title: Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans.
abstract: The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, United Kingdom.
authors.forename: P C
authors.initials: PC
authors.lastname: Guest
authors.forename: M
authors.initials: M
authors.lastname: Ravazzola
authors.forename: H W
authors.initials: HW
authors.lastname: Davidson
authors.forename: L
authors.initials: L
authors.lastname: Orci
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000201:enzymology / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology; D007515:Islets of Langerhans / Q000201:enzymology* / Q000648:ultrastructure; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats
chemical_list: D007328:Insulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H