Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon Didymum M-1
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Agric. Biol. Chem., 44 (6), 1383•`1389, 1980 1383 Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M-1 Nobuhiro MORI,* Bunsei KAWAKAMI,Yoshiki TANI and Hideaki YAMADA Departmentof AgriculturalChemistry, Kyoto University,Kyoto 606, Japan ReceivedFebruary 5, 1980 Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindro carpon didymum M-1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 run. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag+, Hg2+, Zn2+ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1mM and 1.22ƒÊmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O2+H2O ?? sarcosine+form aldehyde+H2O2. It has been reported that dimethylglycine The present paper deals with the purifica and sarcosine were metabolized to sarcosine tion and some properties of dimethylglycine and glycine, respectively, by the oxidative oxidase from C. didymum M-1. demethylation reaction in liver mitochondria.1) Dimethylglycine dehydrogenase and sarco sine dehydrogenase have been partially purfied MATERIALS AND METHODS from liver mitochondria of rat2,3) and Rhesus Materials. Hydroxyapatite was prepared accord monkey.4) On the microbial oxidation of ing to the method of Tiselius et al.9) Reference pro dimethylglycine, Shieh reported that dimethyl teins used for molecular weight determination by gel filtration, and pyruvate kinase, myokinase and lactate glycine was oxidized to sarcosine and formal dehydrogenase were purchased from Boehringer Man dehyde by intact cells of Achromobacter choli nheim GmbH. Phosphodiesterase of Crotalus ada nophagum and sarcosine was further oxidized manteus venom was from Sigma Chemicals Co., Ltd. to glycine and formaldehyde by the cell ex Reference proteins used for molecular weight by SDS tract.5) disc gel electrophoresis were purchased from Pharmacia Fine Chemicals. We have previously reported that choline was oxidized to betaine aldehyde by choline Microorganisms and cultivation. Cylindrocarpon oxidase in Cylindrocarpon didymum M-1.6) didymum M-1, which was able to grow on choline as the This enzyme catalyzed the first step of the cho sole carbon source, was used throughout this work. line oxidation. The enzyme was purified and The medium consisted of 1.0g of betaine, 0.1g of meat extract, 0.1g of KH2PO4, 0.1g of K2HPO4, 0.1g of characterized in details.7,8) Subsequently, we NaCl and 0.05g of MgSO4.7H2O in 100ml of tap attempted to purify the related three enzymes in water, pH 7.0. The cultivation was carried out at 28•Ž choline metabolism by C. didymum M-1, be for 50hr under reciprocal shaking. taine aldehyde dehydrogenase, dimethyl Enzyme assay. Dimethylglycine oxidase activity glycine oxidase and sarcosine oxidase, and suc was assayed by the measurement of formaldehyde or ceeded to purify two enzymes, dimethylglycine H2O2 formed and by the measurement of oxygen con oxidase and sarcosine oxidase. sumed. The standard reaction system to estimate * Present address: Department of Agricultural formaldehyde formed contained 300ƒÊmol of Tris-HCl Chemistry, Tottori University, Tottori 680. buffer (pH 9.0), 150ƒÊmol of dimethylglycine and a 1384 N. MORI, B. KAWAKAMI, Y. TANI and H. YAMADA suitable amount of enzyme solution in a total volume RESULTS of 3.0ml. The reaction was carried out with shaking at 30•Ž for 10min. The reaction was stopped by the Purification of dimethylglycine oxidase addition of 0.1 ad of 4 N HCl. The activity to form All steps were performed at 0•`5•Ž. Tris H2O2 was determined by the method coupled with HCl buffer (pH 8.5) containing 0,1mM dithio peroxidase, phenol and 4-aminoantipyrine. The reac theritol (DTT) was used and centrifugation tion mixture contained 300 pmol of Tris-HCl buffer was carried out at 12,000•~g for 20min through (pH 9.0), 6ƒÊmol of phenol, 4.5ƒÊmol of 4-aminoanti the purification procedure, unless otherwise pyrine, 6 unit of peroxidase, 150ƒÊmol of dimethyl glycine and a suitable amount of enzyme solution in a stated. total volume of 3.0ml. The reaction was started by the Step 1. Preparation of cell extract. addition of dimethylglycine, and the increase of absor bance at 500 nm was followed in a Hitachi double Washed cells (about 185g as wet weight) beam spectrophotometer. The oxygen consumption were suspended in 0.8 liter of 0.1M buffer and was measured with an oxygen electrode at 30•Ž. The disrupted for 50min with a Kaijo Denki reaction mixture contained 60ƒÊmol of Tris-HC1 buffer 19kHz ultrasonic oscillator. The cell extract (pH 9.0), 30ƒÊmol of dimethylglycine and a suitable was obtained by centrifugation. amount of enzyme solution in a total volume of 0.6 ml. One unit of dimethylglycine oxidase activity was de Step 2. Ammonium sulfate fractionation. fined as the amount of enzyme which catalyzed the for To 910ml of the cell extract was added 207g mation of 1ƒÊmol of formaldehyde or H2O2 per min and of solid ammonium sulfate to 30% saturation the consumption of 1ƒÊmol of oxygen per min. Speci fic activity was defined as the unit per mg of protein. under stirring, by adjusting pH to 8.5 with 14% ammonium hydroxide solution. After stirring Analytical method. Formaldehyde was determined for 30min, the precipitate formed was re according to the method of Nash.10) Sarcosine was moved by centrifugation and discarded. The determined by an automatic amino acid analyzer. Protein concentration for dimethylglycine oxidase was ammonium sulfate concentration of the super determined by the absorbance at 280 nm, where natant solution was increased 50% saturation. E1%1cm value of 15.0 was used after dry weight determina The precipitate formed was collected by centri tion. AMP was estimated by an enzymatic method fugation and was dissolved in 10mm buffer. with myokinase.11) The solution was dialyzed against four changes Molecular weight determination. The molecular of the same buffer. weight was determined by the method of Andrews. 1V) Step 3. DEAE-cellulose column chromato Sephadex G-200 was packed to a column (0.9•~100cm) and equilibrated with 50mm of Tris-HCI buffer (pH graphy. The dialyzed solution was placed 8.5) containing 0.1M NaCl and 0.1mm dithiothreitol. on a DEAE-cellulose column (5•~50cm) The flow rate was 1ml/hr and each 0.5ml fraction was equilibrated with 10mM buffer. After wash collected. Cytochrome c, chymotrypsinogen A, egg ing the column with 70mM buffer, the enzyme albumin, bovine serum albumin, aldolase, catalase and was eluted with 70mM buffer containing 0.2M ferritin were used as the reference protein. NaCI. Active fractions were pooled and con Disc gel electrophoresis. Electrophoresis in acryl centrated by the addition of ammonium sul amide gel was performed at a current of 2.0 mA per fate to 70% saturation. The precipitate ob gel in Tris-glycine buffer (pH 8.3) according to the me tained by centrifugation was dissolved in a thod of Davis.13) SDS-disc gel electrophoresis was minimal volume of 10mM buffer and dialyzed performed according to the method of Weber and Osborn.l4) Dimethylglycine oxidase was incubated in against the same buffer. 10mM of sodium phosphate buffer (pH 7.0) containing Step 4. Ammonium sulfate fractionation. 1% sodium dodecylsulfate and 2% 2-mercaptoethanol for 3 hr at 60•Ž before the electrophoretic run. To the dialyzed enzyme solution solid am monium sulfate was added to 30% saturation. Ultracentrifugal analysis. The sedimentation velo After stirring for 30min, the precipitate formed city was measured with a Spinco model E analytical was removed by centrifugation. The am ultracentrifuge at 20•Ž and 59,700 rpm. Diffusion constant was measured with the same apparatus operat monium sulfate concentration was then in ing 12,590 rpm with the synthetic boundary cell. creased to 45% saturation. The precipitate Dimethylglycine Oxidase from Cylindrocarpon didymum M-1 1385 formed was collected by centrifugation and Step 7. Gel filtration by Sephadex G-150. dissolved in a minimal volume of lo mm The enzyme solution was placed on a Sephadex buffer and dialyzed against the same buffer. G-150 column (2.5•~120cm) equilibrated with 50mM buffer containing 0.1m NaCI . Step 5. DEAE-Sephadex A-50 column The enzyme was eluted with the same buffer . chromatography. The dialyzed enzyme was The elution pattern of the enzyme is shown in placed on a DEAE-Sephadex A-50 column Fig. 2. The purification procedure is sum (3•~45cm) equilibrated with 0.1M buffer and marized in Table I. the column was washed with the same buffer. The enzyme was eluted with an increasing linear gradient of NaCI concentration from 0 to 0.2M (Fig. 1). Active fractions were combined and concentrated by the addition of ammonium sulfate to 70% saturation. The precipitate formed was collected by centrifugation and was dissolved in 10mM potassium phosphate buffer containing 0.1 mm DTT. FIG. 2. Elution Pattern of Sephadex G-150 Column Chromatography. The flow rate was 15ml per hr and 3ml fractions were collected. (•›), absorbance at 280nm; (•œ), enzyme activity. TABLE 1. SUMMARY OF PURIFICATION OF FIG. 1. Elution Pattern of DEAE-Sephadex A-50 DIMETHYLGLYCINE OXIDASE FROM Column Chromatography. Cylindrocarpon didymunr M-1 Fractions of 5ml were collected. (•›), absorbance at 280nm; (•œ), enzyme activity. Step 6. Hydroxyapatite column chromato graphy.