Differences Between Homogeneous Spermidine Synthases Isolated
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J. Biochem. 96, 1273-1281 (1984) Differences between Homogeneous Spermidine Synthases Isolated from Rat and Pig Liver1 Banri YAMANOHA,* Keijiro SAMEJIMA,*,z Terumi NAKAJIMA,** and Tadashi YASUHARA** *Tokyo Biochemical Research Institute , Takada, Toshima-ku, Tokyo 171, and **Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo 101 Received for publication, May 25, 1984 Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5•Œ)- 3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme. Spermidine synthase [EC 2.5.1.16] catalyzes the On the other hand, a recent report (4) has shown transfer of the propylamine moiety from S-adeno that there were significant differences among sper syl(5•Œ)-3-methylthiopropylamine (decarboxy Ado midine synthases from different bacterial species Met) to putrescine to form spermidine. The en and rat ventral prostate in their responses to two zyme has been purified to homogeneity from potent spermidine synthase inhibitors, S-adenosyl- Escherichia coli (1), rat prostate (2), and bovine 1,8-diamino-3-thiooctane (AdoDATO) (5, 6) and brain (3); each has shown very similar properties dicyclohexylamine (7). It would be, therefore, of in subunit molecular weight, molecular size, etc. interest to investigate whether there is a difference in this enzyme protein among mammalian species. 1 This work was supported in part by a Grant-in-Aid for In the present communication, we report some Scientific Research from the Ministry of Education, evidence of species differences between rat and Science and Culture of Japan. 2 Present address: Department of Analytical Chemis pig using purified enzymes from liver. try, Faculty of Pharmaceutical Sciences, Josai Univer sity, Keyakidai, Sakado-shi, Saitama 350-02. To MATERIALS AND METHODS whom correspondence should be addressed. Abbreviations: decarboxy AdoMet, S-adenosyl(5•Œ)-3- Chemicals-[1,4-14C]Putrescine dihydrochlo methylthiopropylamine; ATPA, S-adenosyl(5•Œ)-3-thio propylamine. ride (107.1mCi/mmol) and [carboxy-14C]S-adeno- Vol. 96, No. 4, 1984 1273 1274 B. YAMANOHA, K. SAMEJIMA, T. NAKAJIMA, and T. YASUHARA Syl-L-methionine (52.6 mCi/mmol) were purchased at 37•Ž. from New England Nuclear, MA, U.S.A. Un- S-Adenosylmethionine decarboxylase was as- labeled putrescine dihydrochloride and S-adenosyl- sayed according to the method of Pegg (11). L-methionine were from Tokyo Kasei, Tokyo, Protein was measured by the method of Low Japan, and Boehringer-Mannheim, Germany, re ry et al. (12) after precipitation with perchloric spectively. S-Adenosyl(5•Œ)-3-thiopropylamine acid, with crystalline bovine serum albumin as a (ATPA) hydrogen sulfate was prepared by the standard. method of Jamieson (8), and S-adenosyl(5•Œ)-3- Enzyme Purification-Male Wistar rats (3-10 methylthiopropylamine (decarboxy AdoMet) dihy months, 200 rats) were killed by decapitation, and drogen sulfate was prepared by the method re the livers (3,230g), supplied by Nippon Bio-Supp. ported in Ref. 9. Concentrations of the synthetic Center, Tokyo, were immediately frozen on dry decarboxy AdoMet in the text are divided by 2 ice and stocked at -20•Ž. Male LWD pigs because of diastereoisomers. AdoDATO was a weighing 10.7kg (6 weeks) were killed by cutting generous gift from Prof. J.K. Coward. Crystal- the carotid artery, and the livers (1,680g) similarly line bovine serum albumin was obtained from frozen were kindly donated by Dr. Y. Kido, Re- Sigma, MO. Trypsin (trypsin-TPCK, 199 U/mg) search Institute for Animal Science in Biochemistry and Chymotrypsin (alpha-chymotrypsin, 53 U/mg) and Toxicology, Sagamihara, Kanagawa. The were purchased from Worthington, NJ, U.S.A. following scale of purification is described for Dithiothreitol and Combithek (a Kit for standard 1,075g of the rat liver or 925 g of the pig liver. proteins) were from Boehringer-Mannheim, and All steps were carried out at 0-4•Ž. The livers sodium dodecyl sulfate-polyacrylamide gel electro were homogenized with 3 volumes of ice-cold phoresis Marker I (RNA-polymerase B) was from 0.25M sucrose, 0.3mM EDTA, and 10mM 2- Seikagaku Kogyo, Tokyo, Japan. Coomassie mercaptoethanol. The homogenate was centri brilliant blue G-250 was purchased from Nakarai, fuged at 105,000•~g for 60min, and then the Kyoto, Japan. All other chemicals and solvents supernatant was immediately fractionated by addi were commercially available samples with the tion of ammonium sulfate. Proteins precipitating highest purity. between 30% and 60% saturation with ammonium Enzyme Assays-Spermidine synthase was as- sulfate were collected by centrifugation at 8,000•~ sayed by the method previously reported (10) with g, and stored at - 20•Ž without a detectable some modifications. Under standard assay con loss of activity. The precipitate, dissolved in 370 ditions, the reaction mixture contained, in a total ml of 25mM sodium phosphate buffer, pH 7.2, volume of 0.5ml, 0.1M sodium phosphate buffer, 0.3mM EDTA, and 0.5mM dithiothreitol (buffer pH 7.2, 0.1ƒÊCi [1,4-14C]putrescine, 1mM putres A) containing 30mM NaCl (buffer B), was cen cine, 0.05mM decarboxy AdoMet, 5mM dithio trifuged at 105,000•~g for 30min, and the super threitol, 0.3mM EDTA, bovine serum albumin 20ƒÊ natant was passed through filter paper to remove the lipid layer. The filtrate (585ml) was applied g, and enzyme solution. After incubation for 30min at 37•Ž, 100ƒÊl of 1.5N perchloric acid was to a Sephadex G-25 column (20cm I.D.•~26cm, added to the incubation mixture. After centrifu Pharmacia) equilibrated with buffer B, and the column was eluted with buffer B at a flow rate gation for 10min at 1,000•~g, 400 ,al of the super natant was diluted with 4ml of 0.05M pyridine. of 1 liter/h. The protein fractions (1.9 liters) were The solution was then applied to a small carbox then applied to a DEAE-Sepharose CL-6B column ymethyl cellulose column (0.75ml, CM 22, What- (9cm I.D.•~24cm, Pharmacia) equilibrated with man) equilibrated with 0.01M pyridinium acetate buffer B. The column was then washed with 8 buffer, pH 5.0. After successive elution with in liters of buffer B and eluted with a linear gradient creasing concentration of pyridinium acetate buffer of 30-500mM NaCl in buffer A (a total volume (2), labeled spermidine eluted with 3ml of 0.5M of 7.5 liters) at a flow rate of 650ml/h. Active buffer was counted. All counts were corrected fractions, eluted in a salt concentration of 54-105 by subtracting the value obtained when no en mMin the case of rat, or 80-122mM in the case of zyme was added. One unit of the activity repre pig, were collected (1.58 liters and 1.25 liters, re sents the formation of 1 nmol spermidine per min spectively), and the proteins precipitating at 80% J. Biochem. SPECIES DIFFERENCE OF SPERMIDINE SYNTHASE 1275 saturation with ammonium sulfate were collected marized in Table I. Specific activities of the two by centrifugation at 8,000•~g, and stored at enzymes were similar to each other. -20•Ž without any loss of the enzyme activity . Polyacrylamide Gel Electrophoresis-Analyti Each precipitate dissolved in 35ml of buffer A cal disc gel electrophoresis was carried out at 4•Ž containing 0.3M NaC1 (buffer C) was then applied as described by Davis (13) using a 7% acrylamide to a Sephadex G-25 column (3.5cm I.D. •~52cm) separating gel (5.5cm long) and a 3% acrylamide equilibrated with buffer C, and protein fractions spacer gel (1cm long). Protein was stained with (114ml) were collected. The enzyme solution was Coomassie brilliant blue (14). For complete puri then applied to ATPA-Sepharose column (capa fication, the purified enzyme from pig liver by city, 0.95ƒÊmol of ATPA/ml; 3.5ml of the Se Sephacryl S-300 was once dialyzed against buffer pharose packed in a glass column of 2cm I.D.•~ A and then applied to the same disc gel electro 20cm) prepared as previously described (2). After phoresis. A portion containing the native enzyme the column was successively washed with 20ml of was cut out and the enzyme was collected by buffer C, 24 ml of buffer C containing 2mM adeno electro-extraction (15). sine, and 24ml of buffer C, the enzyme was eluted Sodium dodecyl sulfate gel electrophoresis was by 12ml of buffer C containing 2.5mM decarboxy carried out at 25•Ž as described by Weber and AdoMet. The effluent was pooled and concen Osborn (14), using 7% acrylamide separating gel trated to a final volume of approximately 1ml by (6.5cm long). For peptide mapping, conditions ultrafiltration in a Centriflo cone (CF 25, Ultra- were the same as described by Cleveland et al. filtration membrane cones, Amicon Far East). (16). The concentrated enzyme solution was finally ap- Isoelectric Focusing-Isoelectric focusing was plied to a Sephacryl S-300 Superfine column (1.2 carried out according to the method described by cm I.D.•~100cm, Pharmacia) equilibrated with the manufacturer by using LKB Ampholine PAG buffer C.