The Binding of NADH to Succinic Semialdehyde Dehydrogenase

Transcription

1 Eur. J. Biochem. 109, (1980) by FEBS 1980 The Binding of NADH to Succinic Semialdehyde Dehydrogenase William S. BLANER and Jorge E. CHURCHICH Department of Biochemistry, University of Tennessee, Knoxville (Received November 13, 1979/April 9, 1980) The enzyme succinic semialdehyde dehydrogenase from pig brain is a tetramer composed of identical subunits of molecular weight Fluorometric titrations conducted on samples of enzyme in pyrophosphate buffer (ph 8.4) reveal the presence of two NADH binding sites characterized by a dissociation constant of 4 pm. The unusual stoichiometry of binding of NADH, i.e. two moles NADH/enzyme tetramer, as demonstrated by emission anisotropy measurements is not due to reversible association-dissociation of the oligomeric structure at ph 8.4. The results of the fluorometric titrations are consistent with a model of extreme negative cooperativity, i.e. the affinity of NADH for the enzyme is influenced by interactions between the protomers. The reaction catalyzed by succinic semialdehyde dehydrogenase is inhibited by the substrate succinic semialdehyde with a K, of 0.09 mm which is fivefold greater than the K, value. The binding of excess substrate to the catalytic site provides a direct and simple mechanism for regulation of the rate of product formation. Succinic semialdehyde dehydrogenase, the enzyme which catalyzes the conversion of succinic semialdehyde to succinic acid, has been isolated from bacteria and mammalian tissues [1,2]. More recently, a report on the purification to homogeneity and characterization of the enzyme from pig brain has appeared [3]. The reaction catalyzed by succinic semialdehyde dehydrogenase from brain tissues is essentially irreversible; steady-state kinetic studies designed to elucidate the mechanism of actin of succinic semialdehyde dehydrogenase have provided information on the order of substrate addition during catalysis [3]. The enzyme is characterized by a molecular weight of [3]. Although there is strong evidence supporting the contention that the enzyme from brain tissue is composed of several subunits [2], the quaternary structure of this protein has not been investigated in detail. Furthermore, there is insufficient information about the interaction of the enzyme with the cofactor NADH and the substrate succinic semialdehyde which are known to exert inhibitory effects at concentrations below 0.1 mm. In the present paper, the specificity of the reaction catalyzed by succinic semialdehyde dehydrogenase is investigated in detail using several semialdehyde substrates. We also examined binding of NADH by the Enzymes. Succinic semialdehyde dehydrogenase (EC ); 4-aminobutyrate aminotransferase (EC ). enzyme in pyrophosphate buffer under conditions where the oligomeric structure of succinic semialdehyde dehydrogenase is known. The results of fluorometric titrations are consistent with a model of extreme negative cooperativity, i.e. the affinity of NADH for the enzyme is influenced by interactions between the protomers. In agreement with other studies [2], we show that excess substrate (succinic semialdehyde) exerts its inhibitory effect by interacting with the catalytic site of the enzyme. EXPERIMENTAL PROCEDURES Enzymes Succinic semialdehyde dehydrogenase from pig brain was purified according to a procedure previously described [3]. The enzyme has a specific activity of 5.3 units/mg. 4-Aminobutyrate aminotransferase from pig brain was purified following the procedure of Beeler and Churchich [4]. The specific activity of the preparation is approximately 6 units/mg. Protein concentration was determined by Lowry s method [5]. Enzymatic Assay For precise kinetic data, the formation of NADH was measured by following the increase in absorbance

2 + 432 NADH Binding to Succinic Semialdehyde Dehydrogenase at 340 nm at which wavelength NADH is known to have a molar absorption coefficient of 6.22 cmpl mmpl. All assays were performed in duplicate and the initial velocity data were correlated with a standard assay mixture containing 50 pm succinic semialdehyde and 5 mm NAD' in 0.1 M sodium pyrophosphate (ph 8.4) at 25 C. Initial rate measurements were carried out by monitoring the change in absorbance at 340 nm for at least 2 min, good straight lines being generally obtained. The initial velocity data were fitted by a least-square method to the Lineweaver-Burk transformation of Eqn (1) Z' = V [S]/'Km + [S] (1) where [S] represents the concentration of the varied substrate, the other substrate being present in fixed amounts. Inhibition constants for substrate inhibition were determined from Eqn (2) 1) = V Km 1 + ~~ [S] [SI Ki where Ki is the inhibition constant for the substrate inhibitor and [S] represents the concentration of substrate. When [S] is small, Eqn (2) is equivalent to Eqn (1). At large substrate concentrations, inhibition takes place and Eqn (3) can be written as V The value of K, can be determined from the slope of a plot of reciprocal velocity versus substrate concentration. A coupled assay system consisting of two purified enzymes, i.e. 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, was used to study the catalytic conversion of 4-aminobutyrate and 5-aminovalerate to succinic and glutaric acids, respectively. Enzymatic assays were performed in 0.1 M sodium pyrophosphate (ph 8.4) containing 1 mm 2-mercaptoethanol, 5 mm NAD+, 5 mm 2-oxoglutarate and 10 mm 4-aminobutyratc. The progress of the reaction was followed in a double-beam spectrophotometer by monitoring changes in absorbance at 340 nm. The rate of the reaction catalyzed by the primer enzyme (aminotransferase), uprim, is related to the maximum velocity of the second enzyme (inicator), Vind, by the following equation [6] : (2) (4) where Q is the ratio Km/[SIs, K,,, being the Michaelis constant of the substrate (succinic semialdehyde) for succinic semialdehyde dehydrogenase and [S], the steady-state concentration of succinic semialdehyde generated during the consecutive reactions. Polyacrylumide Gel Electrophoresis The enzyme preparations were examined by polyacrylainide gel electrophoresis at ph 8.4 according to the original procedure of Davies [7]. Sodium dodecyl sulphate electrophoresis on polyacrylamide gels containing 7.5 % polyacrylamide, 1 % 2-mercaptoethanol and 0.1 sodium dodecyl sulphate was performed according to the method of Weber and Osborn [8]. Electrophoresis was conducted at 25 "C in a Buchler analytical disc electrophoresis apparatus regulated at 8 ma per tube. Protein bands were detected by staining with Coomassie blue dye for an hour and subsequently destained overnight in a solution containing 10 :; methanol and 7% acetic acid in water. The four protein bands used as standards in the determination of molecular weight were bovine serum albumin, ovalbumin, pepsin and lysozyme. Analytical isoelectric focusing on 7.5 '4 polyacrylamide gels containing 8 M urea, 17; 2-mercaptoethanol and 0.4% ampholytes (ph range 3-10) was conducted at 25 "C for 24 h in a Buchler analytical disc electrophoresis apparatus. The gels were stained in a solution of isopropanol/acetic acidlwater (90 : 30 : 30) containing 0.2 %, bromophenol blue. The samples were destained for 48 h in a solution containing isopropanol/acetic acid/water (10: 10 : 80). Gel Chromatography The effect of the ligand NADH on the molecular size of succinic semialdehyde dehydrogenase was tested by gel filtration on a column of Sephacryl S-200 (55 x 0.9 cm) equilibrated with 0.1 M sodium pyrophosphate (ph 8.4) containing 2-mercaptoethanol (1 mm) and NADH (0.08 mm) (buffer I). A sample of enzyme (0.5 ml) at a concentration of 0.5 mg/ml was applied to the column, eluted with buffer I, and the fractions collected (1 ml) were assayed for enzymatic activity and tested for protein fluorescence. The elution profiles of the enzymes beef heart catalase, yeast alcohol dehydrogenase, beef heart malate dehydrogenase, rabbit muscle lactate dehydrogenase, and pig brain succinic semialdehyde dehydrogenase were measured on the same column equilibrated with 0.1 M sodium pyrophosphate (ph 8.4) containing 2-mercaptoethanol (1 mm) (buffer 11). Fluorescence Spectroscopy Polarization of fluorescence measurements were performed in an instrument previously described [9]. Illumination was provided by a xenon lamp (150 W)

3 W. S. Blaner and J. E. Churchich 433 with wavelengths selected by a quartz prism monochromator. The band-width for excitation was 5 nm. Fluoresence polarized light was passed through a C-S-3-72 Corning glass filter. The degree of polarization of fluorescence (P) was measured with a precision of f The emission anisotropy was calculated using Eqn (5): - 2P A = 3-P (5) Fluorescence emission spectra were obtained in a spectrofluorimeter equipped with two Bausch and Lomb monochromators. The slits of the monochromators were set to give a band width of 3 nm. Spectrophotometric measurements were carried out in either a Cary model 15 or an Aminco DW-2 double-beam spectrophotometer. Muterials Glutaric semialdehyde was prepared according to the procedure of Chang and Adams [lo] using 2-aminoadipic acid as starting material. It was separated from unreacted 2-amino-adipic acid by chromatography through Dowex 50x3. The 2,4-dinitrophenylhydrazone derivative of glutaric semialdehyde gives a single spot (RF = 0.55) on silica gel plates developed with the solvent mixture isopropanol/h20/ammonia (120 : 20 : 10). AMP-Sepharose and DEAE-cellulose were purchased from Pharmacia; NADH, NAD+, 4-aminobutyrate, 2-oxoglutarate and succinic semialdehyde from Sigma. RESULTS Substrate Specificit-v It has been previously [3] with a purified preparation of succinic semialdehyde dehydrogenase from pig brain, that the steady rate of succinic semialdehyde oxidation is sensitive to ph, and maximum initial velocity values are observed over the ph range Therefore, most of the initial velocity studies designed to investigate the ability of the enzyme to catalyze the oxidation of other substrates were performed at ph 8.4 using 0.1 M sodium pyrophosphate buffer. The results obtained with the three semialdehydes tested, i.e. inalonic semialdehyde, succinic semialdehyde and glutaric semialdehyde, are included in Table 1. From these results, it may be seen that succhic and glutaric semialdehydes are substrates of the enzyme, whereas malonic semialdehyde is not oxidized to malonic acid. Double-reciprocal plots of initial velocity versus NAD' or semialdehyde concentrations gave rise to an intersecting pattern of straight lines which are Table 1. Kinetic constants for succinic semialdehyde dehydrogenase Values of V are given relative to that for succinic semialdehyde as substrate Substrate PM NAD Succinic semialdehyde Glutaric semialdehyde Malonic semialdehyde - - Table 2. Determination of the steady-stare concentrations ofsic(.( init' semialdehyde in a coupled assay system The concentration of 4-aminobutyrate aminotransferase was kept constant at 3 nm. [S], = steady-state concentration of succinic semialdehyde; Q = Km/[SIr where K, is the Michaelis constant of succinic semialdehyde for succinic semialdehyde dehydrogenase Dehydrogenasel [SlS Q transaminase ILM xx o consistent with either a sequential ordered or a rapid random equilibrium mechanism for addition of the substrates. A coupled assay system consisting of two purified enzymes, 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, was used to study the catalytic conversion of 4-aminobutyrate and 5-aminovalerate to succinate and glutarate, respectively. Since the turnover of the transaminase and dehydrogenase are nearly the same at ph 8.4, and since succinic semialdehyde acts as an inhibitor of the dehydrogenase at concentrations greater than 0.05 mm, it is important to ensure that succinic semialdehyde accumulation does not take place during the enzymatic reactions. Inhibition by succinic semialdehyde is prevented by using concentrations of dehydrogenase at least fivefold greater than the concentration of aminotransferase. Under this set of experimental conditions the concentration of succinic semialdehyde in the steady state [S], is lower than the K, of succinic semialdehyde for the dehydrogenase and the coupled assay system effectively measures the rate of transamination (Table 2). The results obtained using the coupled assay system agree with those obtained by measuring the rate of formation of succinic semialdehyde and glutaric semialdehyde as catalyzed by the amino-

4 434 NADH Binding to Succinic Semialdehyde Dehydrogenase 1 " " " " " l log ([Dehydrogenase]/M) Fig. 1. Couplid enzymatic ussay. Relation between the reciprocal of transient time (z) and the concentration of succinic semialdehyde dehydrogenase. Results obtained using 4-aminobutyrate (0) and 5-aminovalerate (0) as substrates. Enzymatic assays performed in 0.1 M sodium pyrophosphate (ph 8.4) containing 1 mm 2-mercaptoethanol, 5 mm NAD', 5 mm 2-oxoglutarate and 10 mm 4-aminobutyratc or 10 mm 5-aminovalerate. The concentration of 4-aminobutyrate aminotransferase was kept constant (3 nm), whereas succinic semialdehyde dehydrogenase was varied transferase alone; the V for 4-aminobutyrate is fourfold greater than the V for 5-aminovalerate. A transient time (7) was systematically observed during the coupled assay when the rate of formation of NADH was monitored at 340nm. The transient time observed (T) in a consecutive reaction is related to the concentration of the second enzyme[11] [Eqn(6)]. z= 1 kz [dehydrogenase]. A plot of log (1 / T) versus log [dehydrogenase] yields a straight line provided there is no interaction between the enzymes participating in the reaction. As can be seen from the results included in Fig.1, the plot of log (1 / T) versus the log (concentration of succinic dehydrogenase) is linear up to concentration of dehydrogenase of approximately 0.6 pm. Above this concentration range, the transient time is too short to be measured accurately by ordinary spectrophotometric methods. It should be noted that the transient time (T) is not due to any 'hysteresis' effect attributable to a slow association-dissociation of oligomeric structures of the dehydrogenase, since no lag time was observed in the enzymatic assay of succinic semialdehyde dehydrogenase at concentrations of protein below pm. Inhibition by the Substrates The reaction catalyzed by the dehydrogenase is inhibited by the substrates as shown by the results included in Fig.2. The reaction rate increases as substrate is made available, but the rate eventually de- (6) l/[s] (rnm-') Fig. 2. Kinetics of inhibition of succinic, scwiiuldeliyde clcdi~~dwgenuse hy.succinir semialdehyde (0) and glutaric semiuldeliydc. (0). Plots of reciprocal initial velocity versus high substrate concentrations give Ki values of 0.09 mm and 3 nim for succinic semialdehyde and glutaric semialdehyde, respectively. The concentration of NAD' was 5 mm. Initial velocity, 0, was measured as nmol NADH formedimin creases in the presence of excess substrate, providing a direct and simple mechanism for regulation of the rate of product formation. It should be noted that succinic acid is not an inhibitor of the enzyme [3] and that NADH acts as a competitive inhibitor of NAD' at concentrations of the order of 0.1 mm. The initial rate measurements, carried out by monitoring the change in absorbance at 340 nm for at least 2 min, were correlated with a standard assay mixture containing 50 pm succinic semialdehyde and 5 mm NAD' in 0.1 M sodium pyrophosphate (ph 8.4) at 25 'C. A simple model for this behavior is that binding of a second substrate molecule to the catalytic site prevents the reaction catalyzed by succinic semialdehyde dehydrogenase. Accordingly, the steadystate rate expressions as given in Experimental Procedures, can be used to determine K, and Ki. Plots of reciprocal initial velocity versus high substrate concentrations yielded the Ki values of 0.09 mm and 3 mm for succinic and glutaric semialdehyde, respectively. Thus, the inhibitory effect exerted by succinic semialdehyde is more pronounced than that observed for glutaric semialdehyde. In addition, the Ki value for succinic semialdehyde is only fivefold greater than the K,,, value. Oligomeric Structure It has been reported by our laboratory [3] that the molecular weight of succinic semialdehyde dehydrogenase is when examined by polyacrylamide gradient gel electrophoresis. Similar results are obtained by means of gel filtration on Sephacryl S-200

5 W. S. Blaner and J. E. Churchich u 5 9c 4 L cn " Fraction number Fig. 3. Elution projile of succinic sernialdehyde dehydrogenu.se upplied to a Sepliacryl 920 column (55 x 0.9 cm) equilibrated with 0.1 M sodium pyrophosphale (ph 8.4) containing NADH (0.08 mm). Fractions collected were tested for enzymatic activity (0) and protein fluorescence (0) excited at 280 nm. The column was calibrated with the following standards in the absence of NADH; (1) blue dextran; (2) catalase; (3) yeast alcohol dehydrogenase; (4) lactic dehydrogenase; (5) malate dehydrogenase 1 0 previously calibrated with enzymes of known molecular weight (Fig. 3). As shown in Fig.3, the addition of NADH to a concentration of 0.08 mm has no effect on the elution profile of succinic semialdehyde dehydrogenase. Hence, succinic semialdehyde dehydrogenase behaves as a stable oligomeric structure at protein concentrations of around 1 pm, even in the presence of excess NADH. When the enzyme is subjected to sodium dodecyl sulphate/polyacrylamide gel electrophoresis, only one protein band characterized by a molecular weight of was detected when compared to standards of known molecular weight (Fig. 4). Although these results indicate that succinic semialdehyde dehydrogenase is made up of four subunits of similar size, it was desirable to know if the subunits are identical. The results obtained using the technique of isoelectric focusing on polyacrylamide gels containing 8 M urea and 17; 2-mercaptoethanol indicate the presence of only one protein band of pi around 7.1. Thus, it seems reasonable to conclude that succinic semialdehyde dehydrogenase is composed of four identical subunits (Fig. 4). Binding oj NADH The strong binding of NADH to the dehydrogenase can be easily demonstrated by fluorescence emission spectroscopy. At an enzyme concentration of 3 pm and NADH concentration of 6 pm, the binding of NADH to the dehydrogenase is accompanied by an enhancement of the fluorescence emitted over the spectral range nm (Fig. 5). The binding of NADH also brings about a blue shift in the band position of the emission spectrum. Fig. 4. Determination of the molecular weight of succinic semialdehyde deliydrogenuse by sodium dodecyl.~ulfh~e~polyacrylumide gel electrophoresis. (A) The following proteins were used as standards: (1) bovine serum albumin, (2) ovalbumin, (3) pepsin and (4) lysozyme. The relative mobility ofsuccinic semialdehyde dehydrogenase corresponds to a molecular weight of (B) Isoelectric focusing pattern of succinic semialdehyde dehydrogenase on polyacrylamide gels containing 8 M urea The addition of succinic semialdehyde to the binary complex at concentrations higher than those required to inhibit enzymatic activity results in further fluorescence enhancement of bound NADH (Fig. 5). The interaction of NADH with succinic semialdehyde dehydrogenase is paralleled by an increase in the emission anisotropy. In the presence of excess enzyme ([enzyme]/ [NADH] = 5) the emission anisotropy of bound NADH approaches the limiting value of 0.33, which is significantly higher than the emission anisotropy of free NADH (AF = 0.11). This change in emission anisotropy values associated with binding of NADH to the dehydrogenase

6 436 NADH Binding to Succinic Semialdehyde Dehydrogenase was used to determine the stoichiometry of binding. To this end, the emission anisotrpoy (A) of samples containing a fixed concentration of enzyme (1 pm) and varying concentrations of NADH was measured at constant temperature (10 "C) using an excitation wavelength of 340 nm. The results of these measurements are included in Fig. 6. Since the emission anisotropy values at varying concentrations of NADH reflect the relative contribution of free and bound NADH to the fluorescence properties of the system, it is possible to calculate the fraction (m) of bound NADH by resorting to the following equations : - AbFb. CI + Af(1 - E)Fr A = MFb + (1- a)ff A-Af a= (Ab - 2) p + (d --j? (7) where Ab and Ar are the emission anisotropies Of bound and free NADH, respectively. p = Eb/Ff is " Wavelength (nm) (8) Fig. 5. Fluorescence spectra of (I) NADH (6 pm), (2) NADH (6 plm) + enzyme (3 pm) and (3) NADH (6 pm) + enzyme (3 pm) -+ succinic semialdehyde (2 mm) at ph 8.4, excitation wavelength 340 nrn the ratio of fluorescence yields of bound and free NADH (D = 3.0). The average number of NADH molecules bound per molecule of enzyme protein (M, ), V, was determined by the following equation [Eqn (9)] : where [LO] and [Po] are initial concentrations of NADH and protein. When the titration results were analyzed by plotting V/[L] versus V, where [L] is the concentration of free NADH, the linear plot depicted in Fig.6 was obtained. Thus, the analysis of the binding results yield the following information : two out of four subunits of succinic semialdehyde dehydrogenase display a strong affinity for the coenzyme NADH (K = 4 pm). However, the dissociation constant determined by fluorometric titrations is considerably lower than the inhibition constant (Ki = 0.1 mm) determined by enzymatic methods [3]. DISCUSSION The strong binding of NADH to succinic semialdehyde dehydrogenase can be demonstrated by fluorescence spectroscopy. The fluorescence emitted by the binary complex, enzyme-nadh, is influenced by addition of succinic semialdehyde (0.2 mm), a finding which is consistent with the concept that succinic semialdehyde exerts its inhibitory effect by binding at the catalytic site of the dehydrogenase. The equilibrium state of the oligomeric structure of succinic semialdehyde dehydrogenase and the coenzyme NADH was quantitatively investigated by means of emission anisotropy measurements at protein concentrations of around 1 pm. The results of the titration experiments revealed that two molecules of NADH bind with the same dissociation constant to " v Fig.6. (A) Changes in emission unisotropy (A) upon addition of NADH to u fixed concentration of enzyme (I pm) in 0.1 M sodium pyrophosphate (ph 8.4) containing 1 mm, 2-mercriptoerhanol ai 10 C. (B) PI01 of Vl[L] versus V. A dissociation constant of 4 pm for two binding sites per tetramer is obtained

7 W. S. Blaner and J. E. Churchich 431 one molecule of the enzyme at ph 8.4. This unusual stoichiometry of binding can be explained in terms of two distinct mechanisms : (a) a reversible dissociation of the tetrameric structure into a dimeric structure occurs upon addition of the ligand and (b) protomer interactions in succinic semialdehyde dehydrogenase bring about extreme negative cooperativity between the binding sites. A reversible dissociation of the tetrameric structure into dimers may play an important role in the binding of the coenzyme NADH as well as in the regulation of the total enzyme activity. Examples of dissociation of oligomeric structures have been described in the literature [ and a kinetic analysis of the association process has been developed by Kurganov et al. [15]. However, the results of polyacrylamide gradient gel electrophoresis [3], together with the gel filtration experiments described in the present work, strongly suggest that the tetrameric structure of succinic semialdehyde dehydrogenase remains essentially stable over the concentration range pm. Thus, no dissociation of the tetrameric structure in the absence and presence of NADH could be detected at a protein concentration (1 pm) identical to that used in the fluorometric titrations. Furthermore, succinic semialdehyde dehydrogenase at concentrations as low as pm does not exhibit any slow response to rapid changes in ligand concentrations as revealed by enzymatic assays conducted in 0.1 M sodium pyrophosphate (ph 8.4). In view of these considerations, it appears unlikely that the asymmetry of NADH binding can be explained in terms of a dimeric-tetrameric equilibrium, although such dissociation might exist under experimental conditions which differ from those chosen in the present studies. The alternative mechanism, i.e. the binding of two molecules of NADH to one molecule of protein induces a conformational change which affects the binding characteristics of the remaining unoccupied binding sites, could well explain the apparent asymmetry of binding as revealed by fluorometric titrations. Direct binding studies by equilibrium dialysis designed to determine whether there is a set of binding sites characterized by a low affinity constant for NADH at ph 8.4 are complicated by the tendency of the enzyme (20 pm) to undergo polymerization and denaturation after prolonged dialysis at 4 "C. This complication is avoided by measuring the inhibition of dehydrogenase activity by addition of increasing concentrations of NADH. Initial velocity studies performed at ph 8.4 have shown NADH to be a competitive inhibitor with respect to NAD with an apparent Ki of 100 pm [3], which is 25-fold greater than the dissociation constant determined by fluorometric titrations. Thus, it appears that a different set of binding sites on succinic semialdehyde dehydrogenase becomes saturated at NADH concentrations above 0.1 mm leading to reversible inhibition of enzymatic activity. It should be noted that the reaction catalyzed by succinic semialdehyde dehydrogenase proceeds in the direction of formation of succinic acid and NADH and that any inhibitory effect exerted by NADH would result in accumulation of succinic semialdehyde with further inhibition of enzymatic activity. Hence, the combined inhibitory effect of the substrate succinic semialdehyde and the product NADH might be used to modulate the 4-aminobutyrate shunt. The inhibition of succinic semialdehyde dehydrogenase is reversed by conversion of succinic semialdehyde to 4-aminobutyrate, a reaction catalyzed by 4-aminobutyrate aminotransferase. The accumulation of succinic semialdehyde can also be prevented by an NADPH-linked dehydrogenase [ 161 which interconverts succinic semialdehyde to 4-hydroxybutyrate. This work was supported by a grant from the National Institutes of Health. R REFERENCES 1. Callenwaert, D. M., Rosemblatt, M. S., Suzuki, K. & Tchen, T. T. (1973) J. Biol. Cl7em Kammerat, C. & Veldstra, H. (1968) Biochim. Biophys. Actu, 151, Blaner, W. S. & Churchich, J. E. (1979) J. Biol. Chem. 254, Beeler, T. & Churchich, J. E. (1978) Eur. J. Biochem. 85, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Cliem. 193, Bergmeyer, H. U. 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