Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity.
Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology
D003902:Detergents; D011480:Protease Inhibitors; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D014357:Trypsin; D004229:Dithiothreitol