Abstract
Abstract Low concentrations of calcium (l100µm) cause a marked inhibition of 14C-carbon dioxide production from carboxyl-labeled pyruvate by isolated rat liver mitochondria. This effect disappears in the presence of agents which preclude pyruvate carboxylase activity such as dinitrophenol plus oligomycin. Adding ATP in combination with these agents restores carboxylase activity and the inhibitory effect of calcium returns. If malate is added, the system again becomes less dependent on pyruvate carboxylase activity and calcium loses its effectiveness. Malate can also overcome inhibition caused by dinitrophenol. Pyruvate carboxylase activity can be monitored more directly by measuring the net accumulation of Krebs cycle intermediates formed from pyruvate or incorporation of pyruvate-carboxyl-14C to the cycle intermediates. Calcium (100µm) inhibited both of these processes by as much as 75%. Studies with partially purified carboxylase confirm the concept that calcium is an effective inhibitor, and indicate that the effect may be due to competition with magnesium ion. Mitochondria from vitamin D-deficient or cortisone-treated animals exhibited higher carboxylase activities than those of control animals as measured by the indirect 14CO2 assay method. These changes are consistent with the known effects of those agents on calcium metabolism in vivo. Results are discussed in terms of the possibility that calcium may play an important role in regulating the initial steps of gluconeogenesis.
Highlights
Low concentrations of calcium (
It shows that pyruvate may either be decarboxylated to acetyl-CoA by the pyruvate dehydrogenase enzyme complex, with concomitant formation of DPNH and carbon dioxide, or that it may be carboxylated via pyruvate carboxylase to oxaloacetate
The dashed lines indicate the requirements of the enzyme for the magnesium and acetyl-CoA cofactors [6]
Summary
All experiments were performed with mitochondria prepared from the livers or kidneys of adult male white rats of the Wistar strain. The isolation procedure used was a modification of the method of Schneider [13]. 0.37 M, was used in place of isotonic sucrose, and 100’ pM ethylene glycol bis(&aminoethyl ether)-l\i ,N’-tetraacetic acid was included initially to bind calcium released during homogenization resulting from calcium carried over in the vascular space of the tissue. Lipid on the walls of the centrifuge tube was removed after each spin by absorbing it on cleansing tissue. Rasmussen iu 0.37 M sucrose after preparation (approximately 30 mg of protein per ml), and used as soon as possible thereafter. Protein concentration was determined by the biuret method [14]
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