Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver golgi apparatus

Neuraminidase and galactosyltransferase were investigated in total Golgi apparatus and in the three fractions of increasing densities (GF₁, GF₂ and GF₂) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45–72). About 50% decreases in neuraminidase content (units/g liver) and specifixc activity (units/ mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (intraperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123–125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed in Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.     

CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF₁, GF₂, GF₃) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF₁ and GF₂, and along the outside of the cisternal membranes in GF₃. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF₁ and within many VLDL-filled vacuoles in GF₁ and GF₂, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF₃ and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF₁ and GF₂, and was not found in the cisternae in GF₃. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF₁ and GF₃, representing primarily trans-Golgi elements from the secretory Golgi face, and GF₃ consisting largely of cis-Golgi components from the opposite face.     

Subcellular distribution of the mannan-binding protein and its endogenous inhibitors in rat liver

The subcellular distribution of the mannan-binding protein from rat liver, a lectin specific for mannose and N-acetylglucosamine, was studied. Approximately 75% of the binding activity of the homogenate was recovered in microsomes, approximately 76% of which was accounted for by rough microsomes. Rough microsomes had the highest specific activity of binding, followed by the Golgi apparatus and smooth microsomes, whereas plasma membranes, lysosomes, mitochondria, and the soluble fraction had little or no binding activity. A topographical survey indicated that the binding protein was localized exclusively on the cisternal surface of microsomal vesicles. Thus, the binding protein of microsomal vesicles was protected from protease digestion and was released from the vesicles by mild detergent treatment. Competitive inhibitors, which presumably represent endogenous ligands of the binding protein, were found among subcellular fractions. More than 50% of the inhibitory activity of the homogenate was recovered in rough microsomes, while the highest specific activity of inhibition was found in lysosomes. The K_i values estimated for rough microsomes and lysosomes were 25.9 and 8.67 μg/ml, respectively. The distribution profiles of inhibitors were correlated roughly with those of the binding protein, resulting in masking of the binding activity in organelles up to the level of 86%. On the basis of the known localization and topology of the binding protein and endogenous inhibitors (ligands), possible physiological functions of the binding protein relevant to the transport of biosynthetic intermediates of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus and from the Golgi apparatus to lysosomes were discussed.     

GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES : I. Isolation Procedure and Morphological Characterization

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ~90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ~13 x 10⁶ g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.     

Subcellular distribution of α1-adrenergic receptors in rat liver

The distribution of α₁-adrenergic receptors in rat liver subcellular fractions was studied using the α₁-adrenergic receptor ligand [³H]prazosin. The highest number of [³H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane ‘marker’ enzymes in the same fractions a relative enrichment of [³H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with ¹²⁵I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [³H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED₅₀ values for displacement of [³H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10⁻⁶, 10⁻⁵ and 10⁻⁴ mol/l, respectively. On the basis of lack of correlation between distribution of α₁-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of α₁-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [³H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that α₁-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Isolation and Characterization of Golgi Membranes from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

A simple and rapid technique was developed for the isolationof the vesicular Golgi membranes from suspension-cultured cellsof sycamore (Acer pseudoplatanus L.). The procedure involvespreparation of protoplasts and differential centrifugation ofdisrupted protoplasts followed by the sucrose density gradientcentrifugation. Starting from broken protoplasts, sedimentableat two different centrifugal forces (10,000g and 100,000 g),two Golgi-enriched fractions of lower density, GF₁ and GF'₁,and higher density, GF₂ and GF'₂, were separated. Purity ofthe fraction was assessed by determining the marker enzyme activitiesas well as the electron microscopy of the specimens obtained. Inosine diphosphatase was enriched about 15- and 6-fold, respectively,in the GF₂ fraction from 10,000g and the GF'₂ one from 100,000gpellets, whereas the enrichment in GF₁ and GF'₁ was approximately6–7 fold. Galactosyl-transferase in GF₂ was enriched about25-fold. GF₁ and GF₂ account for 3–4% of the total proteinof 10,000g pellets, and GF'₁ and GF'₂ for about 6–7%of the total protein of 100,000g pellets. Electron microscopicobservations show that GF₂ and GF'₂ consisted principally ofvesicular Golgi membranes without an internal matrix althoughGF₁ and GF'₁ were contaminated with ER membranes and ribosomes. (Received March 11, 1985; Accepted June 17, 1985)     

Distribution of different forms of sterols in three cellular subfractions of Calendula officinalis leaves

From Calendula officinalis leaves three cellular subtractions (mitochondrial, Golgi membranes and microsomal) were obtained and enzymatically characterized. The contents of Δ⁰, Δ⁵, Δ⁷, Δ^{5, 22} sterols, as well as those of 24-methylenecholesterol and clerosterol, in the free and bound in the form of esters, glucosides and acylated glucosides were determined in these fractions. The results revealed the predominance of free sterols in the microsomal fraction, of esters in the mitochondrial fraction and of steryl glucosides and acylated glucosides in the Golgi fraction.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.     

Properties of a NADH-dependent N-hydroxy amine reductase isolated from pig liver microsomes

A multicomponent enzyme system that catalyzes the reduction of hydroxylamine and a number of its mono- and disubstituted derivatives by NADH has been isolated from microsomes. Three protein fractions isolated from pig liver microsomes are required to reconstitute NADH-hydroxylamine reductase activity. Two of the proteins appear identical with detergent-extracted cytochrome b₅, and its flavoprotein reductase. The third protein fraction required for activity differs from previously isolated microsomal proteins. This fraction is free from detectable chromophores that absorb in the visible region of the spectrum and also appears free from metals. The properties of the NADH-hydroxylamine reductase reconstituted with the three components isolated from microsomes appears similar to the particle-bound system with respect to nucleotide and N-hydroxylamine substrate specificity.     

The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.     

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F₀ to F₁ ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N′-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.     

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

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 Mg²⁺-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.