Purification of human hepatic arginase and its manganese (II)-dependent and pH-dependent interconversion between active and inactive forms: a possible pH-sensing function of the enzyme on the ornithine cycle.
Purification of human liver arginase by chromatography on DEAE-Sepharose, CM-Sepharose, hydroxylapatite, and MonoS yielded protein of greater than 95% purity by sodium dodecyl sulfate-gel electrophoresis. Detailed kinetic studies of the interconversion of active and inactive forms of arginase showed the effects of metal ion addition and withdrawal, metal ion type, time, temperature, and pH. At pH 7 and 37 degrees C, removal of Mn2+ caused a first-order deactivation with half-life of 1 h. Reactivation was completed within 0.5 min (1 mM Mn2+) or 90 min (ca. 6 nM Mn2+). Activation by Mn2+ showed a hyperbolic response, with Kd for Mn2+ of about 36 nM. Mn2+ apparently displaced about 2 H+, resulting in sigmoid dependence upon concentration of OH-. Both the maximal velocity of catalysis and the Km toward arginine were markedly pH-dependent in the physiological range. The findings lead to a model where Mn2+ allosterically activates arginase by a sequential, and pH-sensitive, mechanism. The combined pH sensitivities of activation, Vmax, and Km are likely to give arginase a role in mediating the demonstrated pH control of the ornithine cycle and hence in the regulation of body pH.
Kuhn
N J
NJ
School of Biochemistry, University of Birmingham, United Kingdom.
Ward
S
S
Piponski
M
M
Young
T W
TW
D001119:Arginase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008954:Models, Biological; D009952:Ornithine / Q000378:metabolism
D008670:Metals; D008345:Manganese; D009952:Ornithine; D001119:Arginase