J. Biochem. 105, 588-593 (1989)

Characterization of NADH Kinase from Saccharomyces cerevisiae

Yumiko Iwahashi, Akio Hitoshio,Nobuyuki Tajima, and Taro Nakamura1 Departmentof Agricultural Chemistry, Faculty of Agriculture, HokkaidoUniversity, Kita-ku, Sapporo, Hokkaido060 Receivedfor publication, October 14, 1988

At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mito chondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1,041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105ƒÊM and 2.1mM, respectively. The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.

In most organisms, NADPH formation occurs principally tion of 0.5%, supplemented by 2% sodium succinate hexa by reduction of NADP+, catalyzed by NADP+-linked hydrate. dehydrogenases or by energy-linked hydrogen transfer by Preparation of Spheroplasts-The cells grown in the means of NAD(P)+ transhydrogenase (1, 2). Diphos early logarithmic phase were harvested by centrifugation phopyridine nucleotide kinase [EC 2.7.1.23], which cata for 5min at 1,800•~g. After washing twice with deionized lyzes the synthesis of NADP+, was originally detected from water, and then once with 1.2M sorbitol buffered with 10 yeast autolyzates by Kornberg (3), who reported that the mM Tris-HCl, pH 7.4, the cell paste was suspended in the enzyme was able to phosphorylate both NAD+ and NADH. same buffer solution to give 0.05g wet weight of cells per Bernofsky and Utter (4) demonstrated that when NAD+ ml. To 100ml of cell suspension, 0.25ml of 2-mercapto was the substrate, NADP+ production occurred only to a ethanol and 5mg of Zymolyase 100,000 were added. The limited extent; with NADH, however, the reaction pro mixture was incubated for 1 h at 30•Ž with gentle shaking. ceeded to a much greater extent in baker's yeast. Although The suspension was centrifuged for 5 min at 3,000•~g, and they did not determine the actual products. Apps (5) then washed twice with buffered 1.2M sorbitol solution. reported that the mitochondrial fraction of Saccharomyces Through this treatment 80% or more of the cells were cerevisiae contained both NAD+ and NADH-phosphoryla recovered as spheroplasts. tion enzymes besides cytoplasmic NAD+-specific kinase, Subcellular Fractionation-The spheroplasts were sus and both of the mitochondrial enzymes showed non-linear pended in 0.6M mannitol buffered with 10mM Tris-HCl, Michaelis plots for ATP. NADH kinase of mitochondria pH 7.4 (mannitol-containing buffer), supplemented with [EC 2.7.1.89] was also purified 127-fold by Griffiths and 0.1% bovine serum albumin and 1mM phenylmethyl Bernofsky (6). However, the substrate specificity and the sulfonyl fluoride (PMSF), a proteinase inhibitor, and kinetic properties of these enzymes remained unclear. In homogenized in Potter-Elvehjem glass-Teflon homogenizer the present paper we report on the detailed enzymatic by repeated up-and-down strokes. The homogenate was properties of NADH kinase, and compare them with those employed for fractionation of the cell components. To of cytosolic and microsomal NAD+ kinase. minimize damage to organelles all subsequent manipula tions were carefully done at 0-4•Ž.

MATERIALS AND METHODS i) Preparation of mitochondrial fraction: The super natant solution was freed from large particles by centrif Yeast Strain and Cultural Conditions-Saccharomyces ugation for 5 min at 1,000•~g. The resulting supernatant cerevisiae X2181-1A (Yeast Genetic Stock Center, the solution was saved for fractionation of cytosol and mi University of California, Berkeley, Calif.) was used, unless crosomes. The mitochondria) pellets were carefully resus otherwise specified. It has the genotype his2 gall trp1 pended in the mannitol-containing buffer and washed twice ade1. The medium for preculture contained 1% yeast by repeated centrifugations. The mitochondria were finally extract, 0.5% peptone, 0.9% KH2PO4, 0.033% CaC12•E suspended in mannitol-containing buffer to give approxi 2H2O, 0.05% MgSO4•E7H2O, 0.6% (NH4)2SO4, and 2% mately 20mg of protein per ml, and were referred to as mitochondrial fraction. glucose. An aerobically grown inoculum (for 24h at 30•Ž) was transferred to fresh medium with a glucose concentra ii) Preparations of microsomes and cytosol: The super natant solution (20min, 20,000•~g) free of mitochondria was centrifuged for 60min at 105,000•~g. The resulting 1 To whom correspondence should be addressed. supernatant solution was used as cytosol fraction. The Abbreviations: PMSF, phenylmethylsulfonyl fluoride; NEM, N ethylmaleimide. pellets were resuspended in the above described homoge

588 J. Biochem. Characterization of NADH Kinase from Yeast 589

nization buffer, and the larger particles in the suspension reaction mixture consisted of 90mM Tris-HCl buffer, pH were removed by centrifugation for 20min at 45 ,000•~g. Af 7.8, 2mM NADH, 3mM ATP, 10mM MgO2, 200mM ter washing twice with mannitol-containing buffer , the fi sodium acetate, 0.1% bovine serum albumin, and an nal microsomal preparation was suspended in the same appropriate amount of the enzyme in a final volume of 1.0 buffer to give a concentration of about 15 mg of protein per ml. For measurement of the activity of crude enzyme ml. preparations an ATP-generating system consisting of 10 Cell Disruption with French Pressure Cell-In the later mM phosphoenolpyruvate, 10mM KCL, and 5 units of experiments for preparation of large amounts of the pyruvate kinase was introduced into the assay mixtures, mitochondria, pressed baker's yeast was suspended in an and the reaction was conducted in Thunberg tubes under an equal volume (w/v) of mannitol-containing buffer sup N2 atmosphere. Reactions were started by introduction of plemented with 0.1% bovine serum albumin and 1mM the enzyme solution and were allowed to proceed for 20min PMSF. The suspension was disrupted by passing it through at 30•Ž. The reactions were terminated by addition of 2.0 a French pressure cell at 1,100kg/cm2, and the turbid fluid ml of 0.15M NaOH, followed by heating for 5min at 60•Ž was centrifuged at 1,000•~g for 5min. The supernatant with occasional stirring. After chilling in ice-cold water, 2.0 fluid freed from unbroken cells and large debris was further ml of 0.22M Tris-HCl buffer, pH 7.8, containing 0.275M fractionated as described in the procedure for preparation triethanolamine-HCl adjusted in advance to pH 7.8 with of mitochondria from spheroplasts. 1.5M NaOH, was added. To measure NADPH, 0.2ml of 3 Purification of NADH Kinase-The purification of mM N-ethylphenazinium ethylsulfate was added to the NADH kinase was done at 0 to 4•Ž except for the solubiliza final mixtures, and the mixtures were aerated in a vortex tion. mixer for 90 s to ensure complete oxidation of pyridine i) Solubilization: The mitochondrial pellets (about 3.4 nucleotides. After centrifugation at 12,000•~g for 5min, mg of protein) were suspended in a medium consisting of residual phenazium salt in the supernatant was decom

0.5M sucrose, 10mM Tris-HCl, pH 7.8, and 1mM EDTA, posed by exposure to sunlight or fluorescent light until at a final protein concentration of 20mg per ml and placed decolorization was completed. The recovery (80%) of in a water-jacketed beaker maintained at 30•Ž so as not to NADPH was taken into account in the calculation of the solidify Lubrol PX, which was added to the suspension to activity.

give a final concentration of 1% with vigorous stirring. For the determination of NADP+, the cycling assay After further stirring for 60min, the suspension was system consisting of 3-(4,5-dimethyl-2-thiazolyl)-2,5-di chilled and dialyzed against 20 volumes of a medium phenylterazolium and isocitrate dehydrogenase (NADP+) consisting of 20mM Tris-HCl, pH 7.8, 20mM MgCl2 and 1 [EC 1.1.1.42] was employed according to Jacobson and mM EDTA for 3h. In accordance with Griffiths and Jacobson (7). Bernofsky (6) the purification was routinely carried out in Assay of NAD+ Kinase-The activity of NAD+ kinase the presence of Mg2+. was estimated by the procedure described by Tseng et al. ii) Matrex Gel Green A column chromatography: The (8) with the following modifications. To terminate the supernatant obtained by centrifugation of the dialysate at reaction, the mixtures, in 1.0ml, were acidified with 0.4ml 20,000•~g for 30min, was applied to a Matrex Gel Green A of 2M HClO4 and stirred for 2min at 30•Ž, followed by (1.7•~25cm) column equilibrated with 20mM Tris-HCl, neutralization with 0.4ml of 2M KOH in 0.5M Tris maleate buffer, pH 7.4. The concentration of NADP+ was pH 7.8, containing 20mM MgCl2 and 1mM EDTA. After washing with 100ml of the same buffered solution, the determined by the cycling assay method described by enzyme was eluted with 600ml of a linear gradient of 0 to Jacobson and Jacobson (7). 1.0M KCl in the same solution at a flow rate of 0.2ml/min. Determination of Relative Molecular Mass-For estima Active fractions eluted at about 320mM KCl were pooled. tion of relative molecular mass a Sephacryl S-300 column iii) Hydroxylapatite column chromatography: The en (1.8•~160cm) was equilibrated and developed with 20mM zyme solution from the former step was applied to a Tris-HCl, pH 7.8, buffer containing 20mM MgO2, 1mM hydroxylapatite column (1.7•~10cm) equilibrated with 20 EDTA and 0.2M ammonium sulfate. Following calibration mM Tris-HCl, pH 7.8, containing 1mM EDTA. After of the column with standard proteins, NADH kinase was washing with 100ml of the same buffered solution, the chromatographed at a flow rate of 12ml/h. The void enzyme was chromatographed with 600ml of a linear volume was determined with blue dextran 2000. Two milliliter fractions were collected, and protein peaks were gradient made with 0 to 500mM potassium phosphate located by measuring the absorbance at 280nm. buffer, pH 7.4, at a flow rate of 0.1ml/min. Active frac tions eluted at about 230mM phosphate were pooled and Measurement of Kinetic Parameters-Cleland's proce concentrated with a collodion bag. dure (9) was adopted for the calculation of the steady-state kinetics of the reaction of NADH kinase. Initial velocities iv) Sepharose CL-6B gel filtration: The concentrated were measured with either NADH or ATP as the variable enzyme solution was chromatographed on a column of substrate with three fixed concentrations of the other in the Sepharose CL-6B (1.4 x 100 cm) equilibrated with a medium consisting of 20mM Tris-HCI, pH 7.8, 20mM presence of a constant level of 10mM Mg2+. Chemical Modification of NADH Kinase and Protection MgC12 and 1mM EDTA. The elution was performed with -Kinetic studies on inactivation were carried out as the same buffered solution at a flow rate of 0.5ml/min. The follows. Various concentrations of N,N-dimethylformamide active fractions were used in most experiments and re was added to 50ƒÊl of NADH kinase, and the mixture was ferred to as the purified enzyme preparation. incubated at 0•Ž. The remaining enzyme activity in the Assay of NADH Kinase-The activity of NADH kinase mixtures was measured at intervals by adding 0.445ml of was estimated by the procedure described by Griffiths and an assay mixture consisting of 90mM Tris-HCl, pH 7.8, 2 Bernofsky (6) with some modifications as follows. The

Vol. 105, No. 4, 1989 590 Y. Iwahashi et al.

mM NADH, 3mM ATP, 10mM MgCl2 , 0.1M sodium oxidase for mitochondria (cf. Ref. 15 for review), and acetate, and 1% bovine serum albumin. To estimate the NADPH-cytochrome c reductase for microsomes (12). As site(s) of modification on the enzyme protein by SH reagent shown in Table I, good separation of each fraction was the inactivation by iodoacetic acid was carried out with or achieved. To avoid underestimation of the activities of without protection with either substrate. Five microliters enzymes located in the mitochondria due to the appreciable of a solution of 800mM iodoacetic acid in 100mM BICINE permeability barrier of the mitochondrial inner membrane buffer, pH 8.0, was added to 50ƒÊl of NADH kinase sup to pyridine nucleotide coenzymes (16-18), sonicated plemented with 5ƒÊl of 800mM NADH or ATP with MgCl2. mitochondrial preparations were used. To eliminate an After incubation for the desired time at O•Ž, 0.44ml of the inhibitory factor to NAD+ kinase which is present in assay mixture omitting the preexisting substrate(s) was cytosolic fraction (3), the fraction precipitated by salting added, and the remaining activity was measured. out with 60%-saturated ammonium sulfate was employed Miscellaneous-Published methods were used for the for NAD+ kinase determination. determinations of glucose-6-phosphate dehydrogenase [EC As shown in Table I, NADH kinase existed exclusively in 1.1.1.49] (10), cytochrome c oxidase [EC 1.9.3.1] (11), the mitochondrial fraction, which is practically free of NADPH-cytochrome c reductase [EC 1.6.2.4] (12). Pro contamination from other fraction. NAD+ kinase was, on tein content was determined by the method of Lowry et al. the other hand, entirely absent from the mitochondrial (13) with bovine serum albumin as the standard. fraction, and was distributed in the microsomal and the One unit of enzyme activities was defined as the amount cytosol fractions in almost equal amounts. After thorough of each enzyme necessary to form 1ƒÊmol of the product or washings with 150mM KCl buffered with 10mM Tris to consume 1ƒÊmol of substrate in 1 min under the condi - HCl, pH 7.4, the activity of the microsome-bound NAD+ tions employed. The specific activity was expressed as unit kinase was maintained without measurable loss. Quite sim of enzyme per mg of protein. ilar distribution patterns of both enzymes were observed Chemicals-NAD+, NADH, NADP+, ADP, ATP, glu with S. cerevisiae ATCC 38521 and STX185-14D-12C and cose-6-phosphate dehydrogenase, hexokinase, and isocit also Kluyveromyces lactis IFO 1267 (data not shown). rate dehydrogenase (NADP+) were obtained from Oriental Purification of NADH Kinase-Commercially grown Yeast, Tokyo. PMSF and p-hydroxymercuribenzoate were baker's yeast (Oriental Yeast) was used for large-scale from Sigma Chemical, St. Louis, Mo., U.S.A. Zymolyase purification of NADH kinase. The yield of the mitochon 100,000 was from Seikagaku Kogyo, Tokyo. Horse-heart drial preparation prepared by cell disruption with a French cytochrome c (Type IV) and antimycin A were products of pressure cell was considerably lower than that from proto Boehringer Mannheim, GmbH, Mannheim, F.R.G. N plast lysate. Representative results of the purification from Ethylphenazinium ethylsulfate and Lubrol PX were pur mitochondrial fraction which was obtained from 100g of chased from Nakarai Chemical, Kyoto. Enzymobeads were fresh yeast cells are given in Table II. The enzyme was obtained from Bio-Rad Laboratories. Matrex Gel Green A purified 1,041-fold with a recovery of 4.3%. The final was a product of Amicon, Lexington, Mass., U.S.A. preparation showed a major band on native polyacrylamide gel electrophoresis (Fig. 1). NADH kinase activity assayed RESULTS from the companion gel was located in the slices corre sponding to the major protein band in the stained gel. A Subcellular Distribution of NADH Kinase-To clarify faster moving protein band occasionally emerged. It prob the intracellular localization of NADH kinase, the cell ably represents a degradative product of the NADH kinase, constituents were fractionated to mitochondria, mi because it reacted with the antiserum against NADH crosomes, and cytosol. The following marker enzymes were kinase, although it was enzymatically inactive (data not chosen for estimation of cross-contamination: glucose-6 shown). phosphate dehydrogenase for cytosol (14), cytochrome c Physical and Chemical Properties-The purified enzyme

TABLE I. Intracellular distribution of NADH kinase and NAD+ kinasea

aFreshly harvested cells, about 1.3 g wet weight, were employed for this series of experiments, and the values given are from three repeated experiments. A, units; B, units per mg protein.

TABLE II. Purification profile of NADH kinase.

J. Biochem. Characterization of NADH Kinase from Yeast 591

Fig. 1. Slab polyacrylamide gel electrophoresis of NADH kinase in the absence of sodium dodecyl sulfate; 1ƒÊg of the enzyme was electrophoresed in 5.6% gel.

Protein was located by the silver staining method (24) .

Fig. 3. Lineweaver-Burk plots for NADH kinase. The assays were performed as described in the text. (A) Effect of NADH concentration on the specific activities at three fixed concentrations of Fig. 2. Determination of rel ATP. Symbols: •¡, 500ƒÊM; •£, 1mM; •œ, 3mM. (B) Effect of ATP ative molecular mass by mole concentration on the specific activities at three fixed concentrations of cular sieve chromatography NADH. Symbols: •¡, 50ƒÊM; •£, 100ƒÊM; •œ, 2mM. on Sephacryl S-300. Standard proteins were A, ferritin (horse liver, Mr 440,000); B, NADH kinase; C, glucose-6-phosphate dehydrogenase (yeast, Mr 100,000); D, isocitrate dehy drogenase (yeast, Mr 60,000).

TABLE III. Substrate specificity of NADH kinase.

a NADH or NAD+ was added at 2mM, and ATP or ADP was added at

3mM final concentration. a After preincubation of the enzyme solution with the compound at the indicated concentration at 0•Ž for 1 h the enzyme activity was measured. b3-Acetylpyridine adenine dinucleotide. solution in 20mM Tris-HCl buffer, pH 7.8, containing 0.2 M ammonium sulfate, 20mM MgCl2, and 1mM EDTA could be stored at 0-5•Ž for 7 d, or at -30•Ž for 1 mo or strictly specific for NADH, and NAD+ was completely more. Ammonium sulfate was added as an enzyme ineffective. The absence of NAD(P)+ transhydrogenase stabilizing agent (6). This enzyme solution (20ƒÊg protein/ activity [EC 1.6.1.1] was confirmed by assay with NADH ml) is, however, considerably thermolabile. It was inacti and 3-acetylpyridine adenine dinucleotide according to the

vated about 65% at 35•Ž for 5min, and 95% of the enzyme procedure of Stein et al. (2). The maximum activity was activity was lost at 55•Ž for 5min. These characteristics observed with 4 to 5mM ATP and equimolar MgCl2. An are roughly consistent with the descriptions of Apps (5) and addition of more than 6mM ATP inhibited NADH kinase Griffiths and Bernofsky (6). activity, and approximately 70% inhibition was observed at The relative molecular mass of NADH kinase was 10mM ATP. The efficiency of ADP was 1.7%, compared estimated to be approximately 160,000 by means of with that of equimolar ATP (Table III). molecular sieve chromatography on a calibrated column of Kinetic Parameters-With three fixed concentrations of Sephacryl S-300 (Fig. 2). Standard proteins were ferritin ATP, linear double-reciprocal plots were obtained which (Mr 440,000), glucose-6-phosphate dehydrogenase gave a Km value of 105ƒÊM for NADH (Fig. 3A). When the (100,000), and isocitrate dehydrogenase (60,000). The void ATP concentrations were varied at three fixed concentra volume of the column was determined with blue dextran tions of NADH, double-reciprocal plots were also linear, 2,000. From polyacrylamide gel electrophoresis in the giving a Km value of 2.1mM for ATP (Fig. 3B). Each Km presence of sodium dodecyl sulfate we obtained prelimi value was independent of the concentration of the second nary results which were suggestive of the presence of 2 or substrate. The maximum velocity was calculated as 41.7 more subunits of different size. units/mg protein. Contrary to the description of Apps (5), Dependence on pH-To determine the pH dependence of no sigmoidal curve of velocity against concentration of ATP the enzyme activity, we replaced the standard buffer with was obtained. three different buffers at a final concentration of 200mM: Effectors of the Activity of NADH Kinase-The effects Tris-maleate, pH 6.0 to 7.6; Tris-HCl, pH 7.0 to 9.6; of inhibitors on the activity of NADH kinase are summa glycine-KOH, pH 9.0 to 11.0. The optimum pH for NADH rized in Table IV. The involvement of an SH group in the kinase activity was found at about pH 8.5. activity was suggested by the inhibitory effect of 5mM Substrate Specificity-The purified NADH kinase was iodoacetic acid, NEM, and p-chloromercuribenzoic acid. An

Vol. 105, No. 4, 1989 592 Y. Iwahashi et al.

Fig. 4. Plot of pseudo-first order rate constant ƒÒs. N-ethyl maleimide concentration for the inactivation of NADH kinase by N-ethylmaleimide.

Fig. 5. Protection against iodoacetate inhibition by NADH. Details of the experimental conditions were described in the text. addition of 30mM phosphoenolpyruvate lowered the activ Symbols: •›, with 8mM NADH; •œ, without NADH. ity to 43% of that shown with the standard assay mixture, but no effect was observed with pyruvate. Introduction of an ATP-generating system into the NADH kinase-assay system brought about underestimation of the activity. This DISCUSSION may be attributed to the inhibitory effect of phospho enolpyruvate. Sodium acetate significantly activated the From studies of the subcellular distribution of phospho NADH kinase, as reported by Griffiths and Bernofsky (19). rylation activities of nicotinamide adenine dinucleotide, it We extracted an activating factor from the mitochondrial is important to emphasize that yeast mitochondria contain matrix, and it stimulated NADH kinase by 2.0 to 2.5 times. exclusively NADH kinase and the microsomal fraction The physiological significance of the activating factor, contains a comparable amount of NAD+ kinase to the however, is uncertain. No influence on the NADH kinase cytosol. This is contrary to previous reports which claimed was observed by addition of 3mM NAD+ kinase from yeast that two different kinase activities phosphorylating NAD+ (5) and other sources (20, 21). Eighty-three percent of the and NADH existed in yeast mitochondria (4). Griffiths and full activity was attained in the presence of 3-acetyl Bernofsky (6) described that partially purified mitochon pyridine adenine dinucleotide, an NAD analogue. drial enzyme is highly specific for NADH. These contro Kinetics of Inactivation by NEM-The time course of versies can be attributed to contamination with mi inactivation of the NADH kinase with NEM was followed crosomes and/or cytosol, e.g. NAD+ kinase in mitochon by a discontinuous assay method (29). The mixture for drial fraction, or to the contributions of NADH and NADPH inactivation consisted of 5ƒÊl of 50, 100 or 500mM NEM oxidase activities present in mitochondria (22). Detailed solution and 50ƒÊ1 of the enzyme solution, and was incubat studies on the intramitochondrial localization of NADH ed at 30•Ž. At various intervals, a 5ƒÊl aliquot was kinase will be presented elsewhere (Y. Iwahashi and T. withdrawn and the remaining activity was determined. Nakamura, manuscript submitted). Because the NEM-treated enzyme solution was diluted Activity of NAD+ kinase was found to be present in 100-fold when introduced into the assay mixture, the cytosolic fraction of pigeon liver (23), spinach (21), and rat inactivation of the NADH kinase by NEM during the assay liver (25), but it was found in nuclei of dog thyroid (26), was ignored. The enzyme activity was plotted on a logarith and in chloroplasts from various plants (27). In S. cer mic scale vs. time. The rate constant for inactivation, kobs, evisiae, however, a bimodal distribution, in cytosol and was determined from the slope of the first-order plot (data mitochondria, of NAD+ kinase activity was reported (28). not shown). The values of pseudo-first-order rate constant The localization of this enzyme in microsomes has not been obtained with three different NEM concentrations are reported previously. Our results, however, show that a plotted in Fig. 4. The second-order rate constant for the comparable amount of NAD+ kinase is present in mi inactivation was determined as 4.88mM-1 min-1. crosomal fraction with 3.5 to 9.5 times higher specific Similar inactivation was observed with N-acetylimida activity than that of cytosol fraction, depending on the zole and by iodination (data not shown). It seems reason strain. It is unlikely that the NAD+ kinase activity detected able to conclude, therefore, that a tyrosine residue is in microsomal fraction is artifact due to non-specific ad involved as a functional group of NADH kinase. sorption of cytosolic enzyme, since the cross-contamination Protection against Inactivation by Substrate-Figure 5 of microsomes and cytosol indicated by marker enzymes shows the protective effects of substrates against inactiva was low, and furthermore, no release of the microsomal tion by 8mM iodoacetic acid. NADH kinase was 57% enzyme was observed on repeated washing with 150mM KCl. It is not yet known whether the NAD+ kinase protected against the inactivation by the coexistence of 8 mM NADH. An addition of 8mM ATP was, on the other molecules detected in the two fractions are isozymes. hand, completely ineffective on NEM inhibition, whereas Michaelis constants of NADH kinase were measured by essentially the same protective effect as above was ob the procedure of Cleland (9). The Km values for NADH of served with NADH (data not shown). These results strong NADH kinase were determined in the presence of three ly suggest that a cysteine residue is located at or around the different concentrations of ATP, and vice versa. The Km NADH binding site. value for NADH, 105ƒÊM, is considerably higher than the apparent Km values, 52 and 42ƒÊM, reported by Apps (5) and Griffiths and Bernofsky (6), respectively. Compared

J. Biochem. Characterization of NADH Kinase from Yeast 593 with the reported values (5), the Km for ATP, 2.1mM, was (1951) J. Biol. Chem. 193, 265-275 8.4 or 2.1-fold (6) higher. 14. Newburgh, R.W. & Cheldelin, V.H. (1956) J. Biol. Chem. 218, 89-96 15. Schatz, G. & Mason, T.L. (1974) Annu. Rev. Biochem. 43,51-87 We wish to thank K. Fujiwara for help at various stages of this work. 16. Birt, L.M. & Bartley, W. (1980) Biochem. J. 75, 303-315 17. Purivs, J.L. & Lowenstein, J.M. (1961) J. Biol. Chem. 236, REFERENCES 2794-2803 18. von Jagow, G. & Klingenberg, M. (1970) Eur. J. Biochem. 12, 1. Ernster, L. & Lee, C.P. (1964) Annu. Rev. Biochem. 33, 729-788 583-592 2. Stein, A.M., Kaplan, N.O., & Ciottin, M.M. (1959) J. Biol. Chem. 19. Griffiths, M.M. & Bernofsky, C. (1970) FEBS Lett. 10, 97-100 234,979-986 20. Apps, D.K. (1975) Eur. J. Biochem. 55, 475-483 3. Kornberg, A. (1950) J. Biol. Chem. 182, 805-813 21. Yamamoto, Y. (1966) Plant Physiol. 41, 523-528 4. Bernofsky, C. & Utter, M.F. (1968) Science 159, 1362-1363 22. Djavadi, F.H.S., Moradi, M., & Djavadi-Ohaniance, L. (1980) 5. Apps, D.K. (1970) Eur. J. Biochem. 13, 223-230 Eur. J. Biochem. 107, 501-504 6. Griffiths, M.M. &Bernofsky, C. (1972) J. Biol. Chem. 247,1473 23. Wang, T.P. & Kaplan, N.O. (1954) J. Biol. Chem. 206, 311-325 -1478 24. Merril, C.R., Switzer, R.C., & Van Keuren, M.L. (1979) Proc. 7. Jacobson, E.L. &Jacobson, M.K. (1976) Arch. Biochem. Biophys. Natl. Acad. Sci. U.S. 76, 4335-4339 175,627-634 25. Yero, I.L., Farinas, B., & Dietrich, L.S. (1968) J. Biol. Chem. 8. Tseng, Y.M., Harris, B.G., & Jacobson, M.K. (1979) Biochim. 243,4885-4888 Biophys. Acta 568, 205-214 26. Field, J.B., Epstein, S.M., Remer, A.K., & Boyle, C. (1966) 9. Cleland, W.W. (1970) in The Enzymes (Boyer, P.D., ed.) 3rd Ed., Biochim. Biophys. Acta 121, 241-249 Vol. 8, pp. 1-65, Academic Press, New York 27. Muto, S., Miyachi, S., Usuda, H., Edwards, G.E., & Bassham, 10. Noltmann, E.A., Gubler, C.J., & Kuby, S.A. (1961) J. Biol. J.A. (1981) Plant Physiol. 68, 324-328 Chem. 236, 1225-1230 28. Middleton, B. & Apps, D.K. (1969) Biochim. Biophys. Acta 177, 11. Mason, T.L., Poyton, R.O., Wharton, D.C., & Schatz, G. (1973) 276-285 J. Biol. Chem. 248,1346-1354 29. Anderson, B.M. & Vasini, E.C. (1970) Biochemistry 9, 3348 12. Lu, A.Y.H., Junk, K.W., & Coon, M.J. (1969) J. Biol. Chem. - 3352 244,3714-3721 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J.

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