Golgi Fractions Prepared From Rat Liver Homogenates

215

112

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

Section: Resultsmentioning

confidence: 99%

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Amar-Costesec

Wibo

Thinessempoux

et al. 1974

215

112

“…Since smooth microsomes and Golgi elements present in the same fraction are known to contain enzymes such as glycosyl transferases which modify secretory proteins (Fleischer and Fleischer,804 THE JOURNAL OF CELL BIOLOGY • VOLUME 61,1974 1970; Ehrenreich et al ., 1973 ;Bergeron et al ., 1973), we expected to detect differences in the electrophoretic patterns of content proteins from rough and smooth microsomes . These were not observed .…”

Section: Discussionmentioning

confidence: 99%

Selective Release of Content From Microsomal Vesicles Without Membrane Disassembly

Kreibich

Sabatini

1974

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER) . More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons . The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation . A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content . Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER . Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and crossreacted with antiserum against rat serum . Almost all microsomal glycoproteins were at least partly released with the microsomal content .Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [ 3 H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER . On the other hand, after labeling for 30 min with [ 3 H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes . This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae .The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [ 3 H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue,

“…The Golgi light, intermediate, and heavy fractions and a residual microsomal pellet were isolated from liver homogenates by the method of Ehrenreich et al (4,8).…”

Section: Methodsmentioning

confidence: 99%

Passage of serum-destined proteins through the Golgi apparatus of rat liver. An examination of heavy and light Golgi fractions.

Bergeron¹,

Borts²,

Cruz³

1978

The participation of hepatic Golgi apparatus in the intraceUular transport of blood-destined proteins has been analyzed using Golgi fractions enriched in c/s and trans components of the Golgi apparatus.SDS-polyacrylamide gel electrophoresis of the liver Golgi fractions showed several proteins corresponding in relative proportions and mobilities with serum proteins. After a pulse injection of labeled leucine, the secretory content of the c/s Golgi fraction was labeled earlier than the trans Golgi fraction. Taken together, the results show the participation of the liver Golgi apparatus in the secretion of most of the serum proteins and provide documentation for a sequential progression of secretory protein through the c/s and trans components of the Golgi apparatus.