The membrane-bound basic carboxypeptidase from hog intestinal mucosa

The carboxypeptidase activity occurring in hog intestinal mucose is apparen tly due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-b ound carboxypeptidase activity which occurs at neutral optimum pH levels wa s found to be enhanced by CoCl2 and inhibited by guanidinoethylmercaptosucc inic acid, a-phenanthroline, ethylenediamine tetraacetic acid and cadmium a cetate; whereas the soluble carboxypeptidase activity which occurs at an op timum pH level of 5.0 was not activated by CoCl2 and only slightly inhibite d by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl2 and CdCl2. T he latter activity was presumably due to lysosomal cathepsin B, which is kn own to be present in the soluble fraction of hog intestinal mucosa. Althoug h the membrane-bound enzyme was evenly distributed along the small intestin e, it was not anchored in the phospholipidic bilayer via a glycosyl-phospha tidylinositol moiety, as carboxypeptidase M from human placenta is. The enz yme was not solubilized by phosphatidylinositol-specific phospholipase C, b ut was solubilized to practically the same extent by several detergents. Th e purified trypsin-solubilized form is. glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypept idase M (62 kDa). It was found to cleave lysyl bonds more rapidly than argi nyl bonds, which is not so in the case of carboxypeptidase M, and immunoblo tting analysis provided further evidence that hog intestinal and human plac ental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is s uggested that the former might be carboxypeptidase D, the recently describe d new member of the carboxypeptide B-type family. (C) 1999 Published by Els evier Science B.V. All rights reserved.