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Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys.

ABSTRACT: 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.

SUBMITTER: Fulcher IS

PROVIDER: S-EPMC1154422 | biostudies-other | 1983 Jun

REPOSITORIES: biostudies-other

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Project description:A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3' position. The relationship of this membrane metalloendopeptidase to mouse meprin and human 'PABA peptidase' is discussed.

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Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys.

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