J. Biochem. 86, 1109-1117 (1979)

Creatinine () from Pseudomonas putida

Purification and Some Properties 1

Kaoru RIKITAKE, Imao OKA, Makoto ANDO, Tadashi YOSHIMOTO, and Daisuke TSURU

Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Nagasaki 852

Received for publication, April 11, 1979

Creatinine amidohydrolase (EC 3.5.2.-; creatininase] was purified in an overall yield of 11 from cell-free extract of Pseudomonas putida var. naraensis, strain C-83, by column chro matographies on sarcosine-HM-Sepharose and DEAE-cellulose, and gel filtration on Sephadex G-200. The purified was homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the reversible conversion of creatinine to with an optimal pH of 7-9. Km values for creatinine and creatine were 26 mM and 0.13 M, respectively. The enzyme was also active toward glycocyamidine, though the reaction rate was quite low, but it was completely inert toward hydantoin and its derivatives. The molecular weight of the enzyme was estimated to be 175,000 by ultracentrifugal analysis and the subunit molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 23,000, suggesting that the enzyme is composed of eight subunit monomers. The isoelectric point was 4.7 as judged by iso electric focusing experiments. The enzyme was markedly inactivated by heavy metal ions, N-bromosuccinimide, ethoxyformic anhydride, and dye-sensitized photooxidation, and also partially by metal chelators. The enzyme was found to contain about one gram atom of zinc per subunit monomer. The metal-free, inactive enzyme was prepared and could be reactivated by the addition of Mn2+, Co2+, Mg2+, Fe2+, Ni2+, and Zn2+ in that order of de- creasing effectiveness. These results indicate that metal is intimately involved in the creatini nase activity of P. putida.

In order to establish a new enzymatic method for terium belonging to Pseudomonas putida was diagnostic analysis of creatinine and creative in isolated from soil in Nara Prefecture and designated serum (1, 2), microorganisms capable of rapidly as P. putida var. naraensis C-83 (3). The strain was catabolizing creatinine were screened. A bac- found to catabolize creatinine as follows:

CreatinineA?CreatineB•¨+Sarcosine C•¨ Glycine +FormaldehydeD•¨ Formic acid a This work was supported by a grant from the Ministry of Education, Science and Culture of Japan. Abbreviations: BPB, Bromphenol Blue; HM, hexamethylenediamine; EDTA, ethylenediaminetetraacetic acid; DFP, diisopropylphosphorofluoridate; DTNB, 5, 5•Œ-dithiobis-(2-nitrobenzoic acid) [Ellman reagent]; PCMB, ƒÏ-chloromercuribenzoate; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; NBS, N-bromosuccinimide.

Vol. 86, No. 4, 1979 1109 1110 K. RIKITAKE, I. OKA, M. ANDO, T. YOSHIMOTO, and D. TSURU

The involved are creatinine amido of the mixtures were subjected to electrophoresis (creatininase) [A], creatine amidino in 7.5 % gels at a constant current of 8 mA per gel hydrolase () [B], sarcosine dehydrogenase for 3 h. The gels were stained with 0.25%. Cooma [C], and formaldehyde dehydrogenase [D]. Of ssie Brilliant Blue R-250 and destained by washing these four enzymes, the latter three have already overnight with a mixture of acetic acid-methanol- been purified to homogeneity in our laboratory, water (35 : 105: 315). The molecular weight of and their enzymatic properties were characterized creatininase was estimated by comparison with in detail (4-7). We also attempted to purify the parallel runs of marker proteins, bovine serum remaining enzyme, creatinine amidohydrolase albumin (68,000), catalase (58,000), aldolase (creatininase), and succeeded in obtaining a disc (40,000), chymotrypsinogen (25,700), and horse electrophoretically homogeneous preparation of heart muscle cytochrome c (11,700). the enzyme. The present paper deals with the Estimation of molecular weight by ultracentri purification and characterization of this enzyme fugal analysis and gel filtration: The purified from P. putida C-83. This is the first report enzyme was dialyzed against 20 mm Tris-HCl describing the properties of a homogeneous prepa buffer, pH 7.0, containing 0.15 M NaCl at 4•Ž for ration of creatininase in detail. 48 h, during which time the outer solution was changed every 12 h. The dialyzed solution was

MATERIALS AND METHODS subjected to ultracentrifugal analysis using a Beckman model E ultracentrifuge equipped with

Materials-Rabbit muscle aldolase, beef liver schlieren and interference optics at 25•Ž.. The

catalase, and bovine pancreas chymotrypsinogen molecular weight of the enzyme was estimated

were purchased from Boehringer Mannheim, by the meniscus depletion sedimentation equilib

Germany. Sephadex G-100, G-200, Sepharose 4B, rium method (10) at 12,000 rpm with a 12 mm and DEAE-Sephadex A-50 were from Pharmacia double sector cell after 24 and 26 h.

Fine Chemicals, Sweden, and DEAE-cellulose was The molecular weight of the enzyme was also from Brown Co., U.S.A. Diisopropylphosphoro estimated by the gel filtration method of Andrews

fluoridate (DFP), phenylmethanesulfonyl fluoride (11), using a column (2•~110 cm) of Sephadex (PMSF), 5, 5•Œ-dithio-bis-(2-nitrobenzoic acid) G-200 equilibrated with 50 mm phosphate buffer,'

[Ellman reagent] were obtained from Sigma pH 7.4, containing 0.15 M KCl. Chemical Co., U.S.A., and creatinine was from Isoelectric focusing run: This was done by Tokyo Kasei Co., Tokyo. Sarcosine-hexamethyl the method of Vesterberg and Svensson as described enediamine-Sepharose (sarcosine-HM-Sepharose) by Matsuo and Horio (12) using an LKB apparatus. was prepared as described by Yoshimoto et ƒ¿l. The purified enzyme (1.5 ml) was dialyzed over-

(5). Other chemicals were products of Nakarai night against 0.5 % carrier ampholyte, pH 3.5-10.0, Kagaku Co., Kyoto. applied to a column (110 ml) containing a sucrose

Analytical Methods-Disc gel electrophoresis density gradient, and then subjected to isoelectric and SDS polyacrylamide gel electrophoresis: Disc focusing at 300 volts and 9•Ž for 72 h. gel electrophoresis was carried out using a 7.5 Metal analysis: Zinc, manganese, magne gel of pH 8.4 according to Davis (8). The enzyme sium, and calcium contents of the enzyme prepa preparations (20-100 peg) were loaded on gels and rations were measured using a Hitachi 208 atomic a current of 2 mA per tube was applied for 2 h. absorption spectrophotometer. Cobalt was deter- The gels were stained with 1 % Amidoblack B and mined by the ƒ¿-nitroso-ƒÀ-naphthol method (13). destained with 7 % acetic acid. The enzyme was dialyzed for 2 days against 10 mm SDS-polyacrylamide gel electrophoresis was Tris-HCl buffer, pH 7.2, from which metal ions performed by the method of Weber and Osborn had been removed by extraction with 0.2% dith

(9). Samples of about 100 ƒÊ1 (50-100 ƒÊg as pro izone in carbon-tetrachloride. tein) were mixed with an equal volume of 20 mm Amino acid composition and sugar content: phosphate buffer, pH 7.0, containing 50%. glycerol, 2 % SDS, 0.1 % BPB, and 1 % 2-mercaptoethanol. 2 Potassium phosphate buffer was used throughout the

After incubation at 70•Ž for I h, 100 ƒÊl aliquots present experiments, unless otherwise stated.

J. Biochem. CREATININASE FROM Pseudomonas putida 1111

The amino acid composition of the enzyme was Photooxidation: A mixture of the enzyme and

determined by the method of Spackman et ƒ¿l. (14) 0.01% Rose Bengal in 50 mm Tris-HCl buffer,

with a Nippon Denshi JLC-6AH automatic amino pH 7.1, was placed at a distance of 20 cm from the acid analyzer. The contents of half-cystine and front lens of a 300W slide projector and illuminated

tryptophan were determined by the method of at 15•Ž. Aliquots of the reaction mixture were

Moore (15) and Goodwin and Morton (16), periodically withdrawn and the residual activities respectively. Sugar content of the enzyme was were assayed in the dark (20).

measured by the phenol-sulfuric acid method (17) Cultivation of the Microorganism-Pseudo and expressed as glucose. monas putida C-83 was aerobically grown at 28•Ž

Enzyme activity and protein concentration: for 16 h in a medium containing 5% soybean cake

The enzyme activity was assayed by the following extract, 5% dextrin, 1% KH2PO4, 0.02% each of two methods. (A) Creatinine formed from creatine KCl and MgSO4.7H20, pH 7.0, and then inocu

by the enzyme reaction was measured using the lated into 8 liters of a creatinine-enriched medium

Jaffe reaction. The enzyme solution (0.1 ml) was (3) in a Miniferm MF-1 14 jar fermenter (NBS Co., added to 0.9 ml of 50 mm Tris-HCl buffer, pH 7.0, U.S.A.). The cells were aerobically grown at 28•Ž containing 0.1 M creatine at 37•Ž, and after incu for 12 h, harvested by centrifugation (7,000 rpm,

bation for 10 min, a 0.1 ml aliquot of the reaction 20 min) and then washed twice with 50 mm phos

mixture was withdrawn and mixed with 1 ml each phate buffer, pH 7.4 (buffer A). This buffer A of 1 N NaOH and 1% picric acid and 0.9 ml of was used throughout the purification of the native

deionized water. The absorbance at 520 nm was enzyme. For the preparation of metal-free, inactive measured after 20 min against the blank, in which enzyme, on the other hand, buffer B (50 mM Tris-

heat-denatured enzyme had been used. One unit HCl buffer, pH 7.1, containing 2.7 mM EDTA)

of the activity was defined as the amount of en was used unless otherwise stated.

zyme that converts I ƒÊmol of creatine to creatinine

per min under the conditions used, and the activity RESULTS AND DISCUSSION units per ml of the original enzyme solution were

calculated from the following equation: Preparation of Cell free Extract and Purifica tion of Creatininase-All the procedures were

Units/ml =ƒ¢E520nm•~6.45•~dilution factor carried out in the cold. The washed cells were suspended in buffer A, mixed with an equal volume

The specific activity was defined as the enzyme of glass beads (0.5 mm) and disrupted in a Vibrogen activity (units) per mg of protein. Protein con cell mill (Edmund Biihler, Germany) for 10 min.

centration was estimated spectrophotometrically The suspension of disrupted cells was centrifuged based on Al%1cm,280nm=9.8,which was determined at 8,000 rpm for 20 min. Next, 1% protamine from measurements of absorbance and dry weight sulfate solution (pH 7.0) was added dropwise to the

of the purified enzyme. (B) Creatine formed from supernatant to give a final concentration of 0.2%,

creatinine was measured by the ƒ¿-naphthol and after 1 h, the turbid materials were removed

diacetyl method (18). The enzyme solution (0.1 by centrifugation (7,000 rpm, 20 min). Solid

ml) was added to 0.9 ml of 50 mm Tris-HCl buffer, ammonium sulfate was added to the clear cell-

pH 7.0, containing 10 to 200 mm creatinine at free supernatant to give a 55%. saturation (351 g/ 37•Ž, and after incubation for 10 to 30 min, 0.1 ml liter), and after standing for 6 h, the resulting

of the reaction mixture was withdrawn and mixed precipitate was collected by centrifugation (8,000 with 0.5 ml of 0.05% diacetyl and 5.0 ml of a rpm, 20 min), dissolved in 40 ml of buffer A and

mixture of 0.2 % ƒ¿-naphthol and 0.25 N NaOH. then dialyzed against the same buffer for 2 days, The absorbance at 540 nm was measured after during which time the outer solution was changed

30 min, and creatine formed was calculated from every 12 h. The dialyzed solution was applied to a

the standard curve. Kinetic parameters, Km and column (3•~20 em) of sarcosine-HM-Sepharose

Vmax values, were evaluated from Lineweaver- previously equilibrated with buffer A. The column

Burk plots and K, was obtained from Dixon plots was washed with the buffer, then the adsorbed enzyme was eluted with an increasing linear gradient (19).

Vol. 86, No. 4, 1979 1112 K. RIKITAKE, I. OKA, M. ANDO, T. YOSHIMOTO, and D. TSURU

of NaCl concentration from 0 to 0.6 M in a total elution profile of the enzyme from a Sephadex

of 3 liters of the buffer at a flow rate of 60 ml per G-200 column is shown in Fig. 1. The active

h. Active fractions were combined and concen fractions with constant specific activity (480

trated to 40 ml by ultrafiltration (Amicon PM 10), ?units/mg) were combined, concentrated by ultra- then the concentrate was heated at 60•Ž for 20 min. filtration and then stored in a freezer or freeze-

The resulting precipitate was removed by centrifu dried after dialysis against deionized water. Over-

gation (10,000 rpm, 15 min) and the supernatant all, the enzyme was purified about 440-fold with was dialyzed against buffer A, then applied to a an activity recovery of 11%. The purified enzyme column (4•~25 cm) of DEAE-cellulose equilibrated was found to be nearly homogeneous on disc gel

with the above buffer. The adsorbed enzyme was electrophoretic (Fig. 2) and ultracentrifugal ana

eluted with a linear gradient system of NaCl con lyses. A summary of the enzyme purification is centration from 0 to 0.5 M in a total of 2 liters of shown in Table I.

the buffer. Active fractions were combined, and

the enzyme was salted out by 55 % saturation of ammonium sulfate. After 5 h, the precipitate was

collected by centrifugation, dissolved in 2.0 ml of

buffer A containing 0.15 M KCl and then applied

to a column (1.8•~llO cm) of Sephadex G-200

previously equilibrated with the same buffer. The

Fig. 1. Gel filtration profile of the purified creatininase Fig. 2. Disc gel electrophoretic pattern of the purified on Sephadex G-200. See the text for experimental enzyme. The experimental details are described in;the details. The flow rate was 15 ml per h. text.

TABLE I. Purification of creatininase from P. putida. The enzyme activity was assayed by method A.

J. Biochem. CREATININASE FROM Pseudomonas putida 1113

Some Enzymatic Properties of Creatininase- rations used. The reason for this remains unclear, The enzyme was most active at pH 7-9 in the but will be discussed again later. On the other

forward and reverse reactions, and was stable at hand, SH-blocking reagents, such as sodium

pH values between 6 and 12 for 3 h at 30•Ž. About tetrathionate, DTNB, N-ethylmaleimide, and iodo 75% of the activity was retained after incubation acetate, did not affect the enzyme activity, though

at 75•Ž for 30 min. When stored in a freezer, PCMB did.

the enzyme was stable for more than 6 months. specificity and inhibition by some The effects of chemicals and metal ions on the substrate analogs are summarized in Table ‡V,

activity are shown in Table ‡U. The enzyme was and the basic moieties required for substrate or

markedly inactivated by incubation with 1 mM inhibitor activity with respect to the creatininase

NBS, Hg2+, Fe3+, Cue+, ethoxyformic anhydride are shown in Fig. 3. The results suggest that a

and by dye-sensitized photooxidation for 30 min. good substrate must have a CH3 group as R1 The inhibitory effect of metal chelators, such as and an NH group as R2 in the formula shown in

o-phenanthroline and EDTA, was variable, ranging Fig. 3.

from 10 to 70% inhibition under the present Plots of equilibrium constant versus pH and

conditions, depending upon the enzyme prepa- temperature are shown in Fig. 4. The equilibrium

TABLE ‡U. Effects of some chemicals and metal salts on creatininase. The enzyme was preincubated with 1 mm chemicals or metal salts in 50 mm Tris-HCl buffer,

pH 7.1, at 30•Ž for 30 min, and the residual activities were assayed by method A.

Fig. 3. Basic chemical structure necessary for activity as a substrate or inhibitor for creatininase.

Fig. 4. Equilibrium constant versus temperature and

pH. (A) Effect of temperature. The reaction mixture consisted of 0.003% enzyme and 0.3 mm creatinine in 20 mm Tris-HCl buffer, pH 7.0. It was incubated at the temperatures indicated, and the decrease of creatinine was followed until equilibrium. (B) Effect of pH. Buffers used were: 10 mM acetate for pH 5.0-6.0, 10 mM

a Tested at a concentration of 10 mM. b Preincubated phosphate for pH 6.3-7.6, and 10 mm borate for pH at 50•Ž for 30 min with o-phenanthroline. c Illuminated 7.5-8.6. Other conditions were as in (A) except that under the conditions given in the text for 30 min. the reaction was performed at 30•Ž.

Vol. 86, No. 4, 1979 1114 K. RIKITAKE, 1. OKA, M. ANDO, T. YOSHIMOTO, and D. TSURU

TABLE ‡V. Kinetic parameters of the creatininase-catalyzed hydrolysis of creatinine and related compounds.

Hydrolysis of glycocyamidine was followed by method A, except that the substrate was replaced by glycocyami dine. a See Fig. 3. b The ring is broken at the position indicated by the arrow in Fig. 3. N-1 is replaced by C. d Competitively inhibited . n.d., not determined; n.h., not hydrolyzed; n.i., no inhibition.

constant meniscus depletion method of Yphantis (10). _??_ was determined by Logarithmic plots of fringe displacement, ln(f),

measuring the change in absorbance at 235 nm versus the square of radial distance, ƒÁ2, were almost due to creatinine substrate (molar extinction linear, indicating the homogeneity of the purified coefficient: 6,900) and calculated from the follow- enzyme. The molecular weight of the creatininase ing equation, was estimated to be 175,000,9 assuming the partial specific volume to be 0.74 (cm3/g), which was calculated from the amino acid composition. On

the other hand, SDS-polyacrylamide gel electro

where Eo and Eeq are the absorbance at 235 nm phoresis gave a value of 23,000 for the subunit molecular weight. These results suggest that the of the reaction mixtures at zero time and that after enzyme is composed of eight3 subunit monomers. reaching the equilibrium (about 30 min), respec The isoelectric point of the enzyme was deter- tively, starting from creatinine. Logarithmic plots mined to be 4.7 by isoelectric focusing experiments, of the equilibrium constant, ln(Keq), versus the but the activity recovered was less than 15% of reciprocal of absolute temperature, 1/T, were the original amount, presumably because of the almost linear, as shown in Fig. 4A. On the other instability of the enzyme in the acidic pH region. hand, a constant value of Keq was observed at The amino acid composition of the enzyme is both neutral and alkaline pH, but the equilibrium shown in Table ‡W. The enzyme was composed of leaned markedly toward creatinine at acidic pH, 205 amino acid residues, assuming the molecular as shown in Fig. 4B. weight of the subunit monomer to be 23,000. Some Physicochemical Properties-The mole Determination of the sugar content of the cular weight of the enzyme was estimated by the enzyme by the phenol-sulfuric acid method gave a value of 3.5 % as glucose. 3 Although the molecular weight estimated by the gel filtration method was 140,000, we prefer to adopt a Metal Requirement of Creatininase-Metal value of 175,000, since the gel filtration method is known content of the enzyme: The enzyme was markedly to give incorrect values of molecular weight for some inactivated by heavy metal ions and partially by proteins (21, 22). If the value of 140,000 is accepted, o-phenanthroline, but was rather resistant to the number of subunits of the native enzyme would be 6. treatment with EDTA, as shown in Table ‡U.

J. Biochem. CREATININASE FROM Pseudomonas putida 1115

TABLE ‡W. Amino acid composition of creatininase. TABLE V. Metal content of the native enzyme.

attempted to prepare the stable apo-enzyme by

treating the native enzyme with metal chelators under various conditions, but satisfactory results

were rarely obtained. Finally, however, we suc

ceeded in obtaining the metal-free, inactive enzyme by starting from a lysozyme lysate prepared in the

presence of EDTA as follows: washed cells of P. putida C-83, 150 g wet weight, were suspended in an equal volume of buffer A containing 10 mm

EDTA and then digested with egg-white lysozyme

(1 mg/100 ml) at 37•Ž for I h. Cell debris was removed by centrifugation, and the supernatant

(880 ml) was poured onto DEAE-cellulose (50 g) equilibrated with 50 mm Tris-HC1 buffer, pH 7.1,

containing 2.7 mm EDTA (buffer B). The sus

pension was stirred for I h. After removing the unadsorbed materials by filtration, the DEAE- a The subunit molecular weight was assumed to be cellulose was again suspended in buffer B con 23,000 daltons. b Average value of 21-, 45-, and 69-h taining 0.5 M NaCl and stirred for 1 h. The hydrolysates. c Value extrapolated to zero time of suspension was filtered and the DEAE-cellulose hydrolysis. d Performic acid-oxidized preparation. was washed twice with the same buffer. The

e The maximum value was adopted. f Spectropho- filtrate and the washings were combined, and the tometrically determined. proteins were salted out with 55%. saturated ammonium sulfate. The precipitate was dissolved However, we occasionally observed that some in buffer B, desalted by passage through Sephadex enzyme preparations4 were sensitive to EDTA G-25 and then applied to a column (3.8•~40 cm) treatment, and that the activities lost were fully of DEAE-Sephadex equilibrated with buffer B. restored by the addition of Mn2+, Col l, or Zn2+ The column was washed with the same buffer, These results suggest that the enzyme requires then the proteins adsorbed were eluted with an metal for its activity. Analysis of the metal increasing linear gradient of NaCl concentration content of the purified enzyme showed that the from 0 to 0.5 M in a total of 2 liters of the buffer. enzyme contained about one gram atom of zinc Active fractions' were combined, concentrated by per mol of subunit monomer (Table V). ultrafiltration and then subjected to gel filtration Preparation of metal free enzyme and the on a column (2.3•~ll5 cm) of Sephadex G-150. reactivation by the addition of metal salts:-We

5 Since all the fractions were completely inactive in the

4 The enzyme appeared to become EDTA-resistant after absence of metal ions, the enzyme activity was assayed heat treatment at 60•Ž for 20 min, which is necessary after incubation with 0.5-1.0 mm Zn2+, or Mn2+ for to remove some impurities. 10 min at 30•Ž.

Vol. 86, No. 4, 1979 1116 K. RIKITAKE, I. OKA, M. ANDO, T. YOSHIMOTO, and D. TSURU

Active fractions were again applied to a column compared with those of the native one. The (1.3 x 20 cm) of DEAE-Sephadex and the adsorbed inactive enzyme was activated by the addition of enzyme was eluted with an increasing linear Mn2+, Mg2+, Colt, and Fe 2+, which were all much gradient of NaCl concentration from 0 to 0.4 M more effective than Zn2+, the constituent metal in a total of 500 ml of the buffer B. The enzyme component of the native enzyme. Ni2+ also solution was stored in a refrigerator after concen activated the apo-enzyme. tration by ultrafiltration. A summary of the The results shown in Tables V and ‡Z strongly purification procedure is shown in Table VI. The suggest that the creatininase from Pseudomonas preparation contained no metal and was inactive, putida C-83 is a zinc metalloenzyme. Two points, but could be reactivated by incubation with 0.5 mm however, remain to be clarified. One is why the

Zn2+ to give a specific activity of 416, which was native enzyme purified by the usual method is somewhat lower than that of the native enzyme. rather resistant to EDTA treatment (Table H),

Disc gel electrophoretic analysis indicated that the whereas the metal-free, inactive enzyme can be preparation was contaminated with traces of obtained when the enzyme is extracted by digesting impurities. the whole cells with lysozyme in the presence of Table ‡Z shows the reactivation of the apo EDTA and purified in the presence of metal chelator enzyme by several metal salts and Table ‡[ (Tables ‡Y and ‡Z). The other point is why summarizes some properties of the apoenzyme Mn2+, Colt, and Mg2+ are much more effective

TABLE ‡Y. Summary of the purification of metal-free, inactive enzyme. The activity was assayed by method A after addition of 0.5 mm ZnCl2.

TABLE ‡Z. Reactivation of the apo-enzyme by some TABLE ‡[. Summary of the enzymatic and physico metal salts. The apo-enzyme (0.1 ml) was added to 0.9 chemical properties of the native and apo-enzymes. ml of 50 mm Tris-HCl buffer, pH 7.1, containing 0.1 M substrate and 0.5 mm metal salt. After incubation for 15 min at 37•Ž, the activities were assayed using 0.1 ml aliquots by method A.

a Stable for, 30 min at pH 7.1. b Estimated by ultra- centrifugal analysis, c Estimated by the gel filtration method.

J. Biochem. CREATININASE FROM Pseudomonas putida 1117 for the reactivation of the apo-enzyme than Zn2+, 9. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, though the inhibitory effect of externally added 4406-4412 Zn2+ on the activity of the enzyme (Table II) may 10. Yphantis, D.A. (1964) Biochemistry 3, 297-317 account to some extent for this phenomenon. 11. Andrews, P. (1965) Biochem. J. 96, 595-606 Similar results have also been obtained with some 12. Matsuo, Y. & Horio, T. (1967) Tanpaku, Kakusan, Koso 12, 737-748 (in Japanese) other zinc enzymes: aminopeptidases from bovine 13. Tanaka, L. (1961) Keikinzoku 46,52-58 (in Japanese) lens (23) and from Bacillus licheniformis (24), 14. Spackman, D.M., Stein, W.H., & Moore, S. (1958) Clostridiopeptidase (collagenase) from Clostridium Anal. Chem. 30, 1190-1206 histolyticum (25, 26), collagenase from Achromo 15. Moore, S. (1963) J. Biol. Chem. 238, 235-237 bacter iophagus (27, 28), carboxypeptidases A and 16. Goodwin, T.W. & Morton, R.A. (1946) Biochem. J. B from bovine pancreas (29), and mold amino 40,628-633 acylase (30). Another common property of most 17. Dubois, M., Gilles, K.A., Hamilton, J.K., Rehers, of these zinc metalloenzymes is that they are P.A., & Smith, F. (1956) Anal. Chem. 28, 350-361 sensitive to treatments which attempt to modify 18. Ennor, A.H. & Atocken, L.A. (1948) Biochem. J. the histidyl residue(s) of the enzymes, such as 42, 557-561 19. Dixon, M. (1953) Biochem. J. 55, 170-178 dye-sensitized photooxidation (20, 30, 31), ethoxy 20. Tsuru, D., Hirose, T., & Fukumoto, J. (1971) formic anhydride treatment (30, 32) and bromo J. Biochem. 70, 699-705 acetone treatment (28). The roles of the metal 21. Determann, H. (1968) Gel Chromatography Springer and the amino acid residue(s) involved in the active Verlag, New York site of the present creatininase are now under 22. Whitaker, J.R. (1963) Anal. Chem. 35, 1950-1953 investigation and will be reported elsewhere. 23. Hansen, H. & Frohne, M. (1976) Methods in Enzymology (Jackoby, W.B., ed.) XLV-B, pp. 504-521, Academic Press, New York REFERENCES 24. Rodrigues-Absi, J. & Prescott, J.M. (1978) Arch. 1. Oka, I., Watanabe, S., Ishida, H., & Asano, S. Biochem. Biophys. 186, 383-391 (1979) Nippon Rinsho Kagaku Kaishi in press (in 25. Yagisawa, S., Morita, F., Nagai, Y., Noda, H., & Japanese) Ogura, Y. (1965) J. Biochem. 58, 407-416 2. Tsuru, D. (1978) Rinsho Kensa 22, 1331-1338 (in 26. Yasuda, E. & Noda, H. (1972) Biochim. Biophys. Japanese) Acta 268, 719-723 3. Tsuru, D., Oka, 1., & Yoshimoto, T. (1976) Agric. 27. Keil-Dlouha, V. (1976) Biochim. Biophys. Acta 429, Biol. Chem. 40,1011-1018 239-251 4. Yoshimoto, T., Oka, I., & Tsuru, D. (1976) J. 28. Herry, P. & Keil-Dlouha, V. (1978) FEBS Lett. 95, Biochem. 79, 1381-1383 65-69 5. Yoshimoto, T., Oka, I., & Tsuru, D. (1976) Arch. 29. Zisapel, O.N. & Sokolovsky, M. (1973) Biochem. Biochem. Biophys. 177, 508-515 Biophys. Res. Commun. 53, 722-729 6. Oka, I., Yoshimoto, T., Ogushi, S., Rikitake, K., 30. Ogita, K., Ogushi, S., Fujiwara, K., & Tsuru, D. & Tsuru, D. (1979) Agric. Biol. Chem. 43, 1197-1203 Agric. Biol. Chem. in press 7. Ando, M., Yoshimoto, T., Ogushi, S., Rikitake, K., 31. Abe, T., Takahashi, K., & Ando, T. (1971) J. Shibata, S., & Tsuru, D. (1979) J. Biochem. 85, Biochem. 69, 363-368 1165-1172 32. Pangburn, M.K. & Walsh, K.A. (1975) Biochemistry 8. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427 14,4050-4054

Vol. 86, No. 4, 1979