Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations.
An accurate, precise, and convenient procedure was developed for measurement of the latency of the low-Km mannose-6-phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem. (1976) 251, 4901-4907] and consists of measurement of mannose-6-phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier. Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0 degree C in the presence of 4 mM zwitterionic detergent 3-[(3-cholamido-propyl)dimethyl-ammonio]-2-hydroxy-1-propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50-fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure. Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose-6-phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose-6-phosphatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable. Using these optimized procedures for disruption of microsomes and assay of hexose-6-P phosphohydrolase, we found that the low-Km mannose-6-phosphatase activity of untreated rat liver microsomes consistently was less than 5% of the total enzyme activity in the fully disrupted microsomes. Accurate and precise assay of the structural latency of mannose-6-phosphatase in membrane preparations must be performed under well-controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations.
D064753:Ammonium Hydroxide; D000818:Animals; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006878:Hydroxides / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010539:Permeability; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013010:Sonication; D013501:Surface-Active Agents / Q000494:pharmacology*
D002793:Cholic Acids; D003902:Detergents; D006878:Hydroxides; D013501:Surface-Active Agents; D064753:Ammonium Hydroxide; C048531:chapso; C023553:mannose-6-phosphatase; D010744:Phosphoric Monoester Hydrolases