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Glycyl-L-leucine hydrolase, a versatile 'master' dipeptidase from monkey small intestine.

ABSTRACT: 1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the K(m) and k(0) values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the ;master' dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed.

SUBMITTER: Das M

PROVIDER: S-EPMC1165875 | biostudies-other | 1973 Dec

REPOSITORIES: biostudies-other

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Project description:Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.

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Glycyl-L-leucine hydrolase, a versatile 'master' dipeptidase from monkey small intestine.

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