Abstract
Native rat liver arginase was covalently coupled to Sepharose beads and the resulting matrix‐bound tetramer was subsequently dissociated by acid or EDTA treatment. The immobilized derivatives that remain in the matrix were renatured in the presence of the cofactor Mn2+ and analyzed in terms of recoverable activity before and after incubation with soluble enzyme. The activity of the immobilized enzyme was determined under conditions where it is directly proportional to the enzyme content. Both the acid‐treated and EDTA‐treated and renatured immobilized rat liver arginase have about one‐fourth the activity of the untreated immobilized enzyme. The activity of the renatured immobilized enzyme after reassociation with free enzyme was 85%(acid‐dissociated oligomer) and 60% (EDTA‐dissociated oligomer) of the activity of the initially immobilized oligomer. The acid‐dissociated and renatured immobilized arginase had a Km value (18 mM) that is approximately four times the value of the immobilized tetrameric enzyme (3.4 mM). After renaturation and reassociation with free enzyme the Km value of the immobilized enzyme was found to be equal to the value of the immobilized oligomer before dissociation. This indicates that the dissociated and renatured immobilized enzyme is monomeric. The Mn2+‐free buffer. The immobilized tetramer, on the other hand, does not lose Mn2+ after this procedure, and this ion seems to be bound even more tightly than in the case of the free tetramer. The reconstituted oligomer had a Km value approximating the value of the initially bound oligomer.From these results and binding curves we conclude that rat liver arginase oligomer can be dissociated into monomers, which are catalytically active after renaturation with cofactor (Mn2+) at neutral pH. Thus, for arginase the tertiary structure is sufficient for the expression of the function of this enzyme.The immobilized monomeric arginase has been used to purify crude arginase by subunit exchange (affinity) chromatography.
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