Analytical Study of Microsomes

lsopycnic equilibration and sedimentation rate studies of rat liver microsomes led previously to the assignment of microsomal constituents into group al (monoamine oxidase), group a2 (5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and cholesterol), group a3 (galactosyltransferase), group b (NADH cytochrome ¢ reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5 and P 450), and group c (glucose 6-phosphatase, esterase, nucleoside diphosphatase, 13-glucuronidase and glucuronyltransferase). Confirmation and extension of the assignment into groups has been obtained by studying the differential effect of the reagents digitonin, EDTA, and PPi. Digitonin specifically affected the equilibrium density only of the group a2 and (to a lesser extent) group a3, and not of groups b and c under conditions which preserved the structure-linked latency of nucleoside diphosphatase and galactosyltransferase. Within experimental error the rate of sedimentation of all microsomal constituents was unaffected. The morphological appearance under the electron microscope was indistinguishable from that of nondigitonin-treated microsomes, except that a few smooth membranes (< 10%) exhibited broken-looking profiles. Treatment of microsomes with EDTA or PPi detached a substantial part of RNA and released protein in excess over the amount accountable for by detachment of ribosome constituents. This detachment was confirmed by electron microscopy. EDTA and PPi decreased markedly the equilibrium density and the density dispersion of groups b and c, due mainly to the uncoating of rough THE JOURNAL OF CELL BIOLOGY. VOLUME 62, 1974. pages 717-745 717 on A uust 8, 2017 jcb.rress.org D ow nladed fom elements . E D T A and PPi shifted sl ightly the d is t r ibu t ion profi les of g roups a towards lower densit ies, poss ibly as a result of the release of adsorbed proteins . The combina t ion of E D T A and digi tonin, used subsequently, rendered the average equi l ibr ium densi ty of g roup a2 higher than tha t of groups b and c. Dense subfrac t ions were thus enriched in const i tuents of g roup a2 and showed main ly b rokenlook ing vesicles under the electron microscope . The impor t of our results on the b iochemica l and enzymic proper t ies of the subcel lular componen t s of the mic rosome fract ion is discussed.