Analytical subcellular fractionation of rat liver with special reference to the localisation of putative plasma membrane marker enzymes

Abstract

A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in the sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments. Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. Gamma-Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5'-nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.