THE .JOURNAL OF BIOLOGICAL CHEMISTRY Vol 255, No. 10, Issue of May 25, pp. 4786-4-4592, 1980 Printed in (1.S.A

Proline-specific Endopeptidase from Flavobacterium PURIFICATION AND PROPERTIES*

(Received for publication, November 26, 1979)

Tadashi Yoshimoto, Roderich Walter,$ and Daisuke Tsuru From the Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852 Japan, and the +Department of Physiology and Biophysics, University of Illinois Medical Center, Chicago, Illinois 60612

Proline-specific endopeptidase (EC 3.4.21.26) was pu- rified 1,400-fold in an overall yield of 10% from cell-free extracts of Flavobacterium meningosepticum by CM- cellulose and hydroxyapatite column chromatogra- phies and gel filtration on Sephadex G-150. The purified enzyme was apparently homogeneous as judged by both standard disc gel and sodium dodecyl sulfate (SDS)-gel electrophoresis. The enzyme is an endopep- tidase which catalyzes the hydrolysis of Pro-X peptide bonds (and, more slowly, Ala-X bonds), an activity similar to mammalian post-proline cleaving enzyme (Yoshimoto, T., Fischl, M., Orlowski, R. C., and Walter, R. (1978), J. Biof. Chem 253, 3708-3716). Vasopressin, oxytocin, angiotensin 11, thyroliberin, oxidized insulin B-chain, and neurotensin, as well as a variety of syn- thetic substrates, were all hydrolyzed on the carboxyl side of proline residue. The enzyme was, however, inert toward (?-~-Pro)oxytocin and high molecular weight proteins, even after denaturation. The enzyme was most active at pH 7.0 and had an isoelectric point of 9.6. The molecular weight of the enzyme was estimated to be 74,000 by gel filtration on Sephadex (2-150 and 76,000 by SDS-gel electrophoresis. The agreement be- tween the two methods indicates that the native en- zyme is a monomer. The enzyme was inhibited by diiso- propyl phosphorofluoridate and by the chloromethyl ketone derivative of Z-Gly-Pro, but not by phenylmeth- anesulfonyl fluoride. Kinetic studies of the size and stereospecificity of the active site of the enzyme sug- gested that the enzyme has three subsites (S3, SZ, and SI) on the NH2-terminal side of the hydrolyzed bond, and two subsites (S’l, S Z ) on the COOH-terminal side. High stereospecificity was found for Subsites SI, SZ, and S’l. Antiserum raised against this proline-specific en- dopeptidase did not cross-react with the post-proline cleaving enzyme from lamb kidney in Ouchterlony im- munodiffision.

Post-proline cleaving endopeptidase (EC 3.4.21.26) was dis- covered by Walter et at. (1) in human uterus. It hydrolyzes the Pro7-Leu’ bond of oxytocin. The enzyme has been purified from lamb kidney (2,3) and characterized as a serine protease with strict specificity for cleaving the Pro-X bond (4). The size and stereospecificity of the active site of this enzyme have been studied (5, 6), and the presence of post-proline cleaving enzyme in a variety of vertebrates and its organ distribution

* This work was supported by grants from the Ministry of Educa- tion, Science, and Culture of Japan, the Mishima Kaiun Memorial

by United States Public Health Service Grant AM 18399. The costs Foundation of Japan, the Applied Enzyme Foundation of Japan, and

of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

has also been investigated (7-11). While the activity is ubiq- uitous, levels are low in the tissues of vertebrates.

In an effort to find a good source of post-proline cleaving enzyme, our studies were extended to include microorganisms. After screening more than 500 microorganisms from soil and stock strains available in our laboratory, post-proline cleaving- like activity was found only in Flavobacterium meningosep- tzcum (12). This paper describes the purification and charac- terization of the microbial enzyme and compares some of its physicochemical and enzymological properties with those of post-proline cleaving enzyme isolated from lamb kidney.

EXPERIMENTAL PROCEDURES Preqeracion of 2-Ala-ProZ-NNap: 2-AI-Pro-OH,DCH* (6) (1.15 8. 3.49 mole) Y(LB diasalved i n 15 m1 of Lfhano l . The solut ion was cooled 19 an ice bath and s t i r r ed w i th 3 ml of I N HC1 for 1 h. Hethan01 was rellYed i n vacuo a t 3O.c artd 20 ml of H20 YUI added. The que-

vaehed with H ti vnril neutral. dried w e r anhydrous NqSO4. f i l t e r e d . evaporated LO dry””, DUB 801utiOn vas extracted v i t h e t h y l acetate ( 4 x 15 d). The e thy l ace t a t e extract WUI

and dissolved2in CHCl3 (12 01) and cooled at -5 - -1O’C. N-Methyl-morpholine (0.38 ml) aod i.obutylehlolofo-te (0.49 ml) e r e added. After stirrimg fo r 10 pin, J-naphthgl.pin.

and then s t i r r ed for 44 h. The aolurion x- washed vich Hz0 ( ~ 3 ) . 1 N HC1 (~2) . H?O ( d l , (0.5 8. 3.49 -le) “118 sdded. The react ion w- s i lored to e- zo ro- r ~ e n t u r c s l o r l y

8% NaHC03 ( X 2 ) and U20 (x)). After evaporstion of cLlcl3 the product “118 e r p t a l l i r e d f rm ethanol. Yield, 1.01 8 (652). m. p. 144-146.C. -123.48’ (c 1.15 O w ) .

C26H27N304 Pound C. 69.80; H. 6.32; N. 9.19 Calculated C , 70.09; H, 6.11; N. 9.43

Z-D-Ala-Pr0-2-NNa : This W B L ~ prepared by the BLYC r fhod desc r ibed above usin8 2-D-Ala-Pro (6) and p-nsphthy%dne. Yield. 76.82. m. p. 63-70-C. [&la6. -106.3. (c 1.15 W ) .

C26X27N304.1,,zl120 Calcvlared C, 68.70; H, 6.21; N. 9.24 Pound C , 68.5ti; H, 6.46; N, 9.00

2-Ala-Gly-Pro-2-map: 2-Ala-Gly-ProOH (6) (0.394 g. 1.045 -le) VM dissolved i n 6 m1 of 1 ,2d iae rha rYl e t b e and cooled t o -5 - - 1 O T . N-Methyl-orpnoline (0.12 ml) and ieobutyl- c h l o r o f o m t e (0.15 .I) were added. After st irring for 10 r i m . f - a p h t h y l d n e (0.165 8 ,

s t i r r e d for three day@. 1.2-Di.ethoryl ethane was evaporated i n vacuo d the residue was 1.15 . a l e ) YBB added. The reaction w a s a l lp l ed to come t o r- temperature slowly and

dissolved i n ethanol. The so lu t ion was vashed with H 0 ( x 3 111 H C l (12). Hz0 ( 1 3 ) . 87. NaHCO3 (x3) rM dried over v i t h N+O&. The Iolut ion?wr. e&Orated t o 0 amall v o l e and precipitated f ro . p e t r o l e m e c b r . It w a s r ec rys re l l i r sd frm c thmol . Yie ld , 0.321 g (61.21). 0 . P. 159-160.5’C. [ N I ~ J , -94.3’ (f 1.05 DW).

c28H30N405 Pound Calculated C. 66.85; H, 6.03; N. 11.17

C , 66.60; H. 6.30; N. 11.17

7.-D-Na-Gly-Pro-2-m. : Z-D-Ala-2ly-Pro-OH (6) (0.621 8. 1.65 -le) was diseolved i n 10 ml of 1 . Z - d i r t h o ~ l e& and 215 rl of D w , e d eooled t o -5 - -10.C. N“ethyl-rpholine (0.18 r l ) and i aobv ty leh lo ro foNLe (0.23 11) were added. d a f t e r stirring for 10 r i m .

~ a s p h r h y l . n i n ( 0 . 2 3 4 8, 1.65 r o l e ) w a s fu r the r added. The reaction w u all-d t o c O L

ra ted to drynees and rediesolved i n e thyl ace ta te . Sar Prcducts precipitated out. Tlw to r w f m e r a t u r e slowly .ad then a t i r r a d f o r t h r e e days. The react ion mlilcure IR. tvsPD-

p rec ip i t a t e m e f i l t e r e d , washed r l t h 111 Bc1, 820. 82 NlllCO md 0 .ad C V B C d l i Z s d fTOI ethanol. The products were c d i n e d and recrySCallired f t d C!%213f;hmd and hot eth.nO1. Yield. 0.4949 (59.32). m. P. 198-2W.C. [Wl$6, -89.7’ (c 1. n0).

Calculated C , 66.85; H. 6.03; R. 11.17 C28H30N405 Foud C. 67.09; H. 5.80; N. 11.W

Gly-Pro-2-NNap,’ Ala-Ala-2-NNap, Pro-2-NNap, and Z-Pr0-2- NNap were purchased from Vega-Fox. Fast Garnet GBC salt, PheCH2S02F, oxidized insulin B-chain, bovine serum albumin, a- chymotrypsinogen, and P-naphthylamine were obtained from Sigma. DFP, Freund’s complete adjuvant, Nbs, pC1HgBzO-, 2-iodoacetic acid, EDTA, o-phenanthroline, Tos-Lys-CHgC1, Z-Phe-CHAX, glu-

amino acids were from Nakarai Chemicals. [32P]DFP (specific activ- tamic-oxalacetic transaminase, polyamide layer sheets, and dansyl

ity, 250 pCi/mg) was obtained from Amersham. Z-Gly-Pro-Leu-Gly and Ala-2-NNap were purchased from the Protein Research Foun- dation, Japan. Substance P and thyroliberin were obtained from

The abbreviations used are: 2-NNap, /3-naphthylamide; DFP, diisopropyl phosphorofluoridate; Nbs, 5,5”dithiobis(2-nitrobenzoic acid); pC1HgBzO-, p-chloromercuribenzoic acid; dansyl, 5-dimethyl- aminonaphthalene-I-sulfonyl; ONp, p-nitrophenyl ester; Phe- CH2S02F, phenylmethanesulfonyl fluoride; MCA, 4-methylcoumari- namide; DCHA, dicyclohexane; AVP, (8-Arg)vasopressin; HVE, high voltage electrophoresis; Tos, NO-toluene sulfonyl; SDS, sodium do- decyl sulfate. All optically active amino acids are of L configuration unless otherwise stated.

4786

Proline-specific Endopeptidase 4787

Peninsula Laboratory. Pyronine G and sodium tetrathionate were obtained from Merck, Germany. Rabbit muscle aldolase, beef liver catalase, and phosphorylase A were from Boehringer Mannheim, Germany. Chymostatin, leupeptin, antipain, bestatin, pepstatin, phos- phoramidon, and elastatinal were graciously supplied by Dr. Ume- zawa, Institute of Microbial Chemistry. Streptomyces subtilisin inhib- itor was the generous gift of Dr. Murao, University of Osaka Prefec- ture, and porcine a*-macroglobulin was prepared as described previ- ously (13). Neurotensin was kindly supplied by Dr. Yajima, Kyoto University. Z-Gly-Pro-CHzC1 was prepared as described (4). Other peptides, including Z-Gly-Pro-MCA, were from the same batches used in previous studies (5-7). [9-(l-14C)glycinamide]Vasopressin (specific activity, 30 mCi/mmol) and [9-(l-'4C)glycinamide]oxytocin (specific activity, 100 mCi/mmol) were prepared by the method of Walter and Havran (14). [U-l4C]Ala-Ala (specific activity, 19 mCi/ mmol) was from the same batch used before (5). Other chemicals and enzymes were from Seikagaku Kogyo Co., Japan.

Enzyme Activity Assay-Proline-specic endopeptidase activity was measured by a modification of the method of Yoshimoto and Walter (15). Enzyme solution (0.025 ml) was mixed with 0.25 ml of 2.1 mM Z-Gly-Pro-2-NNap in 40% aqueous, 1,4-dioxane and 1 ml of 0.1 M Tris-HCI, pH 7.0. After incubation at 30°C for 10 min, 0.5 ml of Fast Garnet GBC salt (1 mg/ml in 1 M acetate buffer, pH 4.0, containing 10% Triton X-100) was added to the reaction mixture. The absorbance of the resulting diazo dye was measured at 550 nm. One unit of the enzyme activity is defined as the amount of activity which releases 1 pmol of P-naphthylamine/min at 30°C. Protein concentra- tion was assayed by measuring the absorbance at 280 nm and assum- ing that E% = 10.0.

Kinetic Studies-The concentration of enzyme was calculated on the basis of specific activity. A computer program based on that of Bliss and James (16) was used in the calculation of Km and kcat. Peptide-hydrolyzing activity was assayed by the ninhydrin method (17). Hydrolysis of Z-Gly-Pro-MCA was assayed spectrofluorometri- cally at 25°C in a solution containing 3 ml of 0.1 M phosphate buffer, pH 7.0, 50 pl of 0.5 mM Z-Gly-Pro-MCA in dioxane, and 50 pl of enzyme. The release of 4-methylcoumarinylamine was monitored in a Hitachi 5 spectrofluorometer, using an excitation wavelength of 370 nm and an emission wavelength of 440 nm (11). Hydrolysis of peptide p-nitrophenyl ester was assayed by adding 50 pl of substrate in dioxane to a mixture of 50 pl of enzyme and 1 ml of 10 m~ phosphate buffer, pH 7.0, and incubating the mixture at 25OC for various periods of time. The initial velocity of the reaction was measured by the increase in absorbance at 410 nm using a double beam spectropho- tometer, Shimazu UV-200 (4). Hydrolyses of peptide-p-naphthylam- ides were followed by the standard enzyme activity assay method described above.

Inhibition Tests-Inhibition constants were calculated from Dixon plots (18) using Z-Gly-Pro-ONp and Z-Ala-Ala-ONp as substrates. Two substrate concentrations and four inhibitor concentrations were used for these studies.

Kinetic Studies on Oxytocin and Vasopressin-The hydrolysis of oxytocin and vasopressin was assayed by a modifkation of the method of Simmons and Walter (19) using [I4C]AVP or [14C]oxytocin speci- ficially labeled in the glycinamide moiety and measuring the release of Arg-['4C]GlyNH2 or Leu-[14C]GlyNHz, respectively. Substrate was prepared by neutralizing the 5 p1 of radiolabeled AVP (0.5 nmol) or oxytocin (0.16 nmol) with 5 p1 of 0.14 N NaOH and diluting with 15 pl of different concentrations of unlabeled AVP or oxytocin (2 to 0.062 mM). This solution was added to 20 pl of enzyme (0.6 units/ml) and incubated at 30°C for 2,4,8,16, and 32 min. The reaction was stopped by adding 5 pl of 6 N acetic acid. The reaction solution was spotted on Whatman 3MM paper and subjected to high voltage electrophoresis (Savant Instruments; 60 V/cm for 1 h using 0.5% pyridine, 5% acetic acid buffer, pH 3.5). The relative amounts of labeled product and substrate were determined by scanning the paper strip for radioactiv- ity on a Packard model 7201 radiochromatogram scanner. From the time course of hydrolysis, the initial velocity was calculated and used to determine kinetic parameters.

Identification of NH2-Terminal Residues of Enzyme-digested Peptides-Cleavage points in peptides were identified by the dansylation method (20). The peptide (0.1 pmo1/100pl) was incubated with 50 p1 of enzyme (1.5 uNts/ml) at 30°C. After 20 min, a 5-pl aliquot was removed and subjected to dansylation to identify the newly formed NH2-terminal amino acids.

Cleavage points were also identified by high voltage electrophoresis and amino acid analysis. Peptide substrate (1.1 pmol) in 200 pl of 50 mM phosphate buffer was incubated with proline-specific endopepti-

dase (2 units) a t 30°C for 2 h. The reaction was stopped by heating at 100°C for 2 min, and the mixture was applied to Whatman 3MM fdter paper. After electrophoresis (60 V/cm) for 1 h, both edges of the paper strip were developed with ninhydrin-cadmium solution (21). The portion of the paper corresponding to the colored areas was extracted with 50 m~ HCl. The extract was hydrolyzed in 6 N HCl at 110°C for 24 h and then analyzed for amino acids by the method of Moore (22) using a Durmm amino acid analyzer.

Screening of Microorganisms for Proline-specific Endopeptidase and Cultivation of F. meningosepticum-Each microorganism, iso- lated from soil or stock strains, was cultivated in bouillon medium (1% bouillon, 1% polypeptone, and 0.5% NaCI, pH 7.0) at 30°C for 30 h under reciprocal shaking. After cultivation, cell suspensions were assayed for activity by the standard assay method.

Cultivation of F. meningosepticum for purification of the enzyme was performed at pH 7.2 and 30°C using a Microferm Fermentor (New Brunswick) containing 8 liters of bouillon medium. After 18 h, the cells were harvested by centrifugation (8,000 rpm for 20 min) and washed with cold 50 mM Tris-HC1 buffer, pH 7.0.

Enzyme Purification-All steps were carried out a t 4°C unless otherwise described. Cells (70 g wet weight) were suspended in 100 ml of 50 mM Tris-HC1 buffer, pH 7.0, and disrupted for 10 min with glass beads (0.3-mm diameter) in a Vibrogen Cell Mill (Edmund Buhler, Germany). The pellet was diluted three times with 50 mM Tris-HCI buffer, pH 7.0, and centrifuged (10,000 rpm for 20 mid , and the resultant supernatant was treated with protamine-H2S04, pH 7.0. Any precipitate was removed by centrifugation. The solution was fractionated with ammonium sulfate (65 to 90% saturation) and desalted by gel Ntration using a Sephadex (3-25 column (3.5 X 40 cm) equilibrated with 20 mM phosphate buffer, pH 6.2. Pooled enzyme solution was directly applied to a CM-cellulose column (3.5 X 30 cm) equilibrated with the same buffer. Adsorbed proteins were eluted by a linear gradient of NaCl (0 to 0.25 M in 1 liter of eluent). The proline- specific endopeptidase was eluted at 0.05 M NaCl concentration. Active fractions were combined, concentrated by ultrafiltration using an Amicon apparatus (PM-lo), and applied to a hydroxyapatite column (2 X 20 cm) which was equilibrated with 10 mM potassium phosphate buffer, pH 7.0. Enzyme was eluted with a linear gradient of potassium phosphate buffer with a concentration range of 10 to 500 m ~ . Active fractions were again pooled, concentrated, and sub- jected to gel fdtration on a Sephadex G-150 column (2 X 100 cm) equilibrated with 0.1 M phosphate buffer, pH 6.2, containing 0.1 M NaCI. Proline-specific endopeptidase-containing fractions were pooled, dialyzed against deionized water, and then lyophilized. This enzyme preparation was stored in a freezer at -3OOC until use.

Molecular Weight Determination-The molecular weight of the purified enzyme was estimated fust by gel fdtration on a Sephadex G-150 column (2 X 110 cm) according to the method of Andrews (23). The elution buffer was 20 mM phosphate containing 0.2 M KCl, pH 7.2. Glutamic-oxalacetic transaminase (M? = 91,OOO), bovine serum albumin (M, = 68,000), and subtilisin BPN' (Mr = 27,800) served as markers. As a second method, SDS-polyacrylamide gel electropho- resis as described by Weber and Osborn (24) was used. Native or [32P]DFP-treated enzyme, as well as the marker proteins, phospho- rylase A (Mr = 100,000), bovine serum albumin (My = 68,000), catalase (M, = 58,000), aldolase ( M , = 40,000), and chymotrypsinogen ( M , = 25,000) were mixed separately with 0.1 ml of 20 mM sodium phosphate buffer, pH 7.0, containing 2% SDS and 2% 2-mercaptoethanol and incubated at 37°C for 2 h. Electrophoresis was carried out at a constant current of 8 mA/gel for 4 h using bromphenol blue as running marker. Gels containing native enzyme and marker proteins were stained with 0.28% of Coomassie Brilliant Blue H and destained by washing with a mixture of acetic acid/methanol/water (7.550876, v/v). Gels containing ["P]DFP-treated enzyme were developed by radioautography using no-screen fdm, x-ray developer, and rapid fixer from Kodak.

Disc Gel Electrophoresis-Disc electrophoresis was based on the method of Davis (25). A 7% separation gel in p-alanine and acetate buffer, pH 4.5, was prepared and the enzyme preparation (200 pg) was applied. A current of 2.5 mA/tube was applied for 2 h at 4°C using pyronin G as running marker. Matched pairs of gels were prepared. One of the gels was stained for 30 min for protein with 0.05% Coomassie Brilliant Blue R and destained in acetic acid/meth- anol overnight. The other gel was used to evaluate the location of enzymatic activity by submerging the gel, in the presence of 0.5 mM Z-Gly-Pro-2-NNap and 25 mg of Fast Garnet GBC salt, in 20 ml of 0.2 M Tris-HC1, pH 7.0, containing 10% 1,4-dioxane for 1 h at 30°C.

Effect of Metal Ions and Chemical Inhibitors-Enzyme was dis-

4788 Proline-specific Endopeptidase solved in 0.1 ml of 50 n“ Tris-HCI buffer, pH 7.0, in the absence or presence of increasing concentrations of the respective chemical in- hibitor (DFP, pCIHgBzO-, Nbs, sodium tetrathionate, 2-iodoacet- amide, EDTA, or o-phenanthroline) and preincubated at 25°C for 30 min. Residual peptidase activity was then determined after incubation with 1 mM Z-Gly-Pro-2-NNap following the standard method.

Effect of Active Site-directed Inhibitors on the Arylamidase Ac- tivity-Inhibition experiments using chloromethyl ketone derivatives, DFP, and PheCHaOZF were carried out under conditions identical to those used for the chemical inhibitors, except that in the case of the active site-directed inhibitors, the time courses of residual enzyme activity were followed for as long as 16 h. Control experiments were carried out under identical conditions in the absence of inhibitor. Pseudo-fmt order rate constants for enzyme inactivation (hod were calculated from the initial rates of inhibition according to the equation below, where El and EI are the observed activities a t times TI and Tz.

In (EllEd koh = TI - TI

Preparation of [32PJDFP-inhibited Enzyme-The enzyme was inhibited by treatment with a 10-fold molar excess of [=P]DFP in 50 mM Tris-HCI buffer, pH 7.0, for 1 h at 25°C. The irreversibly labeled enzyme was separated from excess unbound [32P]DFP by gel filtration on a Sephadex G-25 column (2.5 X 60 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7.0.

Titration of Enzyme with [32PJDFP-Enzyme (0.167 nmol) in 250 pl of the above buffer was incubated with 1.5 nmol of [=P]DFP in 50 pl a t 25°C for 20 min. The remaining activity of the enzyme was assayed by the standard method. Free [32P]DFP was removed from a 2004 aliquot of the incubation mixture as described above using a column (2.5 X 60 cm) of Sephadex G-25. The amount of [3ZP]DFP that had reacted with the enzyme was calculated from the radioactiv- ity incorporated into the protein.

Isoelectric Focusing-The enzyme (0.45 mg, 5 ml) was applied to an isoelectric focusing column (250 ml) according to the method of Vesterberg and Svensson (26) using carrier ampholytes in the pH range of 3.5 to 10.0 and sucrose to form the density gradient from 0 to 50%. The column was then subjected to isoelectric focusing by apply- ing 400 V for 40 h at 4OC.

Antiserum against Proline-specific Endopeptidase-The purified proline-specific endopeptidase (0.5 mg) was emulsied with 0.5 ml of Freund‘s complete adjuvant. The emulsion was injected into the hind foot pads of a rabbit weighing about 2.5 kg. Two boosters of 0.25 mg of protein were given subcutaneously at intervals of 2 weeks. The rabbit was fed commercial diet ORGI and bled 6 weeks after the first injection. Immunodiffusion was performed overnight in 1.2% agarose gel in 70 mM phosphate buffer, pH 7.5, containing 0.9% NaCl, and precipitated protein was stained with Amido Black 10B after soaking the agar plate in water for 2 days. The plate was destained with 2% acetic acid (27).

RESULTS

Screening Test for Post-Proline Cleaving Enzyme-like Ac- tivity-Of more than 500 strains of microorganisms from soil and stock strains in our laboratory, F. meningosepticum and related strains of Flavobacterium showed arylamidase activ-

TABLE I Purification ofproline-specific endopeptidase /bm F.

meningosepticum Enzyme activity was assayed by the standard method using 2-Gly-

Pro-2-NNap as substrate.

Purification step Volume activity act,vlty Total S p e ~ i f ~ yield Purifi- cation

ml units units/mg S -fold Supernatant of cell-free 1950 2418 0.087 100 1

Salting out with 85 1440 5.1 60 58

CM-cellulose chromatog- 120 655 33.3 27 383

Hydroxyapatite column 70 370 110 15 1264

Gel filtration on Sepha- 50 250 123 10.3 1414

extract

(NHd801 (6540%)

raPhY

chromatography

dex (3-150

0.1 . - u

FIG. 1. Chromatography of proline-specific endopeptidase on DEAE-Sephadex A-SO. The column (3.5 X 30 cm) was equili- brated with 20 mM phosphate buffer, pH 6.2. Elution was performed using a h e a r gradient of NaCl (0.0 to 0.25 M in 20 m~ phosphate buffer (---). Absorbance was measured at 280 nm (O”-O). En- zyme activity was determined under the standard conditions as de- scribed under “Experimental Procedures” using Z-Gly-Pro-2-NNap as substrate (M).

0.1

B C 0

0 10 70 IO a0 50 60 10 80 90

FlaCl lon ““lber. 5 n1/tut+

FIG. 2. Gel filtration of Sephadex G200 and analysis of pro- line-specific endopeptidase. The column (2 X 100 cm) was equili- brated with 10 mM phosphate buffer, pH 7.0, containing 0.1 M NaCl. Absorbance was measured at 280 nm (0- - -0) and amidase activity was determined using Z-Gly-Pro-2-NNap as substrate (M). A, analytical disc gel electrophoresis of prolie-specific endopeptidase (200 p g ) ; B, determination of proline-specific endopeptidase activity following disc gel electrophoresis as described under “Experimental Procedures”; C, SDS-gel electrophoresis of proline-specific endopep- tidase (the enzyme used was 150 pg in 0.1 ml); D, radioautogram of [32P]DFP-inhibited proline-specific endopeptidase on SDS-gel elec- trophoresis. Radioautography was carried out for 10 h. Other details are described under “Experimental Procedures.”

ity using Z-Gly-Pro-2-NNap as substrate. The enzyme activity was only observed in bacterial cell suspensions, not in culture filtrate. Of several media tested for enzyme production, 1% bouillon medium was found to be the best medium for the cultivation of F. meningosepticum. Addition of proline and proline-containing peptides (subtilisin digests of gelatin) showed no significant effect on enzyme production.

Purification of Proline-specific Endopeptidase-After the cultivation of F. meningosepticum in the fermentor, about 70 g wet weight of cells were obtained. Table I summarizes the purification of proline-specific endopeptidase from the bacte- rial cells. Fig. 1 illustrates the chromatographic profile of the enzyme after ammonium sulfate fractionation, and Fig. 2 shows the gel filtration pattern of the enzyme on Sephadex G-

Proline-specific Endopeptidase 4789

TABLE I1 Some enzymatic and physicochemical properties of proline-specific endopeptidase from F. meningosepticum and post-proline cleaving

enzyme from lamb kidneys

Optimum pH 7.0 7.8 pH stability' 5.0-9.0 5.5-9.5 Optimum temperatured 40°C 47°C Stability for temperature' 42OC 43OC Isoelectric point (PI) 9.6 4.8 Molecular weight by gel filtration 74,000 115,006 Standard substrate used was 2-Gly-Pro-2-NNap. Reported in Ref. 5. Buffer used pH 3.5 to 5.5 in 0.2 M acetate; pH 5.5 to 7.5 in 0.2 M

phosphate; pH 7.5 to 8.5 in 0.2 M Tris-HC1; pH 9.0 to 11 in 0.2 M borate/NaOH. Preincubation was at 30°C for 1 h. Assays for residual activities were carried out under standard conditions.

Enzyme and substrate were incubated at pH 7.0 for 5 min and the amount of substrate hydrolyzed was determined.

Residual activity was % after 15 min of incubation at pH 7.0 and the temperature indicated.

'Reported in Ref. 3.

'rABLE 111 Rate inhibition of proline-specific endopeptidase from F.

meningosepticum, post-proline cleaving enzyme from lamb kidney, trypsin. and chymotrypsin by active site-directed reagents

Proline- Post-pro- specific line Chymo-

endopepti- cleaving trypsin Reagents d& enzyme

k d [ I ] (M-' s") DFP 130 64.6 2.5 40.6 PheCHaOZF 0.00 0.07 2.5 703 Z-Phe-CHzCI 0.00 0.00 0.00 73.0

Z-Gly-Pro-CHzC1 21.0 55.6 0.00 0.00 TOS-LYS-CHZCI 0.00 0.00 7.7 0.00

A C

B

FIG. 3. Double immunodiffusion profile of proline-specific endopeptidase antiserum against several proline-specific pep- tidases in agarose gel. The center well (Ab) contained antiserum (10 p l ) to the purified proline-specific endopeptidase. Wells A, B, and C contained proline-specific endopeptidase (10 p g ) from F. meningo- septicum, post-proline cleaving enzyme (30 p g ) from lamb kidney, and post-proline dipeptidyl aminopeptidase (10 pg) from lamb kidney, respectively.

150 after purification by hydroxyapatite chromatography. The enzyme was purified 1,400-fold with a yield of 10% from the cell-free extract of F. meningosepticum.

Purity of Proline-specific Endopeptidase-After gel filtra- tion on a Sephadex G-150 column, the enzyme gave a single

band of protein upon disc electrophoresis (Fig. 2.4) . Using Z- Gly-Pro-2-NNap as substrate, enzyme activity was only de- tected in the region of the single band (Fig. 2B). A single band was also observed after SDS-gel electrophoresis (Fig. 2 0 . When the enzyme which was inhibited by [:"P]DFP was applied to SDS gels, the radioactivity was observed at the same position as that of the protein stain (Fig. 2D).

Effect of pH and Temperature-The optimum pH of the enzyme was 7.0 using Z-Gly-Pro-Leu-Gly and Z-Gly-Pro-2- NNap as substrates. The enzyme retained more than 90% of the original activity between pH 5.0 and 9.0 after incubation at 30°C for 1 h. The optimum temperature was 40°C for a 10- min reaction and 50% of the initial activity was observed after incubation for 15 min at 42OC and pH 7.0 (Table 11). The lyophilized enzyme retained more than 90% of its activity when kept at 25°C for a week.

Effect of Chemical Reagents, Metal Zons, and Inhibitors- The enzyme was not inhibited by 1 m~ EDTA, o-phenan- throline, dithiothreitol, Nbs, sodium tetrathionate, pC1- HgBzO-, Fez+, Mn2+, Co2+, or H8+. Table I11 summarizes the effect of active site-directed reagent on four enzymes. The bacterial enzyme was completely inhibited by DFP and Z- Gly-Pro-CH&l. Pseudo-first order rate constants (kO~JII]) for DFP and Z-Gly-Pro-CKC1 were 130 and 21.0 M" s-', respec- tively. Titration of proline-specific endopeptidase with r2P]- DFP showed that the enzyme was completely inhibited by the incorporation of 1 mol of ["P]DFP/mol of enzyme. The

TABLE IV Degradation of peptides by proline-specific endopeptidase

Identification methods of each substrate and the digestion products are shown below.

veptldc

*rocin

C~s-Tyr-lle-Cln-Asn-C~s-Pro4Leu-[14C]C1y-NH2

Vasopressin

C~s-Tyr-Phe-Gln-Asn-Cjs-Pro*Arg-[14ClGly-NH2

Thyroliberin (TRH)

<Clu-His-Pro&H2

Angiotensin I1

Asp-Arg-Val-Tyr-Ile-His-Pro*Phe

Neurotensin

cClu-Leu-Tyr-Cln-Asn-Lys-Pr~~~~-Arg-Pro~r-lle-Le~

Oxidized insulin &chain

------Arg 22 -Gly 23 -Phe 24 -Phe 25 -Tyr 26 -Thr27-Pro28kys29-Ala,o

Tetra-alanine

A I ~ - A ~ ~ - A I ~ U A ~ ~ Penta-alanine

[ 14C]Ala-Ala-AlaUAla-Ala

IdanClflEatlm rchode for each eubstrate and the dl~estlon productsa)

orrcoc1n: CIY"). m m c t w c y or 1m-1~'c1cly-m2 ( ~ 4 . 5 1 ) me cPu 1,s; u r ~ r .

G1*-hH2).

a 1 u - u - P r o (nf-0.26). < c ~ ~ - H ~ . - P ~ ~ N R ~ (~f-0.6n.

VUOPC*Uh: R.dlo.ctlviCy of k8-116C1C1y-NH2 (Rf-0.82) m WE (pH 1,s; y r k e r .

Wrolib.rln: WE (PH 3.5; u r k e r . H i - ) a d d.tect.d by Pauly rcmcflm.

h e o C m * l n 11: M n o Ceml~~ll .n.ly#l. by d.nay1 I t h o d .

W.u?DC*D.ltti Am100 acid -1Y.l. after IWC (pR 6.8; marker. br-pknol blue)! b r I1e Iru (Rf-0.20) .12 1 1.03

h r Leu Pro Clu Amp Arm L p (Rf-0.61)

Pro k g (Rf-1.19) 1 1.16

.9S 1 2.2 2.1 1.0 1.8 .90

(kldlud l l u u l l n Amino acld maly.1. a f t w Bpz (pn 6.8; u r h r . br-hmol b l m ) . behala: Lys AI. (W-0.97)

?8C?~-*l~lmR: Bpz (pR 3.S: urker. br-h.nol blue) . Trl-.l.nlne (Rf-0.40).

1.1 1

old- ( " 0 . 1 0

(U10.60) PmCr.l.nll*: Bpz ( u .bo*.) lad r.dlo.crlvlcy. Dl - . l .n los (Rf-0.4S). crl-.hnlnc

a) rbm u ..I- t0llawl~ hllb V01t4. p.wr *1.ctropbon.i. (WE) 1. 81Y.D r.l.rlv.2 t o ciw

b) -0 wid Eoqo.lClm Of p p t l d . . h riven r.l.clve 10 ciw resldm 1dlc.t.d u 1. .ppropri.re u r h r u w1e.r.d.

4790 Proline-specific Endopeptidase

TABLE V Kinetic parameters for proline-specific endopeptidase-catalyzed hydrolysis of peptide esters and amides extended NHz terminally from

the scissile bond P4 P3 Pz PI1 P I [SI [ E 1 K , kcat L l K m

mM X Io-' M mM S" mM" 8"

Pro-2-NNap Z---Pro-2-NNap Z---Pro---ONp Z---Ala---ONp

Gly---Pro-a-NNap Ala---Ala-2-NNap

Z---Gly---Pro---MCA Z---Gly---Pro-2-NNap Z---Gly---Pro---ONp Z---Ala---Ala-2-NNap Z---Ala---Ala---ONp Z---Ala---Pro-2-NNap Z-~-Ala---Pro-2-NNap

Z---Gly---Gly---Pro---ONp Z---Ala---Gly---Pro-2-NNap Z-D-Ala---Gly---Pro-2-NNap

0.006-0.065 0.026-0.83 0.01-0.31

0.005-0.60 0.01-0.31

0.026-0.83 0.026-0.83 0.01-0.31

0.026-0.83 0.026-0.83

1.7 3.6 3.7

276 40

180 3.6

3.7 3.6 3.6

No hydrolysis No hydrolysis No hydrolysis No hydrolysis No hydrolysis No hydrolysis 0.025 0.14 0.125 0.58 0.16 0.08 0.20 0.133 0.29 0.14

115 169 102

5.9 2.7

64.2 0.147

85.8 192 38

4600 1212 816

10.1 16.8

834

645 664 271

0.73

TABLE VI Kinetic parameters for proline-specific endopeptidase-catalyzed hydrolysis ofpeptzdes extended COOH terminally from the scissile bond

Ps P? P,1 P I P r P a 3 [SI [ E 1 K , kcat k c a t / K m

1 r n M X Io-' M mm 8" mM" S"

Z---Gly---Pro---Leu 0.042-0.667 3.35 0.22 23 104 Z---Gly---Pro - - -Phe 0.60-1.70 3.35 0.74 180 250 Z---Gly---Pro---Ala 0.047-0.667 1.50 0.39 240 620 Z---Gly---Pro-D-Ala Negligible rate hydrolysis Z---Gly---Pro---Leu---Gly 0.60-10.7 1.50 0.32 520 1600 Z---Gly---Pro---Leu---Ala 0.60-10.7 1.50 0.47 520 1100 Z---Gly---Pro---Leu-D-Ala 0.60-10.7 1.50 1.5 1600 1070 Z---Gly---Pro- --Leu - - - Gly---Gly 0.60-10.7 2.13 1.4 700 500 Z---Glv---Pro---Leu---Glv---Ala 0.60-10.7 2.13 1.82 loo0 550

TABLE VI1 Kinetic studies on peptide inhibitor of proline-specific enzyme

K.

Peptides

Pro Z---Pro

Z---Gly---Pro Z---Ala---Pro Z---Ala---Ala

Z---Ala---Gly---Pro Z---Ala---Gly---Pro---NHp

Z-D-Ala---Gly---Pro---NH2 Gly-Pro

Ala---Gly---Pro---NH* Z---Pro---Leu

mM

0.074-0.59

0.074-0.59

0.18-1.19 0.074-0.59 0.074-0.59

0.074-0.59 0.074-0.59 0.074-0.59

0.074-0.59

0.074-0.59

0.074-0.59

mM NC" NC 0.43 0.18 0.30 0.10 0.10 0.10 0.50 0.34 0.77 0.58 0.75 0.42 0.78 0.61 NIb NI 0.33 0.42 0.85 0.40

" NC, noncompetitive inhibition. NI, no inhibition.

enzyme was not inhibited by 50 p~ ovomucoid from egg white, pancreatic trypsin inhibitor, Streptomyces subtilisin inhibitor, soybean trypsin inhibitor, az-macroglobulin, leupeptin, anti- pain, chymostatin, bestatin, phosphoramidon, or elastatinal.

Immunochemical Reaction-Fig. 3 shows that antibody raised against proline-specific endopeptidase from F, menin- gosepticum gave a single precipitation line with the purified bacterial enzyme, but did not cross-react with post-proline cleaving enzyme from lamb kidney or post-proline dipeptidyl aminopeptidase from lamb kidney.

Substrate Specificity-As shown in Table IV, the enzyme catalyzes hydrolysis of Pro-X bonds of angiotensin 11, neuro- tensin, thyroliberin, oxidized insulin B-chain, oxytocin, and vasopressin. K,,, values for oxytocin and vasopressin were 0.10

and 0.14 mM, respectively. However, (7-~-Pro)oxytocin was not hydrolyzed. The enzyme was also inert toward denatured proteins such as egg-white lysozyme, cytochrome c from horse muscle, and a-amylase from Bacillus amylolique-faciens.

Proline-specific endopeptidase also hydrolyzed oligoalanine as shown in Table IV. The enzyme acted on tetraalanine to produce trialanine and alanine. Pentaalanine was hydrolyzed to tri- and dialanine.

Kinetic Effect of Residues Located on the NH2-terminal Side of the Scissile Bond of Proline- or Alanine-containing Peptide Derivatives-p-Nitrophenylester or P-naphthylamide derivatives of Pro, Z-Pro and Gly-Pro were not hydrolyzed by proline-specific endopeptidase (Table V ) . When the peptide chain length was elongated, e.g. to Z-Gly-Pro or Z-Ala-Pro, the resulting substrates were easily hydrolyzed. However, when the chain length was increased even more, e.g. to Z-Gly- Gly-Pro or 2-Ala-Gly-Pro, the K,,, value increased and kCat/Km decreased. When the PZ residue was replaced by a D-amino acid, hydrolysis occurred very slowly. However, when position P3 was made of a D-amino acid only, a minor decrease was observed in the kCat/Km value. Alanine-containing peptide derivatives were also hydrolyzed, but the kat/K,,, was only '/50 to %m that of proline-containing peptide derivatives. Of the substrates tested, Z-Gly-Pro-MCA showed the lowest Km value and highest kCat/Km value.

Kinetic Effect of Residues Located at the COOH-terminal Side of the Scissile Bond of Proline-containing Peptides-In these experiments, the chain length of the substrates was increased on the COOH-terminal side of the scissile bond, while keeping the Z-Gly-Pro sequence in positions Ps, Pa, and PI the same (Table VI) . When the substrate containing the terminal residue in position P'l was elongated by one amino acid to fill position P'2, kcat and kCat/Km increased. However, when the chain length was increased by one more amino acid,

Proline-specific Endopeptidase 4791

the K, value increased and kCat/K, decreased. When position P'l was replaced with a D-amino acid, the peptide wm not hydrolyzed at all. In contrast, replacement of the P'z amino acid with a D isomer had only a very small effect.

Inhibition Kinetics of Proline- and Alanine-containing Peptides-Table VI1 shows that a variety of proline- and alanine-containing peptide inhibitors each gave almost the same inhibition constant (KJ regardless of whether Z-Gly- Pro-ONp or Z-Ala-Ala-ONp was used as the substrate.

DISCUSSION

Detailed studies on the substrate specifcity of lamb kidney post-proline cleaving enzyme provided the background which led to the discovery of the same type of enzyme in microor- ganisms; namely, the use of the specific substrate, Z-Gly-Pro- 2-NNap, made it very easy to screen microorganisms capable of producing the endopeptidase specific for proline. After testing more than 500 strains of microorganisms isolated from soil and stock cultures, F. meningosepticum was found to be the best source of proline-specific endopeptidase. Production of the enzyme was not inducible, since addition of proline or proline-containing peptides to the culture medium did not increase enzyme levels. Further, enzyme production paralleled cell growth.

Besides hydrolyzing Z-Gly-Pro-2-NNap, the crude cell-free extract of F. meningosepticum also contained other enzyme activities, presumably aminopeptidasels), which could hydro- lyze arylamide substrates such as Gly-Pro-2-NNap, Ala-2- NNap, Leu-2-NNap and Cys-di-2-NNap (12). Most of these activities were separated from proline-specific endopeptidase by ammonium sulfate fractionation (0 to 65% saturation; see Table I). Any remaining aminopeptidase activity, as well as contaminating colored material, was removed in the break- through peak of the CM-cellulose chromatography. Further purification of proline-specific endopeptidase was achieved by chromatography on hydroxyapatite and Sephadex (2-150 col- umns. The purified enzyme gave a single protein band follow- ing standard disc gel electrophoresis. This band had the same mobility as Z-Gly-Pro-2-NNap-hydrolyzing activity detected histochemically on a companion gel. The enzyme also ap- peared to be homogeneous after SDS-polyacrylamide gel elec- trophoresis. In addition, when ["'PIDFP-treated enzyme was subjected to SDS-gel electrophoresis under the same condi- tions, the position of radioactivity was the same as the single protein band. Thus, only one band of protein was found in two different electrophoretic systems, and this band corre- sponded to proline-specific endopeptidase activity.

With the exception of the substrate specificity, the proper- ties of proline-specific endopeptidase from F. meningosepti- cum and post-proline cleaving enzyme from lamb kidney were quite different. The isoelectric point of the bacterial enzyme was 9.6 while that of post-proline cleaving enzyme was 4.8. This fact necessitated different purification procedures for the two enzymes. In short term stability studies, the bacterial enzyme was somewhat less stable than the kidney enzyme (Table 11). However, the former appeared to be reasonably stable during the course of purification without added stabi- lizer, whereas the kidney enzyme required the presence of both dithiothreitol and EDTA ( 3 ) . Immunochemical experi- ments also showed clear differences between the two enzymes (Fig. 3) . Nevertheless both enzymes were rapidly and stoichi- ometrically inhibited by DFP and, in addition, were specifi- cally inhibited by the chloromethyl ketone derivatives of Z- Gly-Pro (Table 111). This suggests that both enzymes are serine endopeptidases. However, neither enzyme was in- hibited by another serine enzyme inhibitor, PheCHZSOzF.

Although there were some differences in the K,,, values for

several substrates, the substrate specificities of bacterial pro- line-specific endopeptidase and kidney post-proline cleaving enzyme were quite similar. Both enzymes hydrolyzed the Pro- X bond of peptides and arylamides and esters. These two enzymes also split the Ala-X bond, but only at a rate '/so to %OO that of Pro-X (5). The bacterial enzyme hydrolyzed oxytocin, vasopressin, angiotensin 11, neurotensin, and thyroliberin, and oxidized insulin B-chain on the carboxyl side of the prolyl residues, as does post-proline cleaving enzyme (2,3).

Additional similarities between the two enzymes were found in studies on the subsite interaction of proline-specific endo- peptidase with substrates or inhibitors carried out by the method of Schechter and Berger (28).

It was observed that proline-specific endopeptidase was unable to hydrolyze substrates which contained only 1 or 2 residues on the amino side of the scissile bond (positions P I

and P2). However, if the substrate chain was elongated to include a residue on position Ps, it was readily cleaved by the enzyme. Further elongation to P4, on the other hand, caused no further increase in the kCat/K,,, value. (This value actually decreased slightly, but not as much as observed for kidney post-proline cleaving enzyme with the addition of the P4 residue.) These results suggest that the binding site of the enzyme consists of three subsites, Sa, S2, and SI, which interact with residues P3, Pz, and PI, respectively, of the substrate.

Subsite SZ but not SS of the proline-specific endopeptidase was found to be stereospecific, since replacement of a PS L- amino acid with a D isomer drastically reduced the kCat/Km value. Although the stereospecificity of the SI subsite was not systematically investigated with this series of peptides, we observed that (7-~-Pro)oxytocin was not hydrolyzed at all, suggesting that the SI subsite also has high stereospecificity.

When the substrate chain length was increased on the COOH-terminal side of the scissile bond, K, did not change but kCat/K,,, increased in going from P ' I to P'2. When an additional residue was added to position P'3, the kCat/Km value no longer increased, but actually decreased due to an increase in K,. This suggests that the enzyme contains two additional binding sites, Sf1 and S'Z, which interact with residues P', and P I 2 of the substrate. High stereospecificity was observed for the SI subsite, but not for SIS. Thus, the length of the binding site (S3, Sz, SI, S I , S'Z) and the stereospecificity of the subsites (S2, SI, S I ) is quite similar to that observed for kidney post- proline cleaving enzyme.

The enzyme preparation used here was shown to catalyze hydrolysis of various alanine-containing peptides (Table V) as well as oligoalanines (Table IV), although the kCat/Km values were only about %oo that of equivalent proline-containing peptides. There remains a question whether the activity to- ward alanine peptides is an intrinsic property of the proline- specific endopeptidase or not. However, the following experi- mental results seem to prove that the same enzyme protein shows the activities toward both proline and alanine peptides. 1) Post-proline cleaving enzyme and post-proline dipeptidyl aminopeptidase from lamb kidney also catalyze the hydrolysis of alanine-containing peptides (4,5,15). 2) Proline and alanine peptides inhibit the cleavage of one another (Table VII). 3) Similar cross-inhibition results are also obtained for post- proline dipeptidyl aminopeptidase (29) and post-proline cleav- ing endopeptidase from lamb kidney.2

Acknowledgments-We are grateful for the generous gift of pro- tease inhibitors of microbial origin from Dr. H. Umezawa, Institute of Microbial Chemistry, and Dr. S Murao, University of Osaka Prefec- ture, Japan. We also appreciate the gift of neurotensin from Dr. H.

T. Yoshimoto et al., unpublished observations.

4792 Proline-specific Endopeptidase

Yajima, Kyoto University, Japan. We also thank Dr. W' 4 Si- .-.,

for help in the preparation of the manuscript and Mr. K. ugna, Mr. S. Nagata and Ms. S. Hu for their experienced assistan-e.

REFERENCES 1. Walter, R., Shlank, H., Glass, J . D., Schwartz, I. L., and Kerenyi,

2. Walter, R. (1976) Biochim. Biophys. Acta 422, 138-158 3. Koida, M., and Walter, R. (1976) J. Biol. Chem. 251, 7593-7599 4. Yoshimo' r ' Orlowski, R. C., and Walter, R. (1977) Biochem-

5. Yoshimoto, T., 1. '. M., Orlowski, R. C., and Walter, R. (1978)

6. Walter, R., and Yoshimoto, T. (1978) Biochemistry 17,4139-4144 7. Walter, R., Griffiths, E. C., and Hooper, K. C. (1973) Brain Res.

8. Walter, R., and Simmons, W. H. (1977) in Proceedings of the International Conference on the Neurohypophysis, Key Bis- cayne, Flu., Nouember, 1976 (Moses, A. M., and Share, L., eds) pp. 167-188, S. Karger, Basel

T. D. (1971) Science 173,827-829

istry 16, - .I '48

J. Biol. Chem. 253, 9708-3716

60,449-457

9. Orlowski, M., Pearce, S., and Wilk, S. (1978) Fed. Proc. 37,915 10. Hersh, L. B., and Mckelvy, J . F. (1979) Brain Res. 168,553-564 11. Yoshimoto, T., Ogita, K., Walter, R., Koida, M., and Tsuru, D.

(1979) Biochim. Biophys. Acta 569,184-192

12. Yoshimoto, T., and TSUN, D. (1978) Agric. Biol. Chem. 42,2417-

13. Tsuru, D., Tomimatsu, M., Fujiwara, K., and Kawahara, K. (1975)

14. Walter, R., and Havran, R. T. (1971) Experientia (Basel) 27,

15. Yoshimoto, T., and Walter, R. (1977) Biochim. Biophys. Acta

16. Bliss, C. I., and James, A. T. (1966) Biometrics 22, 573-602 17. Moore, S., and Stein, W. H. (1954) J . Biol. Chem. 211, 907-913 18. Dixon, M. (1953) Biochem. J. 55, 170-171 19. Simmons, W. H., and Walter, R. (1980) Biochemistry 19, 39-48 20. Hartley, B. S., and Massey, V. (1956) Biochim. Biophys. Acta 21,

21. Fischer, F. G., and Dorfel, H. (1953) Biochem. 2. 324,544-566 22. Moore, S. (1968) J. Biol. Chem. 243,6281-6283 23. Andrews, P. (1965) Biochem. J . 96,595-606 24. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 25. Davis, B. J . (1964) Ann. N. Y. Acad. Sci. 121,404-427 26. Vesterberg, 0.. and Svensson, H. (1966) Acta Chem. Scand. 20,

27. Ouchterlony, 0. (1958) Prog. Allergy 5, 1-9 28. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res.

29. Barth, A. (1979) Wiss. Z. Martin Luther Uniu. Halle- Wittenberg

2419

d'. Biochem. (Tokyo) 77, 1305-1312

645-646

485,391-401

58-70

820-834

Cornmun. 27,157-162

Math. Naturwiss. Reihe 3,23-48