Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys.
The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.
D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007123:Immunoelectrophoresis, Two-Dimensional; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013552:Swine
D008565:Membrane Proteins; D011480:Protease Inhibitors; D010450:Endopeptidases