Proteolytic activities in plasma membrane preparations from rat liver. 1. Preparation of rat liver plasma membranes and solubilization of membrane bound proteases.
Plasma membranes (PM) were prepared by discontinuous density gradient centrifugation of crude nuclear fractions from 6 rat livers. These "nuclear" PM (PM-n) were 15-fold enriched in plasma membrane marker enzymes and contained an endopeptidase activity degrading azocasein at pH 7. To get larger amounts of plasma membranes, microsomal fractions obtained in large scale subcellular fractionations were subjected to continuous gradient zonal centrifugation. These "microsomal" PM (PM-m) were 22-fold enriched in 5'-nucleotidase, however, the separation of PM from endocellular membranes was not complete. PM-m showed endopeptidase activity degrading azocasein at pH 5.4 faster than at pH 7.5 and exopeptidases degrading Ala-Pro-pNA and Ala-pNA at pH 7.6. The latter two activities were distributed over the gradient similar to PM marker enzymes and can be solubilized by detergent and proteinase treatment. Therefore, dipeptidyl-aminopeptidase IV and Ala-aminopeptidase are intrinsic plasma membrane enzymes and can be used as additional markers for rat liver plasma membranes. The efficiency and selectivity to solubilize plasma membrane bound endopeptidase, DPP IV and aminopeptidase activities are compared.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
D010447:Peptide Hydrolases