Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle.
1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5'-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01-0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01-0.02%) caused a modest increase (30-45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (mumol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05-0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res.39, 586-595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta451, 48-61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D005260:Female; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D007545:Isoproterenol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D011506:Proteins / Q000378:metabolism; D012508:Sarcolemma / Q000201:enzymology*; D012969:Sodium Fluoride / Q000494:pharmacology
D003902:Detergents; D011506:Proteins; D006165:Guanylyl Imidodiphosphate; D012969:Sodium Fluoride; D000262:Adenylyl Cyclases; D007545:Isoproterenol