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Published July 1984 | public
Journal Article

Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil

Abstract

Human neutrophils were disrupted by brief sonication under conditions which preserve the hormone sensitivity of adenylate cyclase and yield minimal granule lysis. Fractions enriched in adenylate cyclase were analysed for hormonal and guanine nucleotide regulation of the enzyme as well as structural proteins. Adenylate cyclase was activated by PGE_1 and isoproterenol in a GTP-dependent fashion, while f-met-leu-phe and C5a gave no stimulation. Cholera toxin treatment, which specifically modifies cyclase-related GTP-binding proteins, caused a dose-dependent enhancement of GTP activation, in which GTP alone activated maximally and PGE1 was without further effect. The following proteins were detected in the cyclase-containing vesicles: (a) a 42 K mol. wt protein labeled selectively by cholera toxin; (b) protein subunits observed in SDS gels at 214, 165, 105 and 47 K, of which the 47 K band was the most prominent and comigrated with actin; (c) prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and (d) a set of proteins and lectin-binding activities in fractions containing β-glucuronidase activity distinct from adenylate cyclase containing vesicles. The identification of receptor-controlled cyclase, GTP-binding regulatory proteins, cytoskeletal elements and unique lectin-binding activities in a single vesicle preparation should contribute to an understanding of receptor-mediated control of neutrophil function.

Additional Information

© 1984 Pergamon Press Ltd. First received 9 November 1983; accepted in revised form 17 February 1984. This work was supported by grant No. 17 from the Department of Education and Research, Kaiser-Permanente, Southern California Permanente Medical Group and in part by a Biomedical Research Support Grant RR 05521-19 from the Kaiser Foundation Research Institute. We thank Aline Alenty for advice and assistance in the preparation of neutrophils and in neutrophil function tests. We are also grateful to Lauren Rosenthal and Bill Frankeberger for secretarial assistance. Publication No. 72 from the Kaiser Regional Research Laboratory.

Additional details

Created:
August 19, 2023
Modified:
October 23, 2023