Studies on Specific Enzyme Inhibitors

Regulation of mitochondrial metabolism by specific cellular substances. I. Isolation of a cytoplasmic fraction and its effect on mitochondrial oxidations

“…AMP does not replace ADP or ATP. The more rapid (and for certain experimental purposes, often preferred) polarographic kinetic assay of initial rates of 02 consumption of mitochondrial suspensions (Kun et al, 1964) did not yield reliable quantitative results suitable for the bioassay of CMF.…”

“…Mitochondria from rat liver were prepared by differential centrifugation of 15% tissue homogenates in 0.25 m sucrose containing 1.34 X 10~3 m EDTA adjusted to pH 7.4 with 0.01 m Tris buffer, as described previously (Kun et al, 1964). Sedimented mitochondria were washed once with 0.25 m sucrose-EDTA, then with 0.15 m KC1.…”

“…The cytoplasmic component responsible for this effect was obtained from rat liver, kidney, and heart by gel filtration on Sephadex G-25 and G-50 as a macromolecular complex of apparent molecular weight of 5000-6000. The metabolically active cyto-Dur ing experiments concerned with the mechanism of action of synthetic fluorocarboxylic acids in tissue homogenates (Kun et al, 1964;Loh and Kun, 1966;Kun et al, 1966a; Kun, 1967), 2,4-dinitrophenol (DNP) was employed in order to simplify metabolic control in this system by eliminating the effects of oxidative phosphorylation. It was assumed that in multienzyme systems kinetic consequences of competitive (i.e., substrate type) inhibitors would be easier to evaluate in the absence of oxidative phosphorylation.…”

Transport of glutamine and glutamate in kidney mitochondria in relation to glutamine deamidation

“…In pig kidney mitochondria at pH7.4, the Pidependent glutaminase has about fivefold the activity of the Pi-independent isoenzyme (Crompton et al, 1973), and it is reasonable to assume that, during glutamine oxidation, most of the glutamine is deamidated by the Pi-dependent glutaminase. Rat kidney mitochondria, like pig kidney mitochondria, oxidize externally added glutamate predominantly by the transaminase pathway (Kun et al, 1964). However, rat kidney mitochondria oxidize glutamine two to three times as fast as glutamate (Kovacevic et al, 1970) and, in spite of this, produce aspartate from glutamine at a slower rate than with glutamate as substrate (Kovacevic, 1971).…”

Regulation of mitochondrial metabolism by specific cellular substances. II. The nature of stimulation of mitochondrial glutamate metabolism by a cytoplasmic component

“…Mitochondrial Preparations and Standard Assay Conditions. Although the preparative methods and handling of rat liver mitochondria were mentioned in previous papers (Kun et al, 1964;Loh et al, 1968), certain details require further emphasis. In all experiments Sprague-Dawley male rats weighing 150-200 g were used.…”