The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b5 and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-fl-glucosaminidase, ~3-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.

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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

Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-fl-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.