The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border.
1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000-96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn(2+) was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn(2+)- activated endopeptidase to be characterized.
D000818:Animals; D002118:Calcium / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003035:Cobalt / Q000032:analysis; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D007457:Iodine Radioisotopes; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008345:Manganese / Q000032:analysis; D008970:Molecular Weight; D011480:Protease Inhibitors; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014461:Ultracentrifugation; D015032:Zinc / Q000032:analysis / Q000378:metabolism
D002241:Carbohydrates; D002614:Chelating Agents; D007328:Insulin; D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D012967:Sodium Dodecyl Sulfate; D003035:Cobalt; D008345:Manganese; D004492:Edetic Acid; D010450:Endopeptidases; D015032:Zinc; D002118:Calcium