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Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

ABSTRACT: Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.

SUBMITTER: Macnair DC

PROVIDER: S-EPMC1186636 | biostudies-other | 1979 May

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor-associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor-associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor-associated angioedema and 176 angiotensin-converting enzyme inhibitor-exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg(9)-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6+/-7.8 versus 29.6+/-7.3 nmol/mL per minute; P=0.026) and antigen (465.8+/-260.8 versus 563.1+/-208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor-associated angioedema compared with angiotensin-converting enzyme inhibitor-exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5+/-4.9 versus 29.8+/-6.7 nmol/mL per minute; P=0.001) and antigen (354.4+/-124.7 versus 559.8+/-163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema.

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Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

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