Golgi-rich Fraction Research Articles

Glucokinase microsomique du foie de rat. Localisation dans une fraction riche en appareil de Golgi

Glucokinase activity was previously found to be associated with a microsomal fraction of rat liver. Zonal centrifugation has been used to determined the localisation of this glucokinase. The fraction which contains 60% of glucokinase activity shows also 37% of UDPgalactose: N-acetylglucosamine galactosyltransferase activity. These enzymic activities are purified 10- and 6.5-fold over the crude microsomal fraction, respectively. Morphologically the fraction consists mainly of smooth membranes with bissacs and tubular elements. These results suggest that this fraction is derived from the Golgi apparatus. It does not contain appreciable amounts of enzymes such as glucose-6-phosphatase and 5′-nucleotidase. The level of thiamine pyrophosphatase is higher (50%). The function of this glucokinase bound to a Golgi-rich fraction of rat liver is discussed.

The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver

1. A cell fraction rich in Golgi apparatus was isolated from the livers of guinea pigs fed on a lipid-rich diet (1.6% cholesterol, 15% corn oil). 2. The Golgi cisternae and secretory vesicles contained electron-dense particles which were tentatively identified as VLD (very-low-density) and LD (low-density) lipoproteins. Particles of moderate electron density, 150-500nm in diameter, were seen associated with membranous elements of the Golgi-apparatus cell fraction. Disruption of this cell fraction permitted the release of these three species of particles, which were separated into particulate lipid, and VLD and LD lipoproteins. 3. The large particles of moderate electron density, isolated as particulate lipid, were distinct from both species of Golgi particles in their chemical composition and in possessing an immunochemically unreactive apolipoprotein(s). Morphological observations suggest that the particulate lipid arose from cytoplasmic lipid droplets which were present as contaminants of the Golgi-rich fraction. 4. The chemical and immunochemical results are consistent with the suggestion that the Golgi LD particles are precursors of the VLD particles, into which they may be transformed by the addition of both triglyceride and cholesteryl ester. The present results provide further support for the proposal that the Golgi VLD particles are precursors of the serum VLD lipoproteins in the guinea pig. 5. Hepatic Golgi VLD particles isolated from guinea pigs fed on the lipid-rich diet contained significantly higher molar amounts (relative to protein) of both cholesteryl ester and triglyceride than similar particles from animals fed on a normal diet. These results suggest that the type of Golgi VLD particle produced from the LD particle is a direct consequence of the amount and composition of the dietary lipid. 6. Hepatic Golgi LD particles isolated from guinea pigs fed on different diets were similar in chemical composition and contained approx. 50% by weight of phospholipid. We conclude that the Golgi LD particle is normally present in the Golgi-apparatus cell fraction from guinea-pig liver, and may represent the end product of lipoprotein biosynthesis in the smooth endoplasmic reticulum. 7. The serum LD lipoproteins and Golgi LD particles were quite distinct in chemical composition. However, these two lipoprotein species were immunochemically identical and exhibited a similar range of flotation rate. It appears unlikely that the Golgi LD particles are secreted as the precursors of the serum LD lipoproteins.

Enzymic characterization of Golgi-rich fractions from rat submaxillary-sublingual glands.

Isolation of Golgi-enriched subcellular fractions from rat submaxillar-sublingual glands was achieved by using a combination of differential and discontinuous sucrose-density-gradient centrifugation. Each fraction was characterized by enzyme markers, including three glycosyltransferaseas: galactosyltransferase, N-acetylglucosaminyltransferase, and N-acetylgalactosaminyltransferase. Golgi fractions enriched in the latter two enzymes were further studied using bovine, porcine, and rat submaxillary mucins and their trifluoroacetic-acid-treated materials as acceptors. Treatment of the intact mucins with trifluoroacetic acid yielded two protein fractions, one with a hexosamine content of about 15%, and the other virtually free of sugars. Only intact bovine mucin and the hexosamine-containing acceptor protein were efficient substrates for either hexosaminyl transfer. Ultrafilterable peptides derived from rat submaxillary-sublingual glands showed high acceptor activity for both N-acetylhexosamines in the presence of Golgi-rich fraction 2.

Intracellular distribution of serum albumin and its possible precursors in rat liver.

1. The fractionation of intracellular albumin labelled with radioactive l-leucine was studied in rat liver by means of isoelectric focusing. 2. Isoelectric fractionation was compared with ion-exchange chromatography for purification of radioactive intracellular albumin obtained by antibody precipitation. Similar results were obtained with both methods of separation. Purified albumin contains only a minor amount of the radioactivity. The remainder is associated with albumin-like protein(s). 3. The albumin-like protein has the properties of a precursor of plasma albumin. 4. The distribution and turnover of radioactive albumin in rough and smooth microsomal fractions and in a Golgi-rich fraction were studied. 5. It is concluded that newly synthesized albumin, as such, appears only momentarily if at all in any intracellular structure before its appearance in the plasma. 6. It is also concluded that the rate-limiting step in the secretion of plasma albumin is the conversion of precursor(s) into albumin. We can find no evidence to suggest that there is any significant transport of albumin, as such, during the course of secretion.

The Biosynthesis of Rat Serum Albumin: IV. APPARENT PASSAGE OF ALBUMIN THROUGH THE GOLGI APPARATUS DURING SECRETION

Abstract The role of the Golgi apparatus in the secretion of serum albumin has been investigated by measurements on albumin isolated immunologically from rough microsomes, smooth microsomes, and Golgi-rich fractions prepared from rat liver. Albumin constituted 6% of the total protein of the Golgi-rich fraction, which was the highest level found among intracellular components. After intravenous injection of 14C-leucine the maximum radioactivity of albumin occurred at about 5 min in rough microsomes, 15 min in smooth microsomes, and 20 min in the Golgi-rich fractions. The results indicate the pathway of albumin secretion to be: rough membranes-smooth membranes-Golgi bodies-blood. On the basis of relative specific radioactivities rough and smooth membrane fractions may contain some albumin not normally in the process of active synthesis and secretion, which could represent albumin in temporarily quiescent membrane channels or in pinocytotic vesicles.

Preparation and characterization of golgi membranes from rat liver

1. 1. A Golgi-rich fraction from rat liver has been isolated by the direct application of the zonal centrifugation method used previously to isolate Golgi-rich membranes from bovine liver. 2. 2. UDP-galactose-N- acetylglucosamine galactosyl transferase is concentrated about 100-fold in this fraction compared to the homogenate and appears to be a useful marker enzyme for this organelle in rat liver as well as beef liver. As in bovine liver Golgi, the fraction from rat liver is only slightly contaminated by other organelles as evidenced by low glucose-6-phosphatase, ATPase and acid phosphatase activities. Thiamine pyrophosphatase activity was found present in endoplasmic reticulum and plasma membranes as well as Golgi membranes from rat liver, and was therefore not a useful marker. 3. 3. Some species differences in the Golgi preparations exist. Rat liver Golgi contain little or no rotenone-insensitive NADH- or NADPH-cytochrome c reductase activity whereas bovine liver Golgi have significant levels of these activities. In addition, the Golgi fraction from rat liver appears to have a unique and characteristic protein profile after electrophoresis in polyacrylamide gels as compared with endoplasmic reticulum, plasma membranes and mitochondria. Bovine liver Golgi preparations, on the other hand, appear very similar to endoplasmic reticulum after electrophoresis on acrylamide gels. 4. 4. A major protein of band mobility 0.456 ± 0.008, relative to ribonuclease A, is present in rat liver Golgi preparations. This band is also characteristic of bovine liver Golgi preparations and may be due either to serum very low density lipoproteins or serum albumin, or both. 5. 5. Using the information obtained from this type of preparation, a simpler, one-step procedure was devised which increases the yield of Golgi membranes from rat liver to 0.15–0.3 mg Golgi protein per g as compared to 0.05 mg Golgi protein per g obtained by the zonal procedure.

Isolation of a Golgi-rich fraction from rat liver

A fractiion rich is membranes of the Golgi apparatus was isolated from rat liver by discontinuous density gradient centrifugation. Electron microscopic analysis of the fraction revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements. The Golgi-rich fraction contained over 90% of the UDP-galactose; N- acetylglucosamine galactosyltransferase, about 2% of the glucose-6-phosphatase and 12% of the AMP phosphohydrolase present in the post-nuclear supernatant of liver homogenates.

Intracellular Localization of Liver Sugar Nucleotide Glycoprotein Glycosyltransferases in a Golgi-rich Fraction

Abstract The following trisaccharide unit terminates the oligosaccharide chains of several plasma glycoproteins: sialic acid → galactose → N-acetylglucosamine → oligosaccharide → protein. Previous studies showed that this trisaccharide unit was synthesized by the sequential action of three glycosyl-transferases isolated from goat colostrum; each glycosyltransferase catalyzed the transfer of a monosaccharide residue (N-acetylglucosamine, galactose, or sialic acid) from its nucleotide derivative to the appropriate glycoprotein acceptor. The present studies are concerned with the intracellular location of these glycosyltransferases in rat liver. Kinetic studies were first performed to establish optimum conditions for determining quantitatively the particle-bound rat liver glycosyltransferases; these enzymes exhibited similar properties to the soluble, partially purified glycosyltransferases previously obtained from goat colostrum. The subcellular localization of the enzymes was then investigated by two techniques, differential centrifugation and discontinuous sucrose density gradient centrifugation. In these studies, the following were used as markers for various subcellular organelles: DNA, RNA, glucose 6-phosphatase, NADPH-cytochrome c reductase, acid phosphatase, 5'-nucleotidase, and glutamic dehydrogenase. The results indicated that the glycosyltransferases were all located in the same membranous subcellular component and that this component was different from organelles containing the above marker substances; the active subcellular particle was characterized by electron microscopy as being rich in Golgi apparatus. The apparent location of the glycosyltransferases in the Golgi apparatus suggests that these enzymes may be involved in terminating the synthesis of plasma glycoproteins by the liver during secretion, and may possibly be required for secretion of these proteins.