Physicochemical properties of isocitrate dehydrogenase from lactating bovine mammary gland: effect of substrates and cofactors.
The influence of substrates and cofactors on the oligomeric structure of the cytosolic form of NADP+-specific isocitrate dehydrogenase (IDH) from lactating bovine mammary gland was investigated using analytical ultracentrifugation and kinetic methods. In guanidine-HCl, the monomer molecular weight for reduced and carboxymethylated IDH was found to be 50,000 to 52,000. In nondenaturing solvents IDH behaves as a homogeneous solute with a molecular weight of 97,200. When added separately, manganous isocitrate, isocitrate, manganous citrate (substrate analog), and a mixture of the substrate analog and NADP+ do not significantly alter the sedimentation coefficient or the molecular weight of IDH as judged by direct observation of the enzyme at 0.1 to 3 microM using sedimentation velocity and equilibrium. Active enzyme sedimentation (AES) was used to assess the degree of dissociation of IDH at lower concentrations, and Kd for the dimer-monomer equilibrium was estimated to be 2 nM. In enzymatic studies, the specific activity at several levels of substrate does not vary as the subunit concentration of enzyme is reduced from 10 to 0.3 nM. Estimates for Kd by AES indicate the presence of a significant fraction of monomer at assay concentrations of 1 nM and below, where the weight fraction of monomer is predicted to be 0.6. If the monomer has a lower activity than the dimer, a drop in specific activity is expected below 1 nM. Significant decreases occur only when the IDH is not protected from denaturation. The concentration of cytoplasmic IDH in bovine mammary tissue is estimated to be 5.7 microM, at least 100-fold greater than our estimates of Kd. Since over 90% of the enzyme is present in the dimeric form, ligand-induced changes in aggregation state cannot play a significant role in the regulation of the cytosolic form of IDH in situ in this tissue.
Seery
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U.S. Department of Agriculture, Eastern Regional Research Center, Philadelphia, Pennsylvania 19118.
Farrell
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D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005260:Female; D005615:Freezing; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP; D011247:Pregnancy; D011309:Preservation, Biological; D013379:Substrate Specificity; D014461:Ultracentrifugation
D009249:NADP; D007521:Isocitrate Dehydrogenase