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Kinetic Studies on Rat Liver and Beef Heart Mitochondrial Adenosine Triphosphatase: The Effects of the Chromium Complexes of Adenosine Triphosphate and Adenosine Diphosphate on the Kinetic Properties’ SHELDON Znstitute

for Enzyme

M. SCHUSTER, Research

and

RICHARD

E. EBEL,2

the Department of Biochemistry, Wisconsin 53706 Received

June

AND

HENRY

University

A. LARDY

of Wisconsin,

Madison,

9, 1975

The kinetics of isolated rat liver and beef heart mitochondrial adenosine triphosphatase (ATPase) were studied by using the chromium ATP and ADP complexes as substrate analogs. It was found that both chromium ATP (CrATP) and chromium ADP (CrADP) are competitive inhibitors of ATP hydrolysis. The presence or absence of ATPase-activating anions, e.g., bisulfite, had little effect on the type or potency of the inhibition by these chromium complexes. Both CrADP and CrATP were noncompetitive inhibitors of the hydrolysis of ITP with both the heart and liver-derived enzymes. It was also found that CrADP was a consistently more effective inhibitor than the ATP complex with the beef heart enzyme. These results are consistent with the existence of two types of nucleotide binding sites on mitochondrial ATPases: One site is regulatory and is rather specific for adenosine polyphosphates, while the other site is relatively nonspecific and serves as the hydrolytic site.

Understanding the regulation of mitochondrial ATPase3 activity can provide insight into the mechanism and control of both ATP synthesis and hydrolysis. Several investigators have attempted to study the ATPase reaction by examining the structure (l-7), the nucleotide- and the inhibitor-binding properties (8-12) and the kinetics (13-16) of various mitochondrial ATPases. One promising area of these recent studies has involved the use of nucleotide analogs as they affect the kinetics of ATP hydrolysis (17-23). These groups have recently shown that the p-y-imidobridged analogs of ATP and GTP are po-

tent inhibitors of the activity of mitochondrial ATPases from both rat liver and beef heart. We have further pointed out that the effectiveness of these analogs as ATPase inhibitors is dependent upon the enzyme source as well as the ionic composition of the assay medium (22). This communication presents data regarding the effect of the chromium complexes of ATP and ADP on the ATPase activity of rat liver and beef heart mitochondrial ATPase in the presence and absence of activating anions. MATERIALS

656 0 1975 by Academic Press, of reproduction in any form

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METHODS

Mitochondrial ATPases from rat liver and beef heart were isolated by the methods of Lambeth and Lardy (3) and Knowles and Penefsky (61, respectively. Enzyme specific activities varied between 80 and 130 pm01 of ATP hydrolyzed per minute per milligram of protein. Protein concentrations were determined by a biuret method (24). Initial velocity experiments were performed at 30°C in a total volume of 1.0 ml containing 50 mM iV,N-bis-(2-hydroxyethylj-2-aminoethane sulfonic acid, pH 7.0, 200 mM sucrose, 1 mM free magnesium as MgCl,, 2 mM phosphoenolpyruvate, 0.3 mM

1 This work was supported by National Institutes of Health Grants No. AM lo-334 and HD 08630. S.M.S. is the recipient of a National Institutes of Health postdoctoral training grant, No. l-F22AM00874-01. 2 Present address: Department of Biochemistry, University of Texas Health Sciences Center at Dallas, Dallas, Tex. 75235. 3 Abbreviations used: ATPase, adenosine triphosphatase; CrATP, chromium ATP; CrADP, chromium ADP; ITPase, inosine triphosphatase. Copyright All rights

AND

KINETICS

OF

MITOCHONDRIAL

NADH, 30 pg each of pyruvate kinase and lactate dehydrogenase, and nucleotides as designated in the figures and tables. The lack of nucleoside diphosphate specificity of pyruvate kinase (25) permits the use of this coupling system for measuring the hydrolysis of GTP and ITP as well as of ATP. Before use, isolated ATPase was centrifuged at room temperature to remove the (NH&SO, and ATP that were included in the storage medium (6) and then resuspended in 200 mM sucrose containing 20 mM TrisCl, pH 8.0, at 30°C. The reactions were initiated by the addition of the ATPase. The reaction was followed by monitoring the disappearance of NADH absorbance at 340 nm with a Beckman DU monochromator, modified with a Gilford Model 2220 adapter and a Hewlett-Packard 7101B strip chart recorder. The chromium complexes of ATP and ADP were prepared as described by DePamphilis and Cleland (26) and Janson and Cleland (27). The concentrations of ATP and ITP were determined by optical density at 259 nm (pH 7) and 248.5 nm (pH 6), assuming millimolar extinction coefficients of 15.4 and 12.2, respectively (28). At the concentrations used, CrATP and CrADP had no effect on the coupled assay system. Linear double-reciprocal plots and Hill plots were derived from the data using a weighted leastsquares tit, with the weighting factor being the reciprocal of the variance (29). Inhibition constants (K,) are reported according to the definitions of Cleland (30). The following compounds were purchased from the indicated sources: ATP and ITP, P-L Biochemicals; pyruvate kinase and lactate dehydrogenase (in glycerol), Boehringer Mannheim; phosphoenolpyruvate, Sigma; and N,N-bis-(2-hydroxyethylj-2-aminoethane sulfonic acid, Nutritional Biochemicals. All other reagents were purchased from common commercial suppliers and were of the highest purity available.

ATPases

657

ATPase assay medium profoundly affects the kinetics of the isolated ATPase and that these anions also could affect the inhibitory action of various ATP analogs. Therefore, the inhibition of rat liver mitochondrial ATPase activity in the presence of the activating anion bisulfite (15) was examined. Figure 2 shows that in the presence of 0.6 mM bisulfite, CrATP is a linear competitive inhibitor of ATPase activity. The Ki under these conditions was 0.14 rnM.

CrATP is an effective, linear, noncompetitive inhibitor of the hydrolysis of an alternative ATPase substrate, namely ITP, both in the absence (Fig. 3) and in the presence of the activating anion, bisulfite (Fig. 4). The inhibition constants for these conditions are summarized in Table I. We have observed that the ATPase from beef heart mitochondria behaves differently than the rat liver enzyme with regard to various inhibitors (23). Therefore, the effect of chromium nucleotides on this enzyme was examined. Beef heart mitochondrial ATPase is competitively inhibited by CrATP with no change in the observed negative cooperativity (Fig. 5). The Ki for CrATP was determined to be 600 PM, which is considerably higher than that determined with the rat liver ATPase (see Table I). 800 r--

RESULTS

The effect of the chromium ATP complex on rat liver mitochondrial ATPase activity is shown in Fig. 1. The negative cooperativity of the ATPase activity is unchanged in the presence of over 1 mM CrATP. With all concentrations of CrATP used, the Hill coefficient remained about 0.5. Within experimental error, the inhibition patterns shown indicate that CrATP is a competitive ATPase inhibitor under the assay conditions used. The Ki for CrATP was determined from a Dixon plot to be 0.18 mM. We have previously shown (15, 23) that the presence of activating anions in the

FIG. 1. The effect of CrATP on rat liver mitochondrial ATPase activity. (a) The millimolar concentrations of CrATP are as indicated. (b) The slope and intercept replots of the data of (a).

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SCHUSTER,

EBEL

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LARDY

O0-O

10 [CrATP]

FIG. 2. (a) The effect of CrATP HSO,-. replots

The millimolar concentrations of the data of (a).

on rat liver mitochondrial ATPase in the presence of 0.6 mM of CrATP are as indicated. (b) The slope and intercept

4 oobdv5e”

OohI/ [MS

ITP]

Kinetic studies on rat liver and beef heart mitochondrial adenosine triphosphatase: the effects of the chromium complexes of adenosine triposphate and adenosine diphosphate on the kinetic properties.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 171, 656-661 (19%) Kinetic Studies on Rat Liver and Beef Heart Mitochondrial Adenosine Triphosphatase:...
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