/. Biochem. 82, 1741-1749 (1977)

Purification and Characterization of Choline Oxidase from Arthrobacter globiformis Shigeru IKUTA, Shigeyuki IMAMURA, Hideo MISAKL, and Yoshifumi HORIUTI Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-23 Received for publication, June 7, 1977

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate—polyacrylamide disc gel electrophoresis) and an isoelectric point (p/) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline+O,—kbetaine aldehyde + H,O,, betaine aldehyde+O,+H,O^ betaine+H,O,. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; JV,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, Af-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.

In a previous report from this laboratory (/), the existence of choline oxidase was discussed in relation to the oxidative pathway of choline to betaine found in A. globiformis cells. The enzyme appeared to catalyze the oxidations of both choline and betaine aldehyde coupled with H,O, generation and oxygen consumption. The present paper reports on the purification and characterization of this choline oxidase.

Abbreviations: p/, isoelectric point; SDS, sodium dodecyl sulfate. Vol. 82, No. 6, 1977

MATERIALS AND METHODS Culture of the Bacterium—Cells of A. globiformis were grown aerobically in culture medium for 40 h, as described previously (/). Assay—H,Ot-generating activity, betaine aldehyde-forming activity and oxygen-consumption were determined as described previously (/). Identification and Estimation of Betaine in the Incubation Mixture—The reaction mixture for the production of betaine contained 20 mM Tris-HCI buffer (pH8), 1.5 mM 4-aminoantipyrine, 2.1 mM phenol, 30 ftmol of choline chloride, 40 units of

1741

1742

S. 1K.UTA, S. IMAMURA, H. MISAKI, and Y. HORIUTl

peroxidase, and 200 units of choline oxidase in a final volume of 100 ml. The reaction was carried out for 80 min at 37°C and stopped by adding sufficient cone. H O to give a final pH of 1.0. The amount of betaine formed was determined by the method of Barabanov et al. (2) with some modifications as follows. To the mixture, 5g of charcoal, previously washed with 0.1 N HCI, were added to remove the quinoneimine dye formed. The mixture was filtered, and the filtrate concentrated to 5 ml with a rotary evaporator at 40°C. A portion (0.5 ml) of the concentrated solution was mixed with 1 ml of reineckate solution: the reineckate solution was freshly prepared by dissolving 1.5 g of the monohydrate in 100 ml of distilled water, adjusting the pH to 1.0 with cone. HCI, and filtering the mixture. After addition of reineckate, the mixture was stood for 30 min at room temperature (25°C), and the resulting precipitate was collected by centrifugation (7,000 Xg, 5 min) and washed twice with 2 ml of ethyl ether. The washed precipitate was dissolved in 2.5 ml of distilled water, and the absorbance of the solution at 525 nm was measured. The amount of betaine was calculated from a standard curve obtained with authentic betaine.

Co., Kyoto) at room temperature (25°C). Materials—Choline chloride, betaine, 4-aminoantipyrine, phenol, 2,4-dinitrophenylhydrazine, monoethanolamine, diethanolamine, triethanolamine, N-methylethanolamine, formaldehyde, acetaldehyde, and propionaldehyde were obtained from Wako Pure Chemical Industries Co., Osaka. N,N-dimethylethanolamine was from Tokyo Kasei Organic Chemicals Co., Tokyo, and peroxidase and Coomassie Brilliant Blue R were from Sigma Chemical Co., St. Louis. Sephadex G-200 and DEAE-cellulose were products of Pharmacia Fine Chemicals, Uppsala, and Brown Co., Berlin, respectively. The reference proteins used for molecular weight determinations were aldolase, chymotrypsinogen A, ovalbumin, bovine serum albumin, RNA polymerase, and trypsin inhibitor from Boehringer Mannheim GmbH, Mannheim. RESULTS

Purification of Choline Oxidase from the Cells —The bacterial cells were harvested from 2 liters of culture medium and washed with 10 mM phosphate-2mM EDTA-0.1% KC1 buffer (pH 7) (EDTA-KCI-Pi buffer) by centrifugation. The Determination of Protein—Protein concen- washed cells were suspended in 400 ml of the same trations were determined by the method of Lowry buffer containing 0.05% lysozyme, and the suset al. (5) with bovine serum albumin as a standard. pension incubated for 30 min at 37°C with stirring. Isoelectric Focusing—Isoelectric focusing was The resulting lyzed cell suspension was centrifuged carried out at 5°C for 40 h with Ampholine carrier (7,000X0, 20 min) to remove cell debris, and the ampholytes giving a pH gradient of 3.5 to 10 in a supernatant was mixed with 10 ml of 5% protamine 110 ml electrofocusing column, according to the sulfate solution (pH 7). The precipitate formed was removed by centrifugation, and the clear method of Vesterberg {4). Polyacrylamide Disc Gel Electrophoresis— supernatant mixed with an equal volume of cold Polyacrylamide disc gel electophoresis was carried acetone, stood for 20 min at 25°C and centrifuged out in 50 HIM Tris-glycine buffer (pH 8.3) at a (7,000 x q, 10 min). The resulting supernatant was constant current of 2 mA per column (5 x 80 mm) mixed with acetone to 75% (v/v), and the mixture for 150 min at 15°C, as described by Davis (5). stood for 20 min at 20°C and then centrifuged Disc gel electophoresis in the presence of sodium (7,000 x Q, 10 min). The precipitate was dissolved dodecyl sulfate (SDS) was performed by the method in 50 ml of EDTA-KCI-Pi buffer and fractionated of Weber et al. (6) in 0.1 M phosphate buffer (pH by adding a saturated solution of ammonium 7.2) containing 0.1% SDS on 5% polyacrylamide sulfate (pH8); the fraction which precipitated gel with 0.14% N.N'-methylenebisacrylamide. between 40% and 60% saturation was collected Electrophoresis was carried out at 8 mA per by centrifugation (12,000xg, 15 min) and dissolved column and at 25°C for 4 h. The gel was stained in 20 ml of EDTA-KCI-Pj buffer. The solution was desalted on a Sephadex G-25 column, mixed with Coomassie Brilliant Blue R (6). Absorption Spectrum—The absorption spec- with acetone to 60% (v/v) and centrifuged (7,000 xg, 10 min). The resulting supernatant was trum was measured with a Shimadzu doublemixed with acetone to 75% and the mixture stood beam spectrophotometer UV-210 A (Shimadzu /. Biochem.

PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE for 20min at 20°C. The precipitate formed was collected by centrifugation (7,000xg, lOmin) and dissolved in 2 ml of the buffer. This solution was applied to a column of DEAE-cellulose (Fig. 1). The column was washed with 60 ml of EDTAKCl-Pi buffer containing 0.2 M KCI and then eluted with a linear gradient of KCI (0.2-0.5 M) in the same buffer. Fractions showing the enzymic activity (Nos. 59-76) were combined, mixed with 2 volumes of cold acetone and centrifuged (7,000

1743

xg, lOmin). The precipitate was dissolved in 2 ml of EDTA-KCl-Pi buffer, and the solution rechromatographed on a column of DEAE-cellulose in a similar manner to that described above (Fig. 2). The fractions containing most of the activity (Nos. 59-75) were combined and again subjected to acetone precipitation in the way described above. The precipitate was dissolved in 2 ml of EDTA-KCl-Pi buffer, and chromatographed on a Sephadex G-200 column (Fig. 3).

1.00 •

activity ( u / m l )

20

~ - 0.6

A

28O

1 0.75 o

s

Activity KCI gradient

>• - 0.4

" 0501-

•I

2 075

d°o°d a.

UJ

_o

rwv-«v->rir*(-(T30000nr*V->n00^ 20 40

0.2 D

0 J

60 Fraction number

80

Fig. I. Column chromatography on DEAE-cellulose. The enzyme solution (2 ml) after acetone fractionation (60-75%) was applied to a DEAE-cellulose column (2x15 cm) previously equilibrated with 10 mM phosphate buffer (pH 7) containing 2 mM EDTA and 0.1 % KCI (EDTA-KCl-Pi buffer). The column was washed with 60 ml of the same buffer containing 0.2 M KCI and then eluted with 500 ml of a linear gradient of 0.2 to 0.5 M KCI in the same buffer at a flow rate of about 30 ml per h, and fractions of 6 ml were collected. All procedures were carried out at 20°C. Other experimental conditions are described in the text.

40 60 Fraction number

80

Fig. 2. Column chromatography on DEAE-cellulose. The enzyme solution (2 ml) from thefirstDEAE-cellulose column was fractionated with acetone and then applied to a DEAEcellulose column (2x15 cm). Other experimental conditions were the same as for Fig. 1. Vol. 82, No. 6, 1977

S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI

1744

The fractions constituting the enzyme peak (Nos. 46-54) were collected and lyophilized. The purification procedure is summarized in Table I. The lyophilized powder, having a specific activity of 12.5 units per mg protein, gave a single protein band on disc gel electrophoresis in the presence and absence of SDS (Fig. 4). Absorption Spectrum—The absorption spectrum of the purified choline oxidase showed maxima at 363 nm and 450 nm in the visible region, having a shoulder at 480 nm (Fig. 5). This in-

dicates that the enzyme has a typical flavo-protein spectrum. The flavo-protein enzyme is easily reduced by choline, the substrate, or by sodium hydrosulfite as also shown in the absorption spectra: reduction is reversible. Isoelectric Point and Molecular Weight— Isoelectric separation with Ampholine carrier ampholytes showed that the enzyme has a p / of 4.5 (Fig. 6), although the recovery on isoelectric focusing was low (15%). The molecular weight of the enzyme was determined to be about 83,000 by gel filtration on Sephadex G-150 (7) and about 71,000 by SDS-polyacrylamide disc gel electrophoresis (Fig. 7-a & b). + SDS

90

-I- Nona

60

Friction number

Fig. 3. Column chromatography on Sephadex G-200. The enzyme solution (2 ml) from the second DEAEcellulose column was fractionated with acetone and then applied to a Sephadex G-200 column (2.7x90 cm) at 20°C previously equilibrated with EDTA-KCI-P| buffer. Fractions of 6 ml were collected at a flow rate of about 15 ml per h. Other experimental conditions are described in the text. TABLE I.

Fig. 4. Polyacrylamide disc gel electrophoresis of the purified enzyme. The experimental conditions were as described in the text, except that 12 fig and 10 fig of the enzyme protein (the "lyophilized powder" of Table I) were applied to columns in the presence and absence of SDS, respectively.

Summary of the purification of choline oxidase from A. globiformts. Step

Supernatant from lyzed cell suspension Acetone fractionation (50-75%) (NH,),SO 1 fractionation (40-60%) Acetone fractionation (60-75%) DEAE-cellulose 1st 2nd Sephadex G-200 Lyophilized powder

Total protein Total activity (units) (mg) 1,970 510 260 90 38 3§ 32 32

1,690 1,030 920 660 580 450 410 400

Specific activity (units/mg protein) 0.86 2.01 3.54 7.33 10.35 11.54 12.80 12.50

Recovery (%) 100.0 60.9 54.4 39.1 34.3 26.6 24.2 23.6

/ . Biochem.

PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE

1745

TABLE II. Estimation of the reaction products of choline oxidation with choline oxidase. Experimental conditions are described in the text. The enzyme source was the "lyophilized powder" of Table I. 8

I

Amount (/imol)

Compound

30.0 59.6 30.1

Choline added HiO, generated Betaine formed 390

450 500 Wavt length ( nm }

400

550

Fig. 5. Absorption spectrum of choline oxidase. Enzyme concentration, 9.2 mg protein per ml EDTA-KC1Pi buffer. Curve 1, oxidized enzyme; Curve 2, after addition of 0.2 mM choline chloride; Curve 3, after addition of 2 mM choline chloride; Curve 4, after addition of sodium hydrosulfite. Aerobic conditions at 25°C. Other experimental conditions are described in the text. Some Characteristics of Choline Oxidase— Identification and determination of reaction products of the oxidation catalyzed by choline oxidase: The methods used for the identification and determination of betaine, betaine aldehyde, and hydrogen peroxide (quinoneimine dye) produced from the oxidation of choline and betaine aldehyde are described under "MATERIALS AND METHODS." When 30 (imo\ of choline was incubated

with the enzyme (200 units), the resulting reaction mixture contained about 30 /imol of betaine and quinoneimine dye (equivalent to 60 pmol H,O,) (Table II). During the oxidation of choline to betaine, the transient intermediate, betaine aldehyde, accumulated in the reaction mixture and disappeared at the end of the incubation time accompanied with the production of a stoichiometric amount of H t Oi per choline: using 0.1 ftmo\ of choline and 0.25 units of enzyme, the aldehyde accumulated reaching a maximum of about 0.04-0.05 //mol 3 to 8 min after the start of the reaction and after 30 min, when the aldehyde could no longer be detected, about 0.2 ftmol of H S O, was produced (Fig. 8-a). During the betaine aldehyde oxidation with the enzyme (0.25 units),

12

*280 10 •

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^J3O3O - 0.5

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Activity

^^~rCP^^^

a« Z

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?

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- 0.3 > 0.2.

4

03300

&

- 0.1

2 • |O05 0

f

10

20 Fraction

30

40

50

numbtr

Fig. 6. Isoelectric focusing of choline oxidase. The experimental conditions were as described in the text, except that 12.5 units of the enzyme (the "lyophilized powder" of Table I) was applied to the electrofocusing column. The total recovery of enzyme activity was 15%. Vol. 82, No. 6, 1977

S. TKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI

1746

Aldolui .Cholln*

•S 10 -o • ~ 6

I«

oxtdaft*

Ovalbumln Chymotrypslnoff

I« 1-0

1.2

1.*

1.6

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0

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b

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oildat*

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f*

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s

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005

I 2 X

1

0-2

4

0.6 Mobility

• 0.10 H

S«rum albumin

- 0.05 B»t4ln»

aldthyd*

1X1

Fig. 7-a & b. Determination of the molecular weight of choline oxidase by gel filtration (a) and SDS-polyacrylamide gel electrophoresis (b). (a) The enzyme (0.5 units) in EDTA-KCl-Pi buffer was applied to a Sephadex G-150 column (1 x 100 cm) equilibrated with the same buffer. Elution was performed with the same buffer at 20°C. The flow rate was adjusted to approx. 3 ml/h, and fractions of 0.9 ml were collected. The reference proteins were run under similar conditions {Vo; void volume, V,\ elution volume). Other experimental conditions are described in the text, (b) The enzyme protein (10 /jg) was subjected to electrophoresis, and the reference proteins were run under similar conditions. Other experimental conditions are described in the text. The enzyme source was the "lyophilized powder" of Table I. the rate of disappearance of the substrate aldehyde (0.1 ^zmol) corresponded to the rate of generation of H , O , and by the end of the incubation period (30 min), a stoichiometric amount (0.1 ;/mol) of H , O , had been generated (Fig. 8-b). These results indicate that the enzyme can catalyze two-step oxidation of choline to betaine aldehyde to betaine in reactions coupled with H , O , generation, and that the first step is probably faster than the second step. The respective rates of H , O , generation in the

0

/ 10

•*"—

•—

20

30

Tim* (min )

Fig. 8-a & b. Time courses of oxidation of (a) choline and (b) betaine aldehyde coupled with H,Ot generation by choline oxidase. The reaction for H,O, generation was carried out for the indicated periods in the same reaction mixture as for the assay of H,Ot-generating activity, except that 0.2 mM choline chloride or betaine aldehyde was used. The reaction for formation or disappearance of betaine aldehyde was carried out in the same reaction mixture as for the assay of betaine aldehyde-forming activity, except that 0.2 mM betaine aldehyde, instead of choline chloride, was added to the mixture for disappearance of betaine aldehyde. The "lyophilized powder" of Table I was used as the enzyme source, and 0.25 units enzyme were added for each reaction.

oxidations of choline and betaine aldehyde corresponded quantitatively to those of oxygen consumption (Table III). Based on the above results, it is concluded that choline oxidase catalyzes the following reactions: Choline+O,--Betaine aldehyde + H,O, Betaine a l d e h y d e + O , + H , O ^ B e t a i n e + H , O , /. Biochem.

PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE Substrate specificity and effects of substrate concentrations: The substrate specificity was examined using derivatives of aminoethanol, aldehydes, and alcohols (Table IV). Choline and betaine aldehyde served as substrates for the enzyme, but very low activities were found towards the other compounds: the activities relative to that of choline were about 50% for betaine aldehyde and below 6% for the other compounds. The Michaelis constants (Km) were found to be 1.2 ITIM for choline and 8.7 mM for betaine aldehyde, when the rates of substrate oxidation were measured as the formation of quinoneimine dye due to generation of H t O , (Fig. 9). TABLE III. Activities of H t Oi generation and oxygen consumption with choline oxidase. Experimental conditions are described in the text. The enzyme source was the "lyophilized powder" of Table I.

40 • Bettln* aldthydt

0.47 0.23

0.49 0.24

TABLE IV. Substrate specificity of choline oxidase. Experimental conditions for the activity assay of H,O, generation were as described in the text, except that various substrates were used. The enzyme source was the "lyophilized powder" of Table I. Substrate Choline Monoethanolamine Diethanolamine Triethanolamine N-methylaminoethanol N, N-dimethylaminoethanol Methanol Ethanol Propanol Betaine aldehyde Formaldehyde Acetaldehydc Propionaldehyde

Vol. 82, No. 6, 1977

•

/

30 •

V

^20

.

Choline 10

H,O, generation Ot consumption (unit) (unit)

Choline Betaine aldehyde

•

.£0-00—

Activity Substrate

Effect ofpH on activity: The maximal enzyme activity was observed in the range of pHs 7-8, when choline was used as a substrate (Fig. 10); since the molar extinction of quinoneimine dye was significantly influenced by pH, the activity was assayed by measuring the formation of betaine aldehyde. Determinations of Choline and Betaine Aldehyde with Choline Oxidase—The purified enzyme

025

0.50 075 1/S (mM' 1 )

too

Fig. 9. Lineweaver-Burk plot of the activity of choline oxidase. Experimental conditions were as described in the text, except that various concentrations of choline and betaine aldehyde were used. The enzyme source was the "lyophilized powder" of Table I.

100

Relative activity 100. 0ft 0.0 0.8 2.6 0.0 5.2 0.0 0.0 0.0 46.2 0.0 0.0 0.0

10

Fig. 10. Effect of pH on the activity of choline oxidase. The enzyme activities at various pH values were assayed in the reaction mixture for the assay of betaine aldehydeforming activity described in the text. Dimethylglutarate buffer was used for pH 4 to 6 ( • ) ; phosphate buffer for pH 6 to 8 (O); glycylglycine buffer for pH 8 to 9 (D); and glycine buffer for pH 9 to 10 ( A ) . The enzyme source was the "lyophilized powder" of Table I.

S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI

1748

0

10 20 30 40 50 Amount of additives (nmoles)

Fig. 11. Estimation of choline and betainc aldehyde with choline oxidase. Experimental conditions were as described in the text, except that 0.5 units of the enzyme and various concentrations of substrates (choline and betaine aldehyde) were used and the reaction period was 20 min. The enzyme source was the "lyophilized powder" of Table I. was tested for the quantitative estimations of choline and betaine aldehyde by measuring the formation of quinoneimine dye (Fig. 11). The formation of quinoneimine dye was proportional to the amounts of these substrates up to 50 nmol. Thus, this enzyme can be used for the determination of choline and betaine aldehyde. DISCUSSION Three kinds of enzymes that oxidize choline to betaine aldehyde or betaine aldehyde to betaine have been found in animal tissues and bacterial cells by various investigators. These three kinds of enzymes are choline dehydrogenase [EC 1.1.99.1, choline: (acceptor) oxidoreductase], NAD(P)-dependent betaine aldehyde dehydrogenase [EC 1.2.1.8, betaine-aldehyde: N A D + oxidoreductase] and NAD(P>independent betaine aldehyde dehydrogenase. Choline dehydrogenase has been demonstrated to be localized in mitochondria or bacterial membranes as a particulate enzyme and has been partially purified in a soluble form from a mammalian liver homogenate (8-15) and from bacterial cells (76). Unlike the findings on the choline dehydrogenase, those on the dehydrogenases for betaine aldehyde are conflicting with respect to the cellular location (mitochondria or cytoplasm), the char-

acteristics of the enzyme reaction (NAD(P)-dependent or -independent and specific or nonspecific for betaine aldehyde), and the conditions necessary for oxidation of aldehyde to betaine. The NAD(P)-dependent betaine aldehyde dehydrogenase that was claimed to be present in the cytoplasm or soluble supernatant fraction (17-21) was partially purified from a liver homogenate (17) and completely from a cell-free extract of bacteria (20). On the other hand, the betaine aldehyde dehydrogenase which was clamined to be associated with mitochondria, as opposed to the above cytoplasmic enzyme (22-27), has not been isolated or characterized. The choline oxidase purified and characterized in this work was found to be a new type of flavoprotein enzyme, which can oxidize both choline and betaine aldehyde using molecular oxygen as a primary electron acceptor and producing H,O a : mitochondrial choline dehydrogenase, a flavoprotein enzyme (11, 28-30), is thought to catalyze the oxidation of choline only and to mediate electron transfer from the substrate to oxygen via the electron transport chain involving cytrochromes (28, 31) and ubiquinone (14). In mitochondria or particulate fractions, it is still uncertin whether the oxidation of choline to betaine requires cofactors, such as NAD, and can generate H , O , under suitable conditions. Then, our demonstration of the existence of choline oxidase might provide clues for elucidating the mechanism of the enzymic oxidation of choline to betaine in mitochondria or particulate fractions and also support the scheme we proposed for the oxidation of choline to betaine in A. globiformis cells (1), which has not yet been reported for other bacterial cells or animal tissues.

REFERENCES 1. Ikuta, S., Matsuura, K., Imamura, S., Misaki, H., & Horiuti, V. (1977) /. Biochem. 82, 157-163 2. Barabanov, M.I., Vil'chinskii, S.T., & Litvak, I.M. (1962) Tr. Kievsk. Tekhnol. Inst. Pishchevoi Prom. 25,69-73 3. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 4. Vesterberg, O. (1971) Methods Enzymol. 22, pp. 389-412, Academic Press Inc., New York 5. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 6. Weber, K., Pringle, J.R., & Osborn, M. (1972) / . Biochem.

PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Methods Enzymol. 26, pp. 3-27, Academic Press, Inc., New York Andrews, P. (1965) Biochem. J. 96, 595-606 Williams, J.N., Jr. & Streenivasan, A. (1953) J. Biol. Chem. 203, 899-906 Ebisuzaki, K. & Williams, J.N., Jr. (1955) Biochem. J. 60, 644-646 Kearney, T.B. & Singer, T.P. (1956) J. Biol. Chem. 219, 963-975 Korzenovsky, M. & Auda, B.V. (1958) Biochim. Biophys. Ada 29, 463-464 Rendina, G. & Singer, T.P. (1959) J. Biol. Chem. 234, 1605-1610 Kimura, T. & Singer, T.P. (1962) Methods Enzymol. 5, pp. 562-570, Academic Press Inc., New York Drabikowska, A.K. & Szarkowska, L. (1965) Ada Biochim. Pol. 12, 387-394 Barrett, M.C. & Dawson, A.P. (1975) Biochem. J. 151, 677-683 Nagasawa, T., Mori, N., Tani, Y., & Ogata, K. (1976) Agric. Biol. Chem. 40, 2077-2084 Rothschild, H.A., & Barron, E.S.G. (1954) /. Biol. Chem. 209, 511-523 Jellinek, M., Strength, D.R., & Thayer, S.A. (1959) J. Biol. Chem. 234, 1171-1173 Yue, K.T.N., Russell, P.J., & Mulford, D.J. (1966) Biochim. Biophys. Ada 118, 191-194

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20. Nagasawa, T., Kawabata, Y., Tani, Y., & Ogata, K. (1976) Agric. Biol. Chem. 40, 1743-1749 21. Glenn, J.L. & Vanko, M. (1959) Arch. Biochem. Biophys. 82, 145-152 22. Williams, J.N., Jr. (1952) /. Biol. Chem. 195, 37-41 23. Bianchi, G. & Azzone, G.F. (1964) / . Biol. Chem. 239, 3947-3955 24. Kagawa, T., Wilken, D.R., & Lardy, H.A. (1965) J. Biol. Chem. 240, 1836-1842 25. Wilken, D.R., McMacken, M.L., & Rodriquez, A. (1970) Biochim. Biophys. Ada 216, 305-317 26. De Ridder, J.J.M. & Van Dam, K. (1973) Biochim. Biophys. Ada 291, 557-563 27. De Ridder, J.J.M., Kleverlaan, N.T.M., VerdouwChamalaun, C.V.M., Schippers, P.G.M., & Van Dam K. (1973) Biochim. Biophys. Ada 325, 397^05 28. Rothschild, H.A., Cori, O., & Barron, E.S.G. (1954) J. Biol. Chem. 208, 41-53 29. Packer, L., Estabrook, R.W., Singer, T.P., & Kimura, T. (1960) J. Biol. Chem. 235, 535-537 30. Kimura, T. & Singer, T.P. (1962) Methods Enzymol. 5, pp. 562-570, Academic Press Inc., New York 31. Mann, P.J.G., Woodward, H.E., & Quastel, J.H. (1938) Biochem. J. 32, 1024-1032