Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.

Abstract

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates .The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically . Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96% . Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membranecontaining fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase .The endoplasmic reticulum (ER) of rat liver hepatocytes has been studied extensively by use of marker enzymes to identify the presence of these membranes in fractions (17,14,15,3,1,23,10,11,21) . In recent years, the question as to whether a given amount of marker enzyme activity is associated with a similar amount of ER membrane has become one of increasing importance (15,25,13,44,32,8) . Several papers have suggested that (a) marker enzymes are heterogeneously distributed on the ER and that (b) "ER marker enzymes" may also be attached to membranes other than those of the ER (15,46,2,18,22,29,24,14,41,39,4,38,30) . The existence of such marker enzyme heterogeneities would be expected to have important consequences when one is interpreting bio-J. CELL BIOLOGY © The Rockefeller University Press " 0021-9525/80/06/0577/10 $1 .00 Volume 85 June 1980 577-586 chemical data extrapolated from one fraction to the entire liver, or when one is making corrections for contaminations, particularly if the fractionation procedure leads to a sorting of the heterogeneous ER membranes .In an earlier study (9), the surface areas of the hepatic membranes were determined in each of several fractions collected by differential centrifugation . An average 96% fraction-homogenate recovery for the membrane surface areas, accompanied by an average 95% recovery for several membrane marker enzymes, suggested that, for the most part, both membrane surface area and enzyme activity were being similarly conserved during the fractionation procedure . The study showed the extent to which homogenization forms a pool