Beta-lactamase activity of purified and partially characterized human renal dipeptidase

Human renal dipeptidase has been concentrated from kidneys by homogenization, 1-butanol solubilization, and (NH4)2SO4 fractionation. Final purification was achieved by high-pressure liquid chromatography followed by affinity chromatography. The Enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 220,000 by analytical high-pressure liquid chromatography. The molecular weight of human urinary dipeptidase was estimated by Agarose gel filtration to be 218,000. Dissociation of human renal dipeptidase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a single polypeptide (Mr 59,000). These results suggest that the native Enzyme contains four subunits of Mr 59,000. Analysis of the peptidase for zinc content gave 3.9 g atoms of zinc/mol of Enzyme which supports the suggestion of a 4-subunit structure. Carbohydrate analyses of the purified human dipeptidase demonstrated that it was not a glycoprotein, a characteristic that distinguishes it from porcine and rat renal dipeptidase. beta-Lactamase activity of the purified human Enzyme was demonstrated by measuring its activity against the two Beta-lactam Antibiotics, imipenem and SCH 29482. Kinetic analyses indicated that both Antibiotics undergo enzyme-catalyzed hydrolysis at rates which could produce inactivation of the Antibiotics within the human kidney. The beta-lactamase inhibitor, cilastatin, demonstrated reversible competitive inhibition of the peptidase-catalyzed hydrolysis of both Antibiotics with the same Ki of 0.7 microM.