Properties of Choline Oxidase of Cylindrocarpon Didymum M-1
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Agric. Biol. Chem., 43 (10), 2173 - 2177, 1979 2173 Properties of Choline Oxidase of Cylindrocarpon didymum M-1 Hideaki YAMADA, Nobuhiro MORI and Yoshiki TANI Department of Agricultural Chemistry, Kyoto University, Kyoto Received June 7, 1979 Choline oxidase from the cell-free extract of Cylindrocarpon didymum M-1 showed a mole cular weight of 120,000 by the gel filtration method and 145,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 276, 370 and 454 nm and a shoulder at 470nm. Anaerobic addition of choline as well as sodium dithionite to the enzyme produced a disappearance of the peak at 454 nm. Choline oxidase consists of two identical subunits, which have a molecular weight of 64,000, and contains two mol of FAD per mol of enzyme. The flavin was shown to be covalent ly bound to the protein. The enzyme was inactivated by Ag+, Hg2+, Cu2+ and Zn2+. The enzyme oxidized choline, betaine aldehyde and N, N-dimethylaminoethanol and apparent Km values were 1.3mM, 5.8mM and 14mM, respectively. In the previous papers,1,2) the authors re tion mixture contained 125ƒÊmol of potassium phos ported that a new enzyme, choline oxidase, phate buffer, pH 8.0, 6ƒÊmol of phenol, 4.5ƒÊmol of 4-aminoantipyrine, 6 units of peroxidase and appro was formed in the cells of several fungi, when priate amount of choline oxidase in a total volume they were grown on a medium containing of 3.0 ml. The reaction was started by the addition of choline as a sole carbon source. The enzyme 30 umol of choline. The increase of absorbance at was purified in homogeneous state from the 505 nm at 30•Ž was measured with a Hitachi double- cell-free extract of an isolated strain, Cylindro- beam spectrophotometer. One unit of the enzyme activity was defined as the amount of enzyme which carpon didymum M-l, and characterized as a catalyzed the formation of 1ƒÊmol of betaine aldehyde flavoprotein. or H202 per min. Specific activity was defined as the The present paper describes physicochemical unit per mg protein. and enzymatical properties of the purified Analytical method. Protein concentration for cho enzyme. line oxidase was determined by the absorbance at 280nm, where E1%lemvalue of 14.4 was used after dry MATERIALS AND METHODS weight determination. AMP was estimated by an enzymatic method with myokinase.3 Materials. Choline oxidase which was purified from the cell-free extract of C. didymum M-1 as pre Molecular weight determination. The molecular viously described2) was used in this study. Reference weight was determined by the sedimentation velocity proteins used for molecular weight determination, method of Svedberg and Pederson.4 A Spinco model pyruvate kinase, myokinase and lactate dehydrogenase E analytical ultracentrifuge was run at 20•Ž. Diffu were purchased from Boehringer Mannheim GmbH. sion constants were measured with the same apparatus Phosphodiesterase of Crotalus adamanteus venom was operating 12,590rpm with the synthetic boundary cell.5) The molecular weight was also determined by the from Sigma Chemicals Co. ATP was a kind gift of Kyowa Hakko Kogyo Co., Ltd. method of Andrews e) Sephadex G-200 was packed to a column. (2x95cm) and equilibrated with 0.05M Enzyme assay. Choline oxidase activity was as- Tris-HCI buffer, pH 8.5, containing 0.1M NaCl and sayed by the measurement of betaine aldehyde or H202 0.1mM dithiothreitol. The flow rate was 7ml/hr and formed. The activity to form betaine aldehyde was each 2ml fraction was collected. Cytochrome c, egg determined as described previously.2) The activity to albumin, bovine serum albumin, chymotrypsinogen, form H2O2 was determined by the method coupled with catalase, aldolase and ferritin were used as the reference peroxidase, phenol and 4-aminoantipyrine. The reac proteins. 2174 H. YAMADA, N. MORI and Y. TAM SDS-disc gel electrophoresis. Disc gel electro- of the enzyme in the presence of 1% sodium phoresis was performed in 0.1% sodium dodecylsulfate dodecylsulfate and 2% 2-mercaptoethanol. A according to the method of Weber and Osborn.7) single protein band was obtained with comassie Choline oxidase was incubated in 10mM of sodium blue as a stain. The molecular weight of the phosphate buffer, pH 7.0, containing 1% sodium dodecylsulfate and 2% 2-mercaptoethanol for 3 hr at subunit of the enzyme was estimated to be 60•Ž, before the electrophoresis. The electrophoresis 64,000+ 1,000 (Fig. 2). These results showed was carried out with 6mA per column at 25•Ž for 4hr. that choline oxidase might be composed of The relative migrations versus logarithms of the mole two subunits. cular weight were determined with marker proteins which were treated in the same way. Isoelectric focusing. Isoelectric focusing was carried out at 5•Ž for 48hr with carrier ampholite (LKB- Produkter AB) giving the pH gradient of 3 to 10 in a 120ml-column according to the method of Vester berg.8) RESULTS FIG. 2. Determination of the Molecular Weight of Molecular weight Choline Oxidase by SDS-polyacrylamide Gel Electro- The sedimentation coefficient in water at phoresis. 20•Ž (So20,w) was 7.6S, where the protein con Experimental conditions are described in MATERIALS centration was varied from 1.9 to 9.3mg per ml AND METHODS. of 0.1M sodium phosphate buffer, pH 8.0, and the partially specific volume of the enzyme was assumed to be 0.74. The diffusion co efficient in water at 20•Ž (Do20,w) was 5.0 x 10 -7 cm2/sec. From these values, the molecular weight of the enzyme was calculated to be about 145,000 according to the equation of Svedberg and Pederson.4) On the other hand, the molecular weight was estimated to be 120,000 by gel filtration on Sephadex G-200 (Fig. 1). The subunit composition of the enzyme was determined by disc gel electrophoresis analysis FIG. 3. Isoelectric Focusing of Choline Oxidase, The experimental conditions were described in MATERIALS AND METHODS. (•›), Absorbance at 280 nm; (•œ), choline oxidase activity. Isoelectric point Electrofocusing with a carrier ampholite (pH 3 •`10) showed that the enzyme has an isoelectric point around pH 6 .2 (Fig. 3). The total recovery of the enzyme activity was about 85%. FIG. 1. Determination of the Molecular Weight of Choline Oxidase by Gel Filtration. Identification of prosthetic group Experimental conditions are described in MATERIALS The absorption spectrum of choline oxidase AND METHODS. shows maxima at 276, 370 and 454 nm, and Properties of Choline Oxidase 2175 per mol of the enzyme (Table I). Therefore, the enzyme was determined to contain FAD but not FMN, which was covalently bound to the polypeptide chain. Effect of temperature and pH on the stability of the enzyme When the enzyme was incubated in 0.05 M Tris-HC1 buffer, pH 8.5, at various tempera- tures for 10 min, 20% and 96Y. of initial enzyme activity was lost at 40•Ž and 45•Ž, respectively. However, only 10% of the initial FIG. 4. Reduction of Choline Oxidase by Choline activity was lost at 45•Ž in the presence of or Sodium Dithionite under Anaerobic Conditions. 10% glycerol. Curve A represents the native enzyme solution at When the enzyme solution was incubated in a concentration of 0.1% in 0.05M Tris-HCI buffer, buffers of various pH values for 10 min at pH 8.5. B; the reduced enzyme after the addition 40•Ž, the enzyme was stable between pH 7.0 of 4.5ƒÊmol of choline. C; the reduced enzyme after and pH 9.0, but 50% of the initial activity was the addition of 4.5ƒÊmol of sodium dithionite. lost at pHs of 6.0 and 9.5. a shoulder at 470nm. The addition of choline or sodium dithionite to the enzyme under anaerobic conditions produced disappearance of the peak at 454nm (Fig. 4). The peak at 370nm shifted to 350nm by the addition of choline. Treatment of the enzyme in 5% trichloro acetic acid for 10min in a boiling water bath did not show any release of colored material in the soluble fraction. This result evidenced FIG. 5. Effect of Temperature on Choline Oxidase Activity. the presence of a covalently bound flavin in the enzyme. The enzyme was digested by Enzyme activity was measured by the determination of H202 formed under the standard assay conditions pronase and then incubated with phosphodi- at various temperatures. esterase. AMP was found in amounts of 2mol TABLE I. IDENTIFICATION OF FAD BY LIBERATION OF AMP Fourty-five nanomoles of enzyme and 57ƒÊmol of FAD each in 1.6ml of 50mM Tris-HCl buffer, pH 8.5, were incubated with 4mg of pronase at 37°C for 16hr. The heat-inactivated (3min) digest was lyophilized and resolved in 0.4ml of water. The solution was treated with 50ƒÊg of phosphodiesterase for 30 min. The reaction was stopped by heating and AMP was FIG. 6. Effect of pH on Choline Oxidase Activity. assayed enzymatically in each 0.2ml aliquot. Enzyme activity was assayed by the determination of H202 formed under the standard assay conditions with 0.05 M buffer as indicated. Potassium phosphate buffer was used from pH 6.0 to 8.0, Tris-HCI buffer from pH 8.0 to 9.0, and borate buffer from pH 9.0 to 11.0. 2176 H. YAMADA, N. MORI and Y. TAN! Effect of temperature and pH on the enzyme TABLE III. EFFECT OF METAL IONS AND activity CHELATING REAGENTS ON CHOLINE OXIDASE ACTIVITY As shown in Figs. 5 and 6, the maximum The enzyme activity was assayed by the determi activity of the enzyme was obtained at 40•Ž nation of betaine aldehyde formed under the standard and pH 9.0, respectively.