THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 30, Issue of October 25, pp. 15444-15448,1988 Printed in U. S. A.

Purification and Characterization of Acetate Kinase from Acetate- grown Methanosarcina thermophila EVIDENCE FOR REGULATION OF SYNTHESIS*

(Received for publication, May 2, 1988)

David J. Aceti$ and James G. Ferry5 From the DeDartment of Anaerobic Microbioloev. Vireinia Polytechnic Institute and State University, - Blacksburg, Virginia 24061

Acetate kinase was purified 102-fold to a specific activity of 656 pmol of ADP formed/min/mg of protein from acetate-grown Methanosarcina thermophila. The enzyme was not intrinsically membrane bound. The native enzyme (Mr 94,000) was an a2 homodimer with a subunit M, of 53,000. The activity was optimum between pH 7.0 and 7.4. A PI of 4.7 was determined. The enzyme was stable to 0 2 and stable to heating at 70 OC for 15 min but was rapidly inactivated at higher temperatures. The apparent K , for acetate was 22 mM and for ATP was 2.8 mM. The enzyme phosphorylated propionate at 60% of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. The enzyme required one of several divalent cations for activity; the maximum rate was obtained with Mn2+. Western blots of cell extract proteins showed that ace- tate grown cells synthesized higher quantities of the acetate kinase than did methanol grown cells.

Methanogenic organisms are archaobacteria which are bio- chemically and phylogenetically distinct from all other pro- caryotes (1, 2). A limited number of methanogenic species belonging to two genera, Methanosarcina and Methanothrix, are capable of metabolizing acetate to methane. Methunosar- cina thermophila is an acetotrophic methanogen that was isolated from a thermophilic sewage digestor and which grows optimally near 50 "C (3). The pathway in M. thermophila involves transfer of the methyl group of acetate to 2-mercap- toethanesulfonic acid forming methyl P-mercaptoethanesul- fonic acid, which is reductively demethylated to methane in the final step of the pathway (4, 5). It is proposed that breakage of the carbon-carbon bond of acetate is catalyzed by an enzyme complex with CO dehydrogenase activity (6, 7) and that the carbonyl group of acetate binds to a Ni-Fe site in the complex (7). According to the proposed mechanism, the oxidation of the enzyme-bound carbonyl to carbon dioxide would supply electrons for the reductive demethylation of methyl 2-mercaptoethanesulfonic acid. The proposed mech- anism is similar to the reversal of the CO dehydrogenase-

* This investigation was supported by Grant 5082-260-1255 from the Gas Research Institute and Grant DE-FG05-87ER13730 from the Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Current address: Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacks- burg, Va. 24061.

To whom reprint requests should be addressed.

catalyzed synthesis of acetyl-coA' from CoA, a methylated corrinoid compound, and CO in Clostridium thermoaceticum (8). Thermodynamic considerations suggest acetate must be derivatized to an activated form prior to cleavage of the carbon-carbon bond in the methanogenic pathway (9). Acti- vation to acetyl-coA in M. thermophila is supported by EPR studies showing that acetyl-coA is bound by the CO dehydro- genase complex (7). Recently, activation to acetyl phosphate followed by conversion to acetyl-coA has been suggested for the pathway in Methanosarcina barkeri (10, 11).

Evidence for acetyl-coA as an intermediate in the acetate- to-methane pathway prompted us to assay for the presence of enzymes catalyzing acetyl-coA formation in acetate-grown cells of M. thermophila. We have previously reported (7) two enzymes that together may activate acetate to acetyl-coA: acetate kinase (EC 2.7.2.1) CH3C0; + ATP = CH3CO2PO$- + ADP, and phosphate acyltransferase (EC 2.3.1.8) CH3C02PO% + CoA = CH3COSCoA + Pi. Both of these activities are elevated in acetate grown cells when compared to methanol grown cells (7). This report summarizes the purification of the acetate kinase from M. thermophila and describes some of the properties of this enzyme.

EXPERIMENTAL PROCEDURES

Growth of the Organism and Preparation of Cell Extracts-M. therrnophila strain TM-1 was grown in a 10-liter fermentor with acetate, methanol, or acetate plus methanol as the carbon and energy source(s) as described previously (12, 13), and harvested in late log phase. Cell extracts (33-49 mg of protein/ml) were prepared as described previously (5) except the procedure was done aerobically and 2 mM dithiothreitol replaced 2-mercaptoethanol. Extract was stored in liquid nitrogen until use.

Enzyme Assays-All assays were performed at 37 "C. Specific activities are reported as pmoles of product formed/min/mg of pro- tein. A model 552 spectrophotometer (Perkin-Elmer) was used.

Two methods were used to assay acetate kinase in the forward direction, the enzyme-linked assay detecting ADP formation and the hydroxamate assay detecting acetyl phosphate formation. The en- zyme-linked assay couples ADP formation to the oxidation of NADH (e3@ = 6.22 In"' cm") through pyruvate kinase and lactic dehydro-

tions) 100 mM Tricine-KOH buffer, pH 8.2 (or 200 mM Tris-HC1 genase (14). The assay mixture (0.5 ml) contained (final concentra-

buffer, pH 7.3, where noted), 200 mM potassium acetate, 1.5 mM ATP, 2 mM MgC12, 2 mM phosphoenolpyruvate, 0.4 mM NADH, 2 mM dithiothreitol, 9 units of pyruvate kinase, and 26 units of lactic dehydrogenase. The combined activities of the linked enzymes in the mixture were routinely assayed to determine that they were not limiting. Assays were initiated by addition of the indicated amounts of acetate kinase to the assay mixture. The hydroxamate assay, an adaptation of the method of Lipmann and Tuttle (15) and Rose (161,

The abbreviations used are: CoA, coenzyme A; TES, N - tris(hydroxymethyl)methyI-2-aminoethanesulfonic acid; Tricine, N- tris(hydroxymethy1)methylglycine.

15444

Acetate Kinase from Methanosarcina 15445

utilizes the reaction of acetyl phosphate with hydroxylamine to form acetyl hydroxamate, which forms a colored complex with trivalent iron. The following components were combined in a volume of 333 ~1 (final concentrations) 145 mM Tris-HC1, pH 7.4, 200 mM Potassium acetate, 10 mM MgC12, 10 mM ATP, and 705 mM hydroxylamine hydrochloride (neutralized with KOH before addition). The reaction was initiated with the indicated amounts of acetate kinase and stopped after 12 min by the addition of 333 p1 of 10% trichloroacetic acid followed by the addition of 333 pl of 2.5% FeCb in 2.0 N HC1. The mixture was incubated for 5-30 min to allow color development, and the absorbance at 540 nm was measured. Acetate kinase was assayed in the reverse direction by linking ATP formation to the reduction of NADP through hexokinase and glucose-6-phosphate dehydrogenase (17). A reaction mixture (0.5 ml) contained (final concentrations) 100 mM Tris-HC1, pH 7.4,5 mM ADP, 10 mM MgC12, 5.5 mM glucose, 1 mM NADP, 2 mM dithiothreitol, 6 units of hexo- kinase, and 3 units of glucose-6-phosphate dehydrogenase. Acetate kinase (0.28 pg) was added, and the reaction was initiated by the addition of acetyl phosphate (final concentration 20 mM). Reduction of NADP was followed at 340 nm ((340 = 6.22 mM" cm").

Acetate-CoA ligase (EC 6.2.1.1) activity was determined as de- scribed previously (18) but with the following modified final concen- trations: 200 mM potassium acetate, 48 units of pyruvate kinase, 24 units of adenylate kinase, and 5 units of lactic dehydrogenase. The reaction was initiated with 0.03-0.06 mg of cell extract protein.

Purification of Acetate Kinase-All steps were performed aerobi- cally at 4 "C. Activity was assayed in the forward direction using the enzyme-linked assay. Extract (1.5 ml containing 60-70 mg of protein) from acetate grown cells was injected onto a Mono-Q HR 10/10 anion-exchange column (Pharmacia LKB Biotechnology Inc.) equil- ibrated with 50 mM TES, pH 6.8, containing 10% (v/v) ethylene glycol, 10 mM MgC12, and 2 mM dithiothreitol (buffer A). A linear gradient from 0.0 to 1.0 M KC1 was applied at 0.5 ml/min using a fast protein liquid chromatography system (Pharmacia LKB Biotechnol- ogy Inc.) equipped with a model GP-250 gradient programmer. The active fractions from several Mono-Q separations of cell extract were pooled and loaded onto a 5.0 X 0.9-cm ATP affinity column previously equilibrated with 10 mM Tris-acetate, pH 7.4, containing 1.2 M KCI, 1 mM MgC12, and 2 mM dithiothreitol (buffer B) at a flow rate of 0.5 ml/min. Contaminatingproteins and a small amount of acetate kinase were eluted with 15 ml of buffer B, followed by re-equilibration with 10 ml of buffer C (buffer B without KCl). The acetate kinase was then eluted at 0.1 ml/min by the application of 1 ml of buffer C containing 15 mM ATP followed by 10 ml of buffer C without ATP. The enzyme was stored in liquid nitrogen.

Molecular Weight-Polyacrylamide slab gel electrophoresis was performed using the Laemmli buffer system (19) under denaturing (with sodium dodecyl sulfate) or nondenaturing (without sodium dodecyl sulfate) conditions. The molecular weight of the denatured enzyme was estimated on a 12% gel using the following standards (Bio-Rad): lysozyme (14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), bovine serum al- bumin (66,200), phosphorylase b (92,500). A nondenaturing 4-30% linear gradient gel was used to estimate the native molecular weight of the enzyme (20). Molecular weight standards (Behring Diagnostics) were cytochrome c (12,500), chymotrypsinogen A (25,000), egg albu- min (45,000), black albumin (68,000), hexokinase (lOO,OOO), aldolase (158,000), @-glucuronidase (290,000), and ferritin (450,000). Gels were stained for protein with Coomassie Blue R-250 (Bio-Rad).

Gel filtration liquid chromatography was performed at 0.02 ml/ min on a 0.9 X 34.5-cm column of Sephadex G-200 SF (Sigma) equilibrated with 100 mM Tris-HC1 buffer, pH 7.2, containing 200 mM KC1, 1 mM ATP, and 0.1 mM phenylmethylsulfonyl fluoride (Sigma). Molecular weight standards (Pharmacia LKB Biotechnology Inc.) were catalase (232,000), aldolase (158,000), bovine serum albu- min (67,000), ovalbumin (43,000), and chymotrypsinogen A (25,000).

Western Blotting-Polyclonal anti-acetate kinase antibodies were raised in New Zealand White rabbits. Samples of cell extracts or pure enzyme were electrophoresed on a denaturing polyacrylamide gel as described for molecular weight determinations. Conditions for the transfer of proteins to nitrocellulose were as described previously (21). Additional protein-binding sites were blocked with casein and gelatin (0.5% each in 50 mM potassium phosphate buffer, pH 7.4, containing 120 mM NaCl and 0.1% Nonidet P-40 (Sigma)). The protein band corresponding to acetate kinase was detected using a 1:40,000 dilution of anti-acetate kinase antiserum and '251-labeled goat anti-rabbit IgG (0.635 pCi at 7.9 pCi/pg (Du Pont-New England Nuclear).

Determination of PI-Purified enzyme (5 pg of protein) was elec- trophoresed on a pH 3.5-9.3 Ampholine PAGplate polyacrylamide gel (LKB Instruments, Inc., Gaithersburg, MD) using the protocol provided by the manufacturer. Standards (PI) were trypsinogen (9.30), lentil lectin-basic (8.65), lentil lectin-middle (8.45), lentil lectin-acidic (8.15), myoglobin-basic (7.35), myoglobin-acidic (6.851, human car- bonic anhydrase B (6.55) bovine carbonic anhydrase B (5.851, 0- lactoglobin A (5.20), soybean trypsin inhibitor (4.55), and amyloglu- cosidase (3.50). The gel was stained for protein with Coomassie Blue

Analytical-Amino acid composition and the NH2-terminal se- quence were determined at the Commonwealth of Virginia Protein and Nucleic Acid Sequencing Facility, University of Virginia, Char- lottesville, VA. Samples were hydrolyzed in U ~ C U O for 24 h with constant boiling HCI (6 N). The phenylisothiocyanate-derivatized amino acids were chromatographed using a Waters 840 LC System (Waters Associates, Milford, MA) equipped with a Shandon 25 X 4.6- cm C18 reverse-phase 3-ym column, and were eluted in a sodium acetate-acetonitrile gradient. The values obtained for serine and threonine were corrected for degradation during acid hydrolysis by extrapolation from standards. NHz-terminal amino acids were se- quenced using a model 470 A gas-phase peptide sequencer (Applied Biosystems, Inc., Foster City, CA) and the phenylthiohydantoin derivatives identified with an on-line Applied Biosystems liquid chro- matograph.

Protein was determined by the method of Bradford (22) using protein dye reagent (Bio-Rad) and bovine serum albumin (Sigma) as the standard.

Materials-The ATP affinity column matrix, 8-(6-aminohexylam- ino) 5"adenosine triphosphate conjugated to Sepharose 4B, was purchased from Pharmacia LKB Biotechnology Inc. Adenylate kinase (from porcine muscle), pyruvate kinase and lactic dehydrogenase (both from rabbit muscle), hexokinase and glucose-6-phosphate de- hydrogenase (both from baker's yeast), ATP, ADP, NADH, NADP, and phosphoenolpyruvate were purchased from Sigma. All other chemicals were of reagent grade.

R-250.

RESULTS AND DISCUSSION

Localization in Cell Extracts-Sucrose density gradient ul- tracentrifugation of cell extracts of acetate grown M. ther- mophila was used to separate membranes from soluble pro- teins, using previously described procedures (23). Approxi- mately 77% of the applied acetate kinase activity (27 pmol of ADP formed/min) was recovered in the soluble fractions, and no significant activity was detected in the membrane frac- tions. These results suggest that acetate kinase is not an integral membrane protein, however, a loose association can- not be ruled out.

Purification-Table I shows a representative purification of the acetate kinase from cell extracts of acetate grown M. thermophilu using Mono-Q anion-exchange and ATP affinity chromatography. The enzyme was judged to be homogeneous as only one protein band was visible after denaturing (Fig. 1) or native (data not shown) polyacrylamide gel electrophoresis.

Properties-A subunit M, of 53,000 was estimated by de- naturing gel electrophoresis. The M, of the native enzyme was estimated to be 94,000 by gel filtration on a calibrated Seph- adex G-200 SF column and 87,000 by native gradient gel electrophoresis. Only one NH2 terminus was detected; the sequence was Met-Lys-Val-Leu-Val-Ile-Asn-Ala-Gly-Ser- Ser-unknown-Leu-Lys-Tyr-Gln-Leu-. These results suggest that the acetate kinase was isolated as an aP homodimer. Monomeric, dimeric, and tetrameric native forms of acetate kinase have been reported for eubacteria (17, 24, 25).

The amino acid composition (mol %) was as follows: Ala (6.41, Arg (4.51, Asp (8.51, Cys (1.2), Glu (10.0), Gly (14.7), His (2.4), Ile (6.71, Leu (6.7), Lys (5.5), Met (3.1), Pro (4.1), Phe (3.61, Ser (8.1), Thr (5.9), Trp (not determined), Tyr (2.21, and Val (6.6). A PI of 4.7 was determined by isoelectric focusing.

The activity was stable for at least 4 months when frozen

15446 Acetate Kinase from Methanosarcina

TABLE I Purification of acetate kinose from acetate grown M. thmmophila

step Total Total Specific -Fold activity protein activity purification Recovery

units' mg unitsfmg 76 Cell extractb 2799 439.0 6.4 1 100 Mono-Q anion-exchange chromatography 864 4.4 196.4 31 31 ATP affinity chromatography 459 0.7 655.7 102 16

a A unit = 1 pmol of ADP formed/min (enzyme-linked assay). Cell extract was 49 mg of protein/ml.

1 2 3 " - 'R

FIG. 1. Denaturing polyacrylamide gel electrophoresis of M. thermophila acetate kinase at each step of the purification. Lane I , 75 pg of cell extract protein from acetate grown cells; lane 2, 35 pg of protein from the Mono-Q anion-exchange fraction with the highest specific activity; lane 3, 10 pg of protein from the pooled active fractions eluting from the ATP affinity column after applica- tion of ATP (see text). The gel was stained for protein with Coomassie Blue R-250.

in liquid nitrogen and was unaffected by repeated freezing and thawing. Activity was stable to heating for 15 min at temperatures of up to 70 "C, but was inactivated a t higher temperatures (Fig. 2).

Activity in the reverse direction was 2.6-fold greater than in the forward (hydroxamate assay) direction when the assays were performed as described under "Experimental Proce- dures.'' The latter is presumed to be the physiologically im- portant direction and was studied further. Optimum acetate kinase activity (hydroxamate assay) was between pH 7.0-7.4, with little activity below pH 5 or above pH 9 (Fig. 3). The kinetic constants for acetate and Mg2':ATP are shown in Table 11. Normal hyperbolic saturation curves were obtained with both acetate and Mg2':ATP and no substrate inhibition was observed with up to 10 mM Mg2':ATP or 250 mM potas- sium acetate (data not shown). The enzyme phosphorylated propionate but not formate (Table 111). Other triphosphory- lated nucleotides replaced ATP (Table 111). The enzyme did not show a preference for purine nucleotides.

All acetate kinases studied require divalent cations for activity, with M$+ providing maximum activity in most cases (24-28). The M. thermophila enzyme also required divalent cations and utilized Mg2' for activity in either direction.

l o o t O-O ""-9

_.

*O t 0 ' " " ' ~ ' ~ ' ' " \- I

30 40 50 60 70 80

TEMPERATURE ("C)

FIG. 2. Heat stability of M. thermophila acetate kinase. Each sample (0.23 pg of acetate kinase in 5 pl ) was added to 10 pl of prewarmed 100 mM Tris-HCI (pH 7.4 at the indicated temperature) and incubated for 15 min. The enzyme solution was immediately cooled to 4 "C and the activity determined in the forward direction a t 37 'C by the enzyme-linked assay, which was initiated with 0.23 pg of enzyme. Activity was 88 pmol of ADP formed/min/mgof protein (100%) after preincubation a t 30 "C.

n 400 -

F

c 2 200

- 300 '=. -

2 I- - 0 lL- 0

100 - cn

0 4 5 6 7 8 9 1 0

PH

FIG. 3. Influence of pH on activity of acetate kinase from M. thermophila. The hydroxamate assay was used with the follow- ing modifications: Tris buffer was excluded, the potassium acetate concentration was 600 mM, hydroxylamine hydrochloride was ad- justed to the indicated pH with KOH before addition of the remaining assay components. The reaction was initiated with 0.45 pg of enzyme and was stopped after 5 min. The pH of the assay mixture was determined immediately before starting and immediately before stop- ping the reaction, and was unchanged during this time.

Variation of the MgC12 concentration with a constant concen- tration of 10 mM ATP resulted in a broad peak of activity with an optimum at 10 mM MgC12, indicating an optimum Mg2'/ATP ratio of 1.0 (Fig. 4). This data suggests that M$+ is necessary only to complex with ATP and is not required additionally for the function or stability of the enzyme. In contrast, a M F / A T P ratio of 2.0 is optimal for the Esche- richia coli enzyme (29).

Acetate Kinase from Methanosarcina 15447

TABLE I1 Kinetic constants for acetate kinase from M. thermophila

A double reciprocal plot of the MflATP (1:l) concentrations (0.50, 0.67, 0.80, 1.0, 1.25, 1.50, 2.00, 2.50, 3.33, 4.00, 5.00, 6.25, and 8.00 mM) versus the reaction rate (hydroxamate assay) yielded a straight line with r = 0.998; the reactions were initiated with 0.12 pg of enzyme. A double reciprocal plot of the acetate concentration (2, 3, 4, 7, 10, 15, 20, 25, 30, 50, 70, and 100 mM, with potassium held constant a t 200 mM) versus the reaction rate (enzyme-linked assay) yielded a straight line with r = 0.995; the reactions were initiated with 0.12 ug of enzyme.

Substrate K, Turnover VS3.X number

rmol mol of mM product/min/mg productls mol

protein acetate kinase" M&IZ:ATP 2.8 777 1221 Potassium acetate 22.0 668 1050

a Calculation based on M, 94,000 for the dimer of acetate kinase.

TABLE 111 Substrate specificity of acetate kinase from M. thermophila

Substrates Activity"

ATP, acetate' TTP, acetate" ITP, acetate" UTP, acetate' GTP, acetate" CTP, acetate' ATP, acetate' ATP, propionate' ATP. formate'

?6 100 106 83 80 79 53 100 60 0

The hydroxamate assay was used as described in the text except where the sodium salts of the indicated nucleotides (10 mM) replaced ATP. The reactions were initiated with 0.12 pg of enzyme. Activity with ATP (100%) was 412 pmol of acetyl phosphate/min/mg of protein. ' The enzyme-linked assay was used with potassium salts of the

acids at 200 mM. The reactions were initiated with 0.12 pg of enzyme. Activity with acetate (100%) was 569 pmol of ADP produced/min/ mg of protein.

0.0 1 .o 2.0 3.0

M ~ ~ + / A T P RATIO

FIG. 4. Effect of the M8+ concentration on the activity of acetate kinase from M. thermophila. The hydroxamate assay was used with a constant ATP concentration of 10 mM and an amount of MgCIz to provide the indicated ratio of Mg+:ATP. The reaction was initiated with 0.12 pg of enzyme. Maximal (100%) activity was 598 pmol of acetyl phosphate/min/mg of protein.

Substitution of Mn2+ for Mg?+ (hydroxamate assay) yielded a 30% increase in the rate of activity. Substitution with Co2+ or Ca2+ resulted in 30% of the activity with M C , while Cu2+, Ni2+, or Zn2+ resulted in no significant activity.

Methanogens contain intracellular concentrations of K+ that range between 144 and 780 mM (30). M. thermophila

FIG. 5. Western blot analysis of acetate kinase in extracts of M. thermophila grown on acetate or methanol. Lane A , purified acetate kinase (0.3 pg of protein); lane B, extract from acetate grown cells (5 pg of protein); lane C, extract from methanol grown cells (5 pg of protein); lane D, extract from methanol grown cells plus purified enzyme (5 and 0.3 pg of protein, respectively). The blots were probed with anti-acetate kinase antibody.

acetate kinase activity (enzyme-linked assay) was uneffected by omitting potassium from components of the assay mixture. These results do not exclude the possibility that trace amounts of K+ may be required for maximum activity. No stimulation of activity was obtained when KC1 was added to the assay mixture a t concentrations between 0 and 500 mM; instead, activity linearly decreased from 100% (at 0 mM added KC1) to 71% a t 500 mM added KCl.

Regulation-When M. thermophila is presented with both acetate and methanol, the latter is the preferred growth substrate. Methanogenesis from acetate is almost completely repressed in cells grown on methanol in the presence of acetate (31). However, acetate is used as a carbon source under these growth conditions (3). Thus, the regulation of acetate kinase by acetate or methanol was investigated. The specific activity in cells grown on methanol, in the presence of acetate, was 3.9 pmol of ADP formed/min/mg of protein. The activity in cells grown on acetate alone was 6.4 pmol of ADP formed/min/mg of protein (Table I). The previously reported (7) activity in M. thermophila grown on methanol alone (0.12 pmol of ADP formed/min/mg of protein) is sev- eralfold less than the activity reported here for either acetate grown cells or cells grown on methanol in the presence of acetate. These results suggest that acetate induces acetate kinase activity, but activity is not completely suppressed in cells grown on methanol in the presence of acetate; it is likely that other enzymes essential for catabolism of acetate to methane may be repressed by methanol. Although no conclu- sions can be drawn from the data presented, the results are

15448 Acetate Kinase from Methanosarcina

consistent with a dual function for acetate kinase in activation of acetate for both energy and cell carbon.

When equal amounts of protein from extracts of acetate or methanol grown cells were analyzed by Western blotting, the anti-acetate kinase antibody probe strongly reacted with a protein from acetate grown cells that migrated in gels with a M , corresponding to acetate kinase; however, no reaction with proteins from extracts of methanol grown cells was visible (Fig. 5). If the autoradiogram was overexposed, extracts of methanol grown cells showed only a weak reaction at the acetate kinase position (data not shown). These results sug- gest that the synthesis of acetate kinase in M. thermophila was regulated.

Physiological Function-The acetate kinase from M . ther- mophila is proposed to function in the initial activation of acetate for conversion to methane and COZ based on the following results. (i) The acetate kinase was highly enriched in extracts of acetate grown compared with methanol grown cells. (ii) No significant acetate-CoA ligase activity was de- tected in cell extracts. (iii) In combination with the phosphate acyltransferase previously reported to be present in extracts of acetate grown cells (7), acetate kinase is capable of synthe- sizing acetyl-coA, the proposed activated form of acetate converted to methane by this organism (7).

Acknowledgments-We thank Andrew Clements for assistance in the culturing of M. thermophila and Peter Jablonski for assistance with graphics.

1.

2. 3.

4.

5.

REFERENCES Fox, G. E., Stackebrandt, E., Hespell, R. B., Gibson, J., Maniloff,

J., Dryer, T. A., Wolfe, R. S., Balch, W. E., Tanner, R. S., Magnum, L. J., Zablen, L. B., Blakemore, R., Gupta, R., Boman, L., Lewis, B. J., Stahl, D. A., Luehrsen, K. R., Chen, K. N., and Woese, C. R. (1980) Science 209,457-463

Wolfe, R. S. (1985) Trends Biochem. Sci. 10, 396-399 Zinder, S. H., and Mah, R. A. (1979) Appl. Enuiron. Microbiol.

Lovley, D. R., White, R. H., and Ferry, J. G. (1984) J. Bacterid.

Nelson, M. J. K., and Ferry, J. G. (1984) J. Bacteriol. 160, 526-

38,996-1008

160,521-525

532

6. Terlesky, K. C., Nelson, M. J. K., and Ferry, J. G. (1986) J.

7. Terlesky, K. C., Barber, M. J., Aceti, D. J., and Ferry, J. G . (1987)

8. Ragsdale, S. W., and Wood, H. G. (1985) J. Biol. Chem. 260,

9. Eikmanns, B., and Thauer, R. K. (1984) Arch. Microbiol. 1 3 8 ,

10. Laufer, K., Eikmanns, B., Frimmer, U., and Thauer, R. K. (1987)

11. Grahame, D. A., and Stadtman, T. C. (1987) Bwchem. Biophys.

12. Sowers, K. R., Nelson, M. J., and Ferry, J. G. (1984) Curr.

13. Murray, P. A., and Zinder, S. H. (1985) Appl. Enuiron. Microbiol.

14. Allen, S. H. G., Kellermeyer, R. W., Stjernholm, R. L., and Wood,

15. Lipmann, F., and Tuttle, L. C. (1945) J. Biol. Chern. 159,21-28 16. Rose, I. A., Grunberg-Manago, M., Korey, S. R., and Ochoa, S.

17. Bowman, C. M., Valdez, R. O., and Nishimura, J . S. (1976) J.

18. Oberlies, G., Fuchs, G., and Thauer, R. K. (1980) Arch. Microbiol.

19. Laemmli, U. K. (1970) Nature 227,680-685 20. Rothe, G. M., and Purkhanbaba, H. (1982) Electrophoresis. 3,

21. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad.

22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 23. Baron, S. F., Brown, D. P., and Ferry, J. G. (1987) J. Bacteriol.

24. Vigenschow, H., Schwarm, H.-M., and Knobloch, K. (1986) Biol.

25. Nakajima, H., Suzuki, K., and Imahori, K. (1978) J. Biochem.

26. Brown, M. S., and Akagi, J. M. (1966) J. Bacteriol. 9 2 , 1273-

27. Kahane, I., and Muhlrad, A. (1979) J. Bacteriol. 137,764-772 28. Pelroy, R. A., and Whitely, H. R. (1971) J. Bacteriol. 105 , 259-

29. Anthony, R. S., and Spector, L. B. (1971) J. Biol. Chem. 2 4 6 ,

30. Sprott, G. D., and Jarrell, K. F. (1981) Can. J . Microbiol. 2 7 ,

31. Zinder, S. H., and Elias, A. F. (1985) J. Bacteriol. 163: 317-323

Bacteriol. 168, 1053-1058

J. Biol. Chem. 2 6 2 , 15392-15395

3970-3977

365-370

Z. Naturforsch. 42,360-372

Res. Commun. 147,254-258

Microbiol. 11 , 227-230

50,49-55

H. G. (1964) J. Bacteriol. 87, 171-187

(1954) J. Bwl. Chem. 211,737-755

Biol. Chem. 151,3117-3121

128,248-252

33-42

Sci. U. S. A. 76,4350-4354

169,3823-3825

Chem. Hoppe Seykr. 367,961-956

(Tokyo) 84,193-203

1274

267

6129-6135

444-451