Some aspects of the kinetics of rat liver pyruvate carboxylase.
1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 mumol of oxaloacetate formed/min at 30 degrees C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The K(m) towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. K(m) towards MgATP(2-) was 0.14mm. Mg(2+) ions activated the enzyme, in addition to their role in MgATP(2-) formation. From calculations of likely concentrations of free Mg(2+) in the assay medium a K(a) towards Mg(2+) of about 0.25mm was deduced. Mn(2+) also activated the enzyme as well as Mg(2+), but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn(2+) as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg(2+). 6. Both Co(2+) and Zn(2+) were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg(2+) and the inhibition was time-dependent. 7. Ca(2+) inhibited by competition with Mg(2+) (K(i) about 0.38mm). The inhibition due to Ca(2+) was less pronounced when activation was with Mn(2+). Inhibition by Ca(2+) and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg(2+) gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca(2+). 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. K(a) was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with K(i) 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, beta-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, K(i) towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002247:Carbon Isotopes; D002951:Citrates / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003065:Coenzyme A / Q000494:pharmacology; D003080:Cold Temperature; D000242:Cyclic AMP / Q000494:pharmacology; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology; D009705:Nucleosides / Q000494:pharmacology; D010071:Oxaloacetates / Q000096:biosynthesis; D011773:Pyruvates / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry; D013217:Starvation / Q000201:enzymology; D015032:Zinc / Q000494:pharmacology
D000227:Adenine Nucleotides; D001639:Bicarbonates; D002247:Carbon Isotopes; D002951:Citrates; D009705:Nucleosides; D010071:Oxaloacetates; D011773:Pyruvates; D003035:Cobalt; D008345:Manganese; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D008025:Ligases; D008274:Magnesium; D015032:Zinc; D003065:Coenzyme A; D002118:Calcium