This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GUEST, P. C. Articles by HUTTON, J. C. Search for Related Content PubMed PubMed Citation Articles by GUEST, P. C. Articles by HUTTON, J. C.

Molecular Heterogeneity and Cellular Localization of Carboxypeptidase H in the Islets of Langerhans^*

Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital Cambridge CB2 2QR, United Kingdom
Institut d'Histologie et d'Embryologie Geneva 4, Switzerland
Department of Molecular Biology, Research Institute of the Scripps Clinic La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. John C. Hutton, Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QR, United Kingdom.

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.

Footnotes

^* This work was supported by Swiss National Science Foundation Grant 31-26625.89 and by the British Diabetic Association and the Medical Research Council of Great Britain.

Received January 31, 1991.

This article has been cited by other articles:

				K. D. Jeffrey, E. U. Alejandro, D. S. Luciani, T. B. Kalynyak, X. Hu, H. Li, Y. Lin, R. R. Townsend, K. S. Polonsky, and J. D. Johnson Carboxypeptidase E mediates palmitate-induced {beta}-cell ER stress and apoptosis PNAS, June 17, 2008; 105(24): 8452 - 8457. [Abstract] [Full Text] [PDF]

				K. Salim, T. Fenton, J. Bacha, H. Urien-Rodriguez, T. Bonnert, H. A. Skynner, E. Watts, J. Kerby, A. Heald, M. Beer, et al. Oligomerization of G-protein-coupled Receptors Shown by Selective Co-immunoprecipitation J. Biol. Chem., May 3, 2002; 277(18): 15482 - 15485. [Abstract] [Full Text] [PDF]

				E. G. Novikova, F. J. Eng, L. Yan, Y. Qian, and L. D. Fricker Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D J. Biol. Chem., October 8, 1999; 274(41): 28887 - 28892. [Abstract] [Full Text] [PDF]

				O. Varlamov, F. J. Eng, E. G. Novikova, and L. D. Fricker Localization of Metallocarboxypeptidase D in AtT-20 Cells. POTENTIAL ROLE IN PROHORMONE PROCESSING J. Biol. Chem., May 21, 1999; 274(21): 14759 - 14767. [Abstract] [Full Text] [PDF]

				O Varlamov and L. Fricker Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface J. Cell Sci., January 4, 1998; 111(7): 877 - 885. [Abstract] [PDF]

				O. Varlamov and L. D. Fricker The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing J. Biol. Chem., March 15, 1996; 271(11): 6077 - 6083. [Abstract] [Full Text] [PDF]

				S. Milgram and R. Mains Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins J. Cell Sci., January 3, 1994; 107(3): 737 - 745. [Abstract] [PDF]