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. Fractions of 1ml were collected. Ac plate, pH 4.0-6.5 (LKB 1804-102 gel, LKB, Rock tive fractions (Nos. 61-65) were pooled (Fig. 3), ville, MD, U.S.A.), and an ATTO ST-1071 ap

and concentrated by ultrafiltration in a Centriflo paratus (ATTO Co., Tokyo, Japan). The plates to a final volume of 0.9ml at a concentration of were run with a voltage of 600V and current of 1.56mg/ml for the rat enzyme and 3.22mg/ml for 12.5mA for 2.5h at 4•Ž. Protein was stained the pig enzyme. The purified enzymes were stored with Coomassie brilliant blue (17). Samples for on ice and showed a similar stability to that de amino acid analysis were prepared as follows : the scribed for the enzyme from rat prostate (2). The location of each enzyme at the end of the run results of purification from the step of desalted was detected by placing a strip of wet Millipore 30-60% ammonium sulfate precipitate are sum- filter membrane (type HA, pore size 0.45ƒÊm,

TABLE I. Purification of spermidine synthase from rat and pig liver.a

a Starting materials were 1,075g of rat liver and 925g of pig liver. b Including Sephadex G-25 chromatography.

Vol. 96. No. 4, 1984 1276 B. YAMANOHA, K. SAMEJIMA, T. NAKAJIMA, and T. YASUHARA

Millipore Co., Bedford, MA, U.S.A.) on the plate by successive chromatography on DEAE-Sepha for 30s, and staining the strip with Coomassie rose, ATPA-Sepharose, and Sephacryl S-300 (Ta brilliant blue. The gel portion corresponding to ble I). Typical elution profiles of the enzyme the enzyme protein was cut out, put into a small activities from 210g of each liver on DEAE- cellophane bag with 1ml of buffer A at 4•Ž, and Sepharose are shown in Fig. 1. Maximum ac dialyzed against buffer A for 24h, followed by tivity was eluted at 80mM NaCl for rat, while at an extensive dialysis against distilled water for 100mM for pig. An extensive elution with buffer 24h. A control using a gel portion of similar B before starting the salt gradient was advanta size with no protein was dialyzed in parallel. The geous in this chromatography because it removed dialyzed solutions were lyophilized and subjected substances monitored at 410 nm. These impurities to acid hydrolysis. For the determination of reduced the purification efficiency of ATPA-Sepha amino acids, an ATTO amino acid analyzer rose affinity chromatography. In the case of pig,

(MLC-203, ATTO Co.) was used. other colored substances eluted with a peak at 60mM NaCl were partly incorporated in the active fractions. S-Adenosylmethionine decarboxylase RESULTS activity was observed in the active fractions of Purification of Spermidine Synthase from Rat spermidine synthase. The decarboxylase, how- and Pig Liver-Liver enzyme was purified by a ever, was not adsorbed on ATPA-Sepharose under modification of the method previously used for rat the conditions described, and more than 99% of prostate (2). After fractionation with ammonium the activity was recovered in the unadsorbed and sulfate followed by Sephadex G-25 chromatog washing fractions. Before the elution of sper raphy, the enzymes from rat and pig were purified midine synthase with buffer C containing 2.5mM

Fig. 1. Elution patterns of spermidine synthase activities from DEAE-Sepharose. Samples prepared from 210g of liver were applied to the column (2.6cm I.D.•~40cm), and the column was eluted with

1.13 liters of buffer B followed by a linear gradient of 30-500mM NaCl in buffer A (a total volume of

1.5 liters). About 1/5 scale of that described in " MATERIALS AND METHODS" is shown. Frac tions of 10ml were collected. Enzyme activities are shown for rat (•œ) and pig (•›), and colored sub

stances monitored at 410nm for rat (---- ) and pig (...... ).

J. Biochem. SPECIES DIFFERENCE OF SPERMIDINE SYNTHASE 1277 decarboxy AdoMet, the column was once washed purities were different between rat and pig, but with buffer C containing 2mM adenosine. Less spermidine synthase from the two species were than 0.5% of protein applied to the column was eluted at the same fractions. The active fraction eluted in this fraction, but the adenosine washing from rat showed a single protein band on ana seemed to be effective in removing proteins having lytical disc gel electrophoresis, but that from pig a mobility close to that of spermidine synthase on showed a trace contaminating band close to the polyacrylamide gel electrophoresis (Fig. 2b). The enzyme protein (Fig. 2d). This was removed by active fraction thus obtained contained protein disc gel electrophoresis coupled with an electro impurities which migrated slower than spermidine extraction, and the purified enzyme was used for synthase on polyacrylamide gel electrophoresis the following studies. (Fig. 2c). To remove the impurities, the sample Some Properties of Spermidine Synthase from was chromatographed by gel filtration on Sepha Rat and Pig-Molecular weight and subunit com- cryl S-300 (Fig. 3). Elution profiles of the im- position: The estimation of molecular weight of spermidine synthase was made with the calibrated column on Sephacryl S-300 used for purification (Fig. 3). From the calibration graph, a molecular weight of approximately 74,000 was calculated for both the rat and pig enzymes. Polyacrylamide gel electrophoresis of the two enzymes in the presence of sodium dodecyl sulfate showed a single protein band with a mobility corresponding to a molecular weight of approximately 37,000 (Fig. 4). These results agree with those from rat prostate enzyme, and suggest that each enzyme is composed of two subunits with the same or very similar molecular weights. Isoelectric point: The results of isoelectric focusing of each enzyme are shown in Fig. 5. The enzyme preparation from pig showed a single protein band focusing at pH 5.16 coincident with enzyme activity, but that from rat showed a main band accompanied by a trace contaminating band. The main single protein band coincident with en zyme activity focused at pH 5.34. Peptide mapping: Digestion of the two en zymes with trypsin or chymotrypsin according to the method of Cleveland et al. (16) revealed com pletely distinct patterns of peptides (Fig. 6). Sper midine synthase seemed to be more resistant to tryptic digestion than chymotryptic digestion, and

Fig. 2. Polyacrylamide gel electrophoresis of fractions the rat enzyme was more sensitive to the proteases from ATPA-Sepharose and purified enzymes after gel than the pig enzyme. Most of the rat enzyme filtration. Samples from ATPA-Sepharose: a, unad was rapidly hydrolyzed by chymotrypsin to pep sorbed and washing fraction (21.2ƒÊg of protein for rat tides with approximate molecular weights of less and 25.6ƒÊg for pig); b, 2mM adenosine eluate (16.7ƒÊg than 17,000 even at the incubation time of 5min, and 8.1ƒÊg); c, 2.5mM decarboxy AdoMet eluate (26.5ƒÊ while the pig enzyme was hydrolyzed to rather g and 13.9ƒÊg); d, samples from Sephacryl S-300 (7.1ƒÊ large peptides accompanying an increase of smaller g and 5.0ƒÊg, respectively). The arrow shows the peptides during the incubation time of 30min. location of a trace contaminating band in the pig Amino acid composition: The two enzyme enzyme. Conditions are described under " MA TERIALS AND METHODS." preparations purified by isoelectric focusing were

Vol. 96. No. 4. 1984 1278 B. YAMANOHA, K. SAMEJIMA, T. NAKAJIMA, and T. YASUHARA

Fig. 3. Elution patterns of spermidine synthase activities from Sephacryl S-300. For samples and conditions see " MATERIALS AND METHODS." The enzyme activities are shown for rat (•œ)

and pig (•›), and protein concentrations were monitored at 280nm for rat (---- ) and pig (...... ).

as Ser, Gly, Ala, and Met. In addition, the num bers of acidic amino acids in the pig enzyme were larger than those of the rat enzyme, while basic amino acids were nearly the same. The results may explain the difference in the pl values, al though the contents of Gln and Asn were not determined. Inhibition by AdoDATO: The rat enzyme was inhibited by AdoDATO to a larger extent than the

pig enzyme, when assayed in the standard assay Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel medium containing 1mM putrescine and 50ƒÊM electrophoresis of spermidine synthase and RNA-poly merase B. a, purified enzyme from rat (1.4ƒÊg); b, the decarboxy AdoMet (Fig. 7). From the curves,

rat enzyme and RNA-polymerase B; c, purified enzyme the rat enzyme required approximately 1ƒÊM Ado from pig (5ƒÊg); d, the pig enzyme and RNA-poly DATO for 50% inhibition, while the pig enzyme

merase B. Molecular weights for subunits of RNA- required 4ƒÊM. These values were higher than the

polymerase B are 180,000 (ƒÀ•Œ), 140,000 (ƒÀ), 100,000 (x), reported 0.2ƒÊM for the enzyme from rat ventral 42,000 (ƒ¿), and 39,000 (z). Conditions are described prostate (5), but the discrepancy would be ex under " MATERIALS AND METHODS." plained by the higher concentration of substrates in the present assay mixture.

subjected to the classical acid hydrolysis (18), and

their amino acid compositions were compared as- DISCUSSION suming the molecular weight of 74,000 (Table ‡U). The two enzymes seemed to be closely related, but The present work was undertaken prior to prepa significantly different in some amino acids such ration of the antibody against spermidine synthase.

J. Biochem. SPECIES DIFFERENCE OF SPERMIDINE SYNTHASE 1279

Fig. 5. Isoelectric focusing of purified spermidine synthase from rat and pig. A photograph of a separate run stained with Coomassie was set so as to corre spond to the enzyme activity. a, the rat enzyme (3.5 units); b, the pig enzyme (6.0 units); c, the rat and pig enzyme were run together. For measurements of enzyme activities and pH gradient, 7.0 units of the rat enzyme and 12.0 units of the pig enzyme were run together. At the end of the run, the gel was cut into slices 1mM wide, and each piece was immersed in 0.2ml of distilled water for 3h at 4•Ž. The solutions were used for measurements of enzyme activity and pH gradient. Other conditions are described under " MATERIALS AND METHODS."

Fig. 6. Time dependency of digestion of spermidine synthase. All procedures were carried out using a 15% acrylamide SDS gel according to the literature (16). The rat enzyme (12.3ƒÊg) was digested with trypsin (0.25ƒÊg) in a final volume of 20ƒÊl at 37•Ž for 30 (C) and 60min (D), and the pig enzyme (9.4ƒÊg) for 30 (E) and 60min (F). Similarly, chymotrypsine (0.25ƒÊg) digestion was performed with the rat enzyme for the incubation time of 5 (G), 15 (H), and 30min (I), and with Fig. 7. Inhibition of spermidine synthase activity by the pig enzyme for 5 (J), 15 (K), and 30min (L). Con AdoDATO. Incubation was carried out under the trols of the rat enzyme (3.7ƒÊg, A) and the pig enzyme standard assay conditions in the presence of the rat (2.4ƒÊg, B) were similarly treated in the absence of enzyme (6.4 units, •œ) or the pig enzyme (5.7 units, •›) protease. The numbers on the right side are approxi and the indicated concentration of AdoDATO. Re mate molecular weights. sults were expressed as the percentage of the activity in the absence of AdoDATO.

Vol. 96. No. 4. 1984 1280 B. YAMANOHA, K. SAMEJIMA, T. NAKAJIMA, and T. YASUHARA

TABLE ‡U. Amino acid compositions of spermidine affinity on ATPA-Sepharose, was eluted without synthase from rat and pig.a any adsorption on the column. This finding led to omission of purification on a hydroxyapatite column, known to be effective for the separation of spermidine synthase and S-adenosylmethionine decarboxylase (20). Washing with adenosine was adopted to remove proteins which may have an affinity to the adenosyl moiety of ATPA-Sepha rose. A concentration of 2mM adenosine was used after a preliminary elution experiment with 2-20mM adenosine. During the experiment, sper midine synthase was found to be eluted slowly with an increasing concentration of adenosine, but not completely even at 20mM. There was apparently no difference in molec ular weight, subunit composition, or specific ac tivity between the rat and pig enzymes. But a difference was observed in their pl values. The results were also consistent with the elution profiles on DEAE-Sepharose. Although the pl differences did not rule out the possibility of secondary minor changes in the enzyme protein, significant evidence defining the differences between the two enzymes came from their peptide maps and amino acid a Samples (82.5ƒÊg for rat and 61 .3ƒÊg for pig) collected compositions. It was notable that the rat enzyme after isoelectric focusing as described under " MA was more sensitive to both tryptic and chymo TERIALS AND METHODS" were hydrolyzed in 6N tryptic digestion than the pig enzyme, and that HCl for 72h according to the method described in the patterns of the peptides from the two enzymes Ref. 18. The residues were dissolved in 0.5ml of 0.1N were distinct. HCl, and 100ƒÊl of each was analyzed. Coefficients of

variation for the determination of amino acids were in From these results and the inhibitory effects the range of 0.34% and 1.10%. b Not extrapolated to of AdoDATO, one can conclude that hepatic zero hydrolysis time. c Without oxidation with per- spermidine synthase is structurally different be formic acid. d Parentheses show nearest integer calcu tween rat and pig. lated from molar ratios of amino acids to Phe assuming the molecular weight of 74,000. We are grateful to Prof. A.E. Pegg, the Milton S. Hershey Medical Center, and the late Prof. A. Raina, In order to obtain sufficient material for charac University of Kuopio, for critical evaluation of this work and the manuscript. We also thank Prof. S. Takitani terization and future immunization, and with ref and Miss C. Kuragaki, Tokyo Science University, and erence to studies of distribution in rat tissues (19), Dr. M. Okada of this Institute for their support during liver was chosen as the enzyme source. For the this work. large scale purification, DEAE-cellulose previously

used was replaced by DEAF-Sepharose. Evidence REFERENCES for species difference between rat and pig was first suggested in this chromatography. From the 1. Bowman, E.H., Tabor, C.W., & Tabor, H. (1973) J. Biol. Chem. 248, 2480-2486 reproducible elution profiles (Fig. 1), the pig en 2. Samejima, K. & Yamanoha, B. (1982) Arch. Bio zyme was expected to be more acidic than the rat chem. Biophys. 216, 213-222 enzyme. S-Adenosylmethionine decarboxylase, 3. Samejima, K., Raina, A., Yamanoha, B., & which was observed in the active fractions of Eloranta, T. (1983) in Methods in Enzymology spermidine synthase after DEAE-Sepharose chro (Tabor, H. & Tabor, C.W., eds.) Vol. 94, pp. 270- matography and which was supposed to have an 276, Academic Press, Inc., New York

J. Biochem. SPECIES DIFFERENCE OF SPERMIDINE SYNTHASE 1281

4. Pegg, A.E., Bitonti, A.J., McCann, P.P., & Coward, 14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, J.K. (1983) FEBS Lett. 155, 192-196 4406-4412 5. Tang, K.-C., Pegg, A.E., & Coward, J.K. (1980) 15. Horinishi, H., Satoh, S., & Honjoh, T. (1982) in Biochem. Biophys. Res. Commun. 96, 1371-1377 Japan Patent, No. 56-021805 6. Tang, K.-C., Mariuzza, R., & Coward, J.K. (1981) 16. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., J. Med. Chem. 24, 1277-1284 & Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102- 7. Hibasami, H., Tanaka, M., Nagai, J., & Ikeda, T. 1106 (1980) FEBS Lett. 116, 99-101 17. Reisner, A.H., Nemes, P., & Bucholtz, C. (1975) 8. Jamieson, G.A. (1963) J. Org. Chem. 28, 2397-2400 Anal. Biochem. 64, 509-516 9. Samejima, K., Nakazawa, Y., & Matsunaga, 1. 18. Moore, S. & Stein, W.H. (1963) in Methods in (1978) Chem. Pharm. Bull. (Tokyo) 26,1480-1485 Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) 10. Pegg, A.E. & Williams-Ashman, H.G. (1969) Vol. 6, pp. 819-831, Academic Press, Inc., New J. Biol. Chem. 244, 682-693 York 11. Pegg, A.E. (1974) Biochem. J. 141, 581-583 19. Raina, A., Pajula, R.-L., & Eloranta, T. (1976) 12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & FEBS Lett. 67, 252-255 Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 20. Hannonen, P., Janne, J., & Raina, A. (1972) Bio 13. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427 chem. Biophys. Res. Commun. 46, 341-348??