a c t i v i t y . The crude enzyme used i n several of the assays had been kept at -20°C for several months without loss of a c t i v i t y , however i f l e f t to stand at room temperature or 4°C for several hours, appreciable loss of a c t i v i t y occurred. - t De*ceryi.i9&e (poo*, g) So /oo /So — I — zoo 3oo •£~Lur/or>/ vocu/^te (Ve.) Estimation of molecular.weight' of the pooled enzyme fractions from the Sephadex G-200 column (Figure 6b). 28. ( i i i ) S p e c i f i c i t y : At various stages of p u r i f i c a t i o n , the deacetylase enzyme was assayed for s p e c i f i c i t y of a c t i v i t y on [ lhC]-acetylated h i s -tones I l b i , I I b 2 , III and IV. The res u l t s are shown on figures 10a, 10b, and 10c for the 0-30% ( N H O z S O i * d i a l y s a t e , Sephadex G-200 lyophilyzed material and DE-52 lyophilyzed and dialysed f r a c t i o n respectively. As shown, the s p e c i f i c i t y changes quite d r a s t i c a l l y from being very Histone IV s p e c i f i c at the (NHi*) 2 S O i , stage to almost completely histone III s p e c i f i c at the DE-52 stage. The reason for t h i s change i n s p e c i f i c i t y i s unknown. However, i t does seem reasonable to postulate that the histone III and IV fractions may have a more d e f i n i t e part to play i n gene regulation, at the l e v e l of DNA-histone i n t e r a c t i o n , due to the greater extent of acetylation noted here over the other f r a c t i o n s . On the other hand, there might be a s p e c i f i c deacetylase f o r each histone f r a c t i o n owing to t h e i r v a r i a t i o n i n structure and acety1-modifi-cation. I t i s tempting to ask i f such a v a r i a t i o n i n the spec-i f i c i t y of the enzyme towards d i f f e r e n t histones, r e f l e c t s an inherent property of the enzyme(s) involved i n vivo, or i t merely r e f l e c t s a r e s u l t of the s p e c i f i c treatment as reported here. Tor/it c/>/-f.s)tf/:ji.. 0 >Cs «^  Crj Figure 10a, 10b,.10c. I n i t i a l S p e c i f i c i t y "studies of the 0-30% (NH^) 2S0it p r e c i p i t a t e , Sephadex G-200 enzyme pool B (Fig. 6b) and DEAE-cellulose enzyme f r a c t i o n , respectively. The reaction conditions were as described under 'Assay of Enzyme A c t i v i t y ' . 30. V. Bib1i og r aphy 1. Gershey, E.L. , V i d a l i , G., and A l l f r e y , V.G. , J. B i o l . Chem. 243 : 5018-5022, 1968. 2. V i d a l i , G., Gershey, L.E., and A l l f r e y , V.G., J . B i o l . Chem. 243 : 6361-6366, 1968. 3. De Lange, R.J., Fambrough, D.M., Smith, E.L., and Bonner, J . , J . B i o l . Chem. 244 : 5669-5679, 1968. 4. De Lange, R.J., Fambrough, D.M., Smith, E.L., and Bonner, J . , J . B i o l . Chem. 244 : 319-334, 1969. 5. Candido, E.P.M., and Dixon,. G.H., J. B i o l . Chem. 246 : 3182-3188, 1971. 6. Candido, E.P.M., and Dixon, G.H., J . B i o l . Chem. 247 : 3868-3873, 1972. 7. De Lange, R.J., Smith, E.L., and Bonner, J . , Biochem. Biophys. Res. Comm. 40 : 9 89-99 3, 19 70. 8. A l l f r e y , V.G., Foulkner, R., and Mirsky, A.E., Proc. Nat. Acad. S c i . , U.S., 51 : 786, 1964. 9. Frenster, J.H., A l l f r e y , V.G., and Mirsky, A.E., Proc. Nat. Acad. S c i . , U.S., 50 : 1026, 1963. 10. A l l f r e y , V.G., and Mirsky, A.E., Cold Spring Harbour Symp. Quant. B i o l . 2_8 : 247, 1963. 11. Pogo, B.G.T., Pogo, A.O., A l l f r e y , V.G., and Mirsky, A.E., Proc. Nat. Acad. S c i . , U.S., 5_9 : 1337, 1968. 12. Inoue, A., and Fujimoto, D., Biochem. Biophys. Res. Comm. 36_ : 146, 1969. 13. Inoue, A., and Fujimoto, D., Biochim. Biophys. Acta 220 : 307-316, 1970. 14. Boffa, L.C., Gershey, E.L., and V i d a l i , G., i n preparation. 15. Libby, P.R., Biochim. Biophys. Acta 213 : 234-236, 1970. 16. Wigle, D., Ph.D. Thesis, U.B.C., 1970. 17. Bray, G.A., Anal. Biochem. 1 : 279, 1960. 18. Gilmour, R.S., and Dixon, G.H., J. B i o l . Chem. 246 : 4621-4627, 1972. 31. 19. A l l f r e y , V.G. i n Histones and Nucleohistones ( P h i l l i p s , D.M.P. ed.) p. 241-294, Plenum Publishing Corporation, New York, 19 71. 20. Sung, M. , and Smithies, 0., Biopolymers, 1_ : 39, 1969. 21. P h i l l i p s , D.M.P. , Biochem. J. , 87_ : 258, 1963. 22. P h i l l i p s , D.M.P., Biochem. J . , 10 7 : 135, 1968. 

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