Assay of adenylate cyclase in homogenates of control and Duchenne human skeletal muscle.
The wide range of values reported for activity of adenylate cyclase (AC) in human skeletal muscle prompted re-evaluation of conditions used for homogenization and assay. Adenylate cyclase activity in the same normal muscle differed with different techniques of homogenization. In pH 7.5 isotonic Tris buffer, basal and catecholamine-activated activities declined rapidly in homogenates kept at 4 degrees C. Loss of basal activity was prevented by addition of a chelator of divalent cations. Loss of response to isoproterenol was prevented by addition of guanylnucleotides. Enzyme activity was maximal at 37 degrees C and pH 7.6. Enzyme activity was lower when theophylline was used to prevent degradation of labelled 3',5' cyclic adenosine monophosphate (cyclic AMP) than when unlabelled cyclic AMP was used to this purpose. Basal activity increased with increased MgCl2 concentration up to 50 mmol/l, but isoproterenol-activated activity was maximal at 4 mmol/l MgCl2. AC was inhibited by exogenous adenosine, but addition of adenosine deaminase to the assay mixture did not increase AC activity. Based upon these observations, standardized procedures of homogenization and assay were devised and used to measure AC activity in muscles of boys with Duchenne muscular dystrophy: basal and isoproterenol-stimulated activities were abnormally low.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000032:analysis*; D002118:Calcium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000032:analysis; D008722:Methods; D009132:Muscles / Q000201:enzymology*; D009136:Muscular Dystrophies / Q000201:enzymology*; D013696:Temperature
D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D008274:Magnesium; D002118:Calcium