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H A Lardy

Publications and source records attributed to H A Lardy.

At least 91 records · Page 5Linked to original sources
PubMed1981

Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions.

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.

Animals↗
PubMed1981

Isolation from erythrocytes of a green hemoprotein with ferroactivator activity.

A protein that exhibits ferroactivator activity with purified rat liver P-enolpyruvate carboxykinase was purified to apparent homogeneity from bovine erythrocytes. The homogeneous protein has a molecular weight of 82,000 by high speed sedimentation equilibrium and 90,000 by gel exclusion chromatography. A subunit molecular weight of 22,500 was obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its isoelectric focusing indicated that the ferroactivator protein has an isoelectric point at pH 5.7. The reduced pyridine hemochrome of the denatured protein shows absorption maxima at 419, 527, and 557 nm which is characteristic of protoheme IX. The native protein is green in color with absorption maxima at 407, 507, and 543 nm. The amino acid composition and immunological studies show that bovine erythrocyte protein is structurally similar to the P-enolpyruvate carboxykinase ferroactivator protein isolated from rat liver. Its function in the erythrocyte is unknown.

Amino Acids↗
PubMed1979

Evidence for mitochondrial localization of the hormone-responsive pool of Ca2+ in isolated hepatocytes.

Conditions are described that allow chlortetracycline, a fluorescent probe of membrane-associated Ca2+, to monitor the content of the major exchangeable pool of intracellular Ca2+ present in the isolated rat hepatocyte. Chlortetracycline fluorescence is decreased in cells whose Ca2+ content is diminished by treatment either with carbonylcyanide-m-chlorophenylhydrazone or with ionophore A23187. Norepinephrine releases Ca2+ from this exchangeable pool and decreases both the fluorescence signal and its subsequent response to A23187. Previous suggestions that chlortetracycline fluorescence is localized in the mitochondria of liver and other cells is supported by comparison of the fluorescence that follows the addition of chlortetracycline to intact hepatocytes and to isolated hepatic microsomes and mitochondria. Identification of the hormone-responsive pool of Ca2+ with the mitochondria is strengthened by comparison of the total calcium content of mitochondria isolated from control and hormone-treated animals. The uptake and release of Ca2+ in control and hormone-treated hepatocytes rendered permeable by treatment with digitonin is also consistent with this interpretation.

Animals↗