Golgi-rich Fractions Research Articles

Differential solubilization of proteins, phospholipids free and esterified cholesterol of rat liver cellular membranes by sodium deoxycholate

1. 1. Smooth microsomes, Golgi-rich fractions, and light and heavy plasmalemmal subfractions from rat liver were isolated and their purity assessed using enzymic, chemical and morphological criteria. 2. 2. Membranes were prepared by Tris-EDTA washing combined with sonication treatment of the different subcellular fractions. 3. 3. Washed membranes were submitted to differential solubilization with 0.26% sodium deoxycholate. When the deoxycholate/phospholipid molar ratio ( R) is raised, all the membranes showed a maximum protein solubilization occuring at R ≅ 2. The higher the membrane neutral lipid to phospholipid molar ratio is, the lower the solubilized protein plateau lies. 4. 4. Phospholipids are solubilized in slightly greater amounts than proteins and their solubilization is complete at R = 14–16. 5. 5. R < 2, sterols are solubilized in slightly greater amounts than phospholipids. At maximum protein solubilization cholesterol and cholesterol esters completely differ in their behaviour. The whole membrane cholesterol goes into solution for R = 14–16 while the solubilization of esterified cholesterol is never complete. The higher the protein plateau is, the lower the cholesterol esters solubilization curve asymptote lies.

Recovery of galactosyltransferase activity from sucrose gradients during isolation of Golgi membranes.

The purification of Golgi membranes has been characterized in membrane fractions isolated from rat liver by homogenization and by differential centrifugation on a discontinuous sucrose gradient. The purification of membranes, judged from the ratio of galactosyltransferase in the Golgi-rich fractions to that in the liver homogenate, correlated with the proportion of structurally intact isolated membranes. With increasing purification of membrane fractions, there was a corresponding increase in the total recovery of enzyme activity. It is postulated that this may reflect not only an increase in the purification of membranes but may involve the presence of specific galactosyltransferase inhibitors in intracellular membranes of the liver.

An Effect of Puromycin on Galactosyltransferase of Golgi-rich Fractions from Rat Liver

Abstract Some of the properties of galactosyltransferase of Golgi membrane-rich fractions from rat liver were investigated. The kinetic properties of the enzyme were determined from the initial rates of reaction under various conditions of incubation. Puromycyin, a known inhibitor of protein synthesis, was shown to inhibit the galactosyltransferase activity in vitro. The inhibition depended on the concentration of puromycin and on the duration of exposure of Golgi membranes to the drug. A number of compounds, structurally related to puromycin, were unable to produce the inhibition. A combination of the aminonucleoside and amino acid portions of the puromycin molecule was equally ineffective. Binding of puromycin to the membranes was demonstrated with [3H]puromycin. It was concluded that this binding to the membrane disrupted the enzymatic activity.

Studies on the golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by D-galactosamine.

1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100-120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose-N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-(14)C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of galactosamine by: (a) inhibition of protein synthesis leading to a diminution of the endogenous acceptor pool of the galactosyltransferase; (b) inhibition of the galactosyltransferase activity by galactosamine metabolites and (c) replacement of UDP-galactose by UDP-galactosamine.

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg 2+-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate. At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.

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.

N-Acetylglucosaminyltransferase activity in liver, serum, and ovaries of domestic fowl.

Homogenates of the liver, serum, and ovaries of the laying hen and the liver and serum of the immature pullet were examined for N-acetylglucosaminyltransferase activity. The activity in the liver homogenates was higher in the laying hen than in the pullet while their sera showed similar low levels of enzyme activity. The homogenate of the ovary from the laying hen was also found to contain N-acetylglucos-aminyltransferase. Golgi-rich and Golgi-depleted membrane fractions were prepared from the liver homogenates of both types of fowl and examined in the assay system. The Golgi-depleted membrane fractions from the livers of both laying hen and pullet had higher enzyme activities than the corresponding Golgi-rich fractions. The N-acetylglucosaminyltransferase activities in both cellular membrane fractions were higher in the laying hen than in the pullet. The results are compatible with the hypothesis that N-acetylglucosaminyltransferase is involved in synthesis of lipoproteins and/or glycoproteins by the liver and the ovary. In contrast to the rat, the hen enzyme did not show increased activity in the presence of added CDP-choline.

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.