Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil.
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 PGE1 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 42 K mol. wt protein labeled selectively by cholera toxin; 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; prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and a set of proteins and lectin-binding activities in fractions containing beta-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.
D000262:Adenylyl Cyclases / Q000097:blood*; D002458:Cell Fractionation; D002772:Cholera Toxin / Q000494:pharmacology; D004586:Electrophoresis; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins; D006023:Glycoproteins / Q000097:blood*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins / Q000494:pharmacology; D008565:Membrane Proteins / Q000097:blood*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000097:blood* / Q000378:metabolism
D006023:Glycoproteins; D006728:Hormones; D037102:Lectins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases