In vivo incorporation of radioactive metabolites by Golgi apparatus and other cell fractions of onion stem

Incorporation in vivo of various ¹⁴C-labeled substrates into dictyosomes of onion (Allium cepa) stem was determined, and comparisons were made with other cell fractions on a nitrogen basis. Tissue explants were incubated for varying times in the presence of the radioactive metabolites supplied in the external medium. Fractions were then obtained from homogenates stabilized with glutaraldehyde. Purified fractions containing dictyosomes (individual stacks of cisternae) of the Golgi apparatus were obtained by centrifugation in a sucrose gradient also yielding a smooth membrane fraction free of dictyosomes. Dictyosomes were preferentially labeled with choline-1,2-¹⁴C and acetate-2-¹⁴C, suggesting that plant Golgi apparatus participate in the synthesis or modification of membrane lipids. Dictyosomes were also labeled with glucose-U-¹⁴C and leucine-U-¹⁴C, but on a molar basis incorporation was less than with choline or acetate.     

Intracellular localization of N-formyl chemotactic receptor and Mg dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm⁻³. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm^?3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Isolation and Characterization of Components of the Lytic Compartment of Several Plant Tissues Including Separations with a Zonal Rotor

The ultrastructural localization of acid phosphatase in cellsof pea and mung bean root tips, wheat coleoptiles, cauliflowerflorets and potato shoots, occurred in association with theinner surface of membranes of vacuoles, within multivesicularbodies and in the Golgi stacks and associated vesicles. Tissue homogenization techniques were varied and behaviour ofcomponents of the lytic compartment in density gradients ofvarious types was systematically examined. A basic fractionationmethod, employing linear Ficoll density gradients, was developedand modified for routine use with the various plant tissues,and the results compared with those obtained on sucrose gradients.Two predominant bands of components of the lytic compartmentwere isolated; one of these equilibrated at a density of 1·06g cm^–3 and contained vacuoles and tonoplast components,the other band, with an isopycnic equilibrium density of 1·11g cm^–3, was rich in mitochondria, Golgi stacks and associatedvesicles. Sucrose gradients gave broadly similar separations,but of lower resolution, and with increased density of the components.Prior fractionation of homogenates by differential centrifugationgave poor subsequent separations of the lytic compartment indensity gradient ultracentrifugation. The bulk isolation of components of the lytic compartment frompotato shoots and cauliflower curd, using a zonal density gradienttechnique, is described and its possible applications are discussed. An improved method of rapid fixation of fractions enabled theuse of a modified Gomori lead-salt method for ultrastructurallocalization of ß-glycerophosphatase activity in isolatedelements of the lytic compartment. Lytic component, Golgi apparatus, density gradient fractionation, ultrastructure, acid hydrolases, ß-glycerophosphatase, Pisium sativum L., Phaseolus aureus L., Triticum aestivum L., Solanum tuberosum L., Brassica oleracea L.     

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.     

A possible role of Golgi membrane-associated galactosyltransferase in the formation of zymogen granule glycoproteins

A galactosyltransferase activity in smooth microsomes and Golgi membrane-rich fractions from rat pancreas glycosylated endogenous acceptors during incubation with UDP-[¹⁴C]galactose in the absence of exogenous glycoproteins. To evaluate the role of this activity in secretion, the endogenous products were partially characterized. Galactose-labeled fractions were sequentially extracted in 0.2 m NaHCO₃ and 0.25 m NaBr to prepare membranes and soluble acceptors. Bound radioactivity was equally distributed between these two fractions. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the particulate galactose-labeled polypeptides were distinct from the soluble galactose acceptors. Rabbit antisera against highly purified zymogen granule membranes precipitated approximately 40% of the radioactivity of the particulate fraction when solubilized in nonionic detergents. In polyacrylamide gels, the galactose-labeled species of the immunoprecipitate migrated with zymogen granule membrane glycoproteins. Rabbit antisera against secretory proteins cross-reacted with less than 5% of the galactose-labeled soluble acceptors. Mature zymogen granule membranes neither contained detectable galactosyltransferase activity nor served as galactosyltransferase acceptors. These results suggest that galactosyltransferase activity associated with membranes derived from the Golgi complex glycosylated zymogen granule membrane precursors. Analysis of [¹⁴C]galactolipids did not implicate lipid intermediates in this process.     

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.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Effect of Brefeldin A on cell-wall polysaccharide production in the red microalga Porphyridium sp. (Rhodophyta) through its effect on the Golgi apparatus

The cells of the red microalga Porphyridium sp. are encapsulated within a complex sulphated polysaccharide, comprising cell-wall-bound and soluble fractions. The current study investigated the involvement of the Golgi apparatus in the production of the sulphated polysaccharide by treating the cultures with brefeldin A (BFA), a membrane-traffic inhibitor of the Golgi apparatus. Addition of BFA (10–25 μM) upon inoculation (logarithmic-phase cells) decreased the contents of both bound and soluble polysaccharides. Exposure of stationary-phase cultures to BFA (20 μM) inhibited the formation of the cell-wall bound polysaccharide to a greater extent than that of the soluble polysaccharide. Under conditions of nitrate starvation, BFA treatment had a more marked effect on soluble than on bound polysaccharide formation, as was supported by ¹⁴C pulse-chase experiments. BFA addition up to the first 10 h of the cell cycle affected cell division and bound polysaccharide and starch contents. An ultrastructural study showed that exposure of the cells to 20 μM BFA for 16 h disrupted the integrity of the Golgi apparatus. The integrated results of this study demonstrate clearly that BFA affects the architecture of the Golgi apparatus and hence polysaccharide production in algal cells.     

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.     

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Using a polyclonal antiserum specific for the tonoplastic H⁺-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H⁺-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously. Received: 29 June 1998 / Accepted: 9 November 1998     

Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases

Endogenous acceptors for N-acetylglucosamine (GlcNAc), galactose (Gal) or sialic acid (NeuAc) transfer were labeled to high activities when purified hepatic Golgi fractions were incubated with the corresponding radiolabeled nucleotide sugar in the absence of detergent. The in vitro conditions which were optimal for the endogenous glycosylation of GlcNAc and Gal acceptors (Mn²⁺, ATP) also promoted fusion within a subset of Golgi membranes. Electron microscope radioautography revealed that the majority of NeuAc acceptors were associated with unfused Golgi membranes, whereas the majority of Gal acceptors were localized to fused membranes. GlcNAc acceptors were approximately equally distributed between fused and unfused membranes. Under conditions in which Golgi membrane fusion was absent (− Mn²⁺), only NeuAc transfer was active. The majority of endogenous NeuAc acceptors were consequently assigned to the more trans regions of the hepatic Golgi apparatus as concluded from a combination of radioautography (NeuAc transfer) and acid NADPase cytochemistry (reactive medial and trans Golgi saccules). The distribution of NeuAc and Gal transferases was assessed after Percoll gradient centrifugation of disrupted Golgi fractions. The median density of NeuAc transferase was lower than that of Gal transferase. The studies are indicative of distinct Golgi components harboring the majority of acceptors and enzymes for terminal glycosylation.     

Acyl transfer reactions associated with cis Golgi apparatus of rat liver

Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A₂ on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [¹⁴C]lysophosphatidylcholine to [¹⁴C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37°C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine.     

Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles

Membranes of corn (Zea mays, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient centrifugation and the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase was selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelles. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCl, Mg²⁺-ATPase of the PM-rich fraction from a step gradient was 6.0 to 6.5, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To reduce PM contamination, Golgi-rich fractions obtained from step or rate-zonal gradients were recentrifuged isopycnically on linear sucrose gradients. The distribution of KCl, Mg²⁺-ATPase activity was measured at pH 6.5 and 7.5. The pH 6.5 ATPase was coincident with UDPG-sterol glucosyl transferase, a PM marker, while the pH 7.5 ATPase overlapped with latent UDPase, a Golgi marker. These results provide strong evidence for a KCl, Mg²⁺-ATPase, active at pH 7.5, associated with the Golgi membranes of corn coleoptiles.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)     

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.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Distribution of insulin receptors among mouse liver endomembranes

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. ¹²⁵I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10–20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1–2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0 · 10⁸ M^?1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na⁺ + K⁺ + Mg²⁺)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.