Abstract
Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.
Highlights
Kinetic studies of Morris7777 hepatoma mitochon- of liver, regenerating liver, and many other organs, but is drial NAD(P) malic enzyme were consistent with an present in tumor mitochondria in levels proportional to the ordered mechanism where NAD adds to the enzyme rapidity of cell division [7]
In spite of this, when malate is supplied to tumor mitochondria, it can be channeled exclusively into NAD(P) malic enzyme so that pyruvate and COzare the major products and both are produced at equivalent rates [1,7]
We have made a detailed for the known ability of NAD(P) malic enzyme to in- kinetic study of this enzyme, which we recently purified from tercept exogenous malate from malate dehydrogenasethe Morris 7777 hepatoma [13],and compared the results in intact tumor mitochondria
Summary
Squares method as described previously [29]. Non-Michaelis-Menten kinetic results were evaluated as described previously [30] and in the text. NAD(P) malic enzyme purified from 22 aH hepatoma mitochondria [8].Under these conditions and when NAD was the coenzyme, double-reciprocal plots of the velocity of Morris 7777 mitochondrial NAD(P) malicenzyme uersus malate assayed with the same methods (Methods I and I1 [31]plus additional concentration were not linear unless the allosteric activator methods) used in the accompanying article to measure enzyme levels fumarate was present (Fig. 1).These results were conin liver mitochondria [31]. As a test for the strictly NADP-dependent malic enzyme, the Assays were performed in the presence of1.0 mM NAD and either extract was assayed at low concentrations of malate (2 mM) and the presence (curue B ) or absence (curueA ) of 5.0 mM fumarate.
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