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.
D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004150:Dipeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D015032:Zinc / Q000032:analysis; D001618:beta-Lactamases
D002241:Carbohydrates; D046911:Macromolecular Substances; D004150:Dipeptidases; D001618:beta-Lactamases; D015032:Zinc