14CO2 fixation in incubated rat diaphragms.
The time course (0-60 min) of label incorporation from NaH14 CO3 into citric-acid-cycle intermediates and amino acids was investigated in incubations of isolated rat diaphragms. On the basis of these results, 14CO2 exchange by isocitrate dehydrogenase and 14CO2 fixation by propionyl-CoA carboxylation and pyruvate carboxylation could be estimated. Apparent rates amounted to about 30-40, 2, and 35 nmol/min per g of muscle, respectively. About 90 percent of C4-carbon compounds originating from 14CO2 fixation were subsequently removed by decarboxylation. 2-Cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, effectively reduced 14CO2 production from [1-14C]pyruvate but did not affect incorporation of radioactive label from NaH14CO3. In cell-free muscle extracts, 14CO2 fixation was demonstrable under assay conditions suitable for NADP -dependent 'malic' enzyme(s). Addition of hydroxymalonate, an inhibitor of the latter enzyme(s), significantly reduced 14CO2 incorporation. The results provide evidence for a continuous cytosolic replenishment and mitochondrial depletion of citric-acid-cycle carbon skeletons in resting skeletal muscle tissue. The functional role of malic (iso)enzyme activities in these processes is discussed.
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003964:Diaphragm / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D002250:Carbon Radioisotopes; D002245:Carbon Dioxide