Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes.
The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.
D000262:Adenylyl Cyclases / Q000097:blood*; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000097:blood*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008982:Molybdenum / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013601:T-Lymphocytes / Q000201:enzymology*
D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D008982:Molybdenum; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase