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A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.

Although chickens are uricotelic and do not have significant urea-ornithine cycle in any tissue, the kidneys contain a high concentration of arginase which apparently functions to regulate degradation of dietary arginine. A series of investigations has been made to determine the intracellular localization of this arginase in chicken kidney. Tissue fractionation using sucrose density gradients and differential centrifugation showed as association of arginase activity with certain marker enzymes and with fractions identified as mitochondria by electron microscopy. This is consistent with the localization of the arginase in the mitochondrial matrix of chicken kidney cells. Such a finding has significance in understanding the regulation of arginine degradation in chickens.

The affinities of anti-human liver arginase antibodies raised in rabbits to liver arginases from man, bovine, pig, dog, guinea pig, rat and mouse were investigated by Scatchard analysis of the binding of the arginases from crude liver extracts to Sepharose-bound immunoglobulins. All arginases bound with good affinity, but the binding capacities of the immunosorbent for the enzymes from various species decreased with decreasing phylogenetic relationship of the species. Arginase from murine peritoneal macrophages did not bind to the immunosorbent at all. A simple two-step purification method for the liver arginases of all species mentioned above is given. All arginases were purified to electrophoretical homogeneity. The molecular weights of their subunits were estimated.