Purification of endoplasmic reticulum from wheat roots and shoots (Triticum aestivum). Comparison of their blue light-sensitive redox components with those of highly purified plasma membrane

Plasma mebranes (PM) and endoplasmic reticulum (ER) were prepared from 4.5-day-old, light-grown wheat ( Triticum aestivum L. cv. Drabant) shoots and roots, using phase partitioning for the PM, which yields very pure PM preparations, and sucrose gradient centrifugation for the ER. Also the ER fractions were highly purified, being totally free from mitochondria (cytochrome c oxidase, EC 1.9.3.1), PM (glucan synthase II, EC 2.4.1.34) and thylakoid membranes (chlorophyll), and with a low content of tonoplast (nitrate-sensitive ATPase) and Golgi (latent IDPase). Sodium dodecyl sulphate polyacrylmide gel electrophoresis of root ER resulted in a similar polypeptide pattern as for shoot ER, but very different from the crude fraction from which the ER fractions were purified, also indicative of a high purity. The PM and ER preparations were compared with respect to their blue light-sensitive flavoprotein-cytochrome b . About half of the dithionite-reducible cytochrome b of shoots (PM as well as ER) and root PM was light-sensitive, compared to only 10–20% of that of root ER. Shoot PM needed 2–3 times less light compared to the other membranes, for half saturation of the reaction. NADH-cytochrome c reductase activity was found with all four membrane fractions, with ER having activities 5–10 times higher than PM. The relevance of photoreducible components in the PM and the ER is discussed in connection with blue light photobiology.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

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.     

Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.     

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.     

Identification and characterization of Golgi equivalents fromAllomyces macrogynus

Golgi equivalents similar to those described in other fungi were identified in freeze substituted hyphae ofAllomyces macrogynus. These Golgi equivalents were composed of individual or a few loosely associated cisternae, were surrounded by vesicles, and were in a zone relatively free of ribosomes. Certain smooth cisternae in both vegetative hyphae and gametangia stained positively for the Golgi marker enzyme thiamine pyrophosphatase. Subcellular fractionation and biochemical analysis of vegetative hyphae and gametangia revealed endoplasmic reticulum and Golgi membrane fractions with average buoyant densities of 1.09 and 1.15 g/cm³, respectively. Enriched membranes obtained by differential centrifugation were further purified by ultracentrifugation on sucrose step gradients to obtain a presumptive Goldi fraction. Gel electrophoresis of both crude homogenates and fractions prepared by differential centrifugation demonstrated stage-specific glycoproteins that bind the lectin concanavalin A. The results demonstrated thatA. macrogynus has a Golgi complex composed of structurally simple Golgi equivalents and that these Golgi equivalents have physiological functions, such as glycoprotein processing, typically associated with stacked Golgi cisternae from other organisms.     

Characterization,enzymatic and lectin properties of isolated membranes from Phaseolus aureus

Cellular membranes were prepared from the non-extending part of dark grown hypocotyls of Phaseolus aureus. The relative effectiveness of continuous and discontinuous sucrose gradient centrifugation for the separation of membranes was investigated. Characteristic densities of membranes were determined by the localization of enzyme activities on continuous sucrose gradients: NADH-cytochrome c-reductase for endoplasmic reticulum, β-1-3-glucan synthetase for plasma-membrane and IDPase for dictyosomes. The diffuculties involved in the application of ATPase and IDPase as specific membrane markers are discussed. Negative staining of isolated fractions indicated that intact dictyosomes could be prepared from this tissue without the use of chemical fixatives in the homogenization medium.Extraction of isolated membranes showed that carbohydrate-binding proteins (lectins) were present both in an easily removable and in a more strongly bound form. In vivo incorporation of d-[U-¹⁴C]glucose and subsequent isolation and solubilization of the different membranes showed that sugar-containing polymers could be released without hydrolytic techniques and were present in the equivalent extracts that exhibited lectin activity. The possibility of lectin-polysaccharide complexes in endoplasmic reticulum and dictyosomes and their involvement in the synthesis and transport of secretory substances by the membranes is discussed.     

Characterization, enzymatic and lectin properties of isolated membranes from Phaseolus aureus.

Cellular membranes were prepared from the non-extending part of dark grown hypocotyls of Phaseolus aureus. The relative effectiveness of continuous and discontinuous sucrose gradient centrifugation for the separation of membranes was investigated. Characteristic densities of membranes were determined by the localization of enzyme activities on continuous sucrose gradients: NADH-cytochrome c-reductase for endoplasmic reticulum, beta-1-3-glucan synthetase for plasma-membrane and IDPase for dictyosomes. The difficulties involved in the application of ATPase and IDPase as specific membrane markers are discussed. Negative staining of isolated fractions indicated that intact dictyosomes could be prepared from this tissue without the use of chemical fixatives in the homogenization medium. Extraction of isolated membranes showed that carbohydrate-binding proteins (lectins) were present both in an easily removable and in a more strongly bound form. In vivo incorporation of D-[U-14C]glucose and subsequent isolation and solubilization of the different membranes showed that sugar-containing polymers could be released without hydrolytic techniques and were present in the equivalent extracts that exhibited lectin activity. The possibility of lectin-polysaccharide complexes in endoplasmic reticulum and dictyosomes and their involvement in the synthesis and transport of secretory substances by the membranes is discussed.     

Isolation of dictyosomes fromEuglena gracilis

Summary A method for the isolation of dictyosomes fromEuglena gracilis Klebs strain Z (Pringsheim) is described. An extensive Golgi system, with the individual dictyosomes commonly containing ten to twenty cisternae is present. Log phase cells are broken in a French pressure cell at 105 to 120 kg/cm₂ in a breaking mix containing sucrose, sorbitol and ficoll. Addition of 0.3% of glutaraldehyde or formaldehyde to the breaking mix increases the number of stacked cisternae present in the final preparation. In addition to membrane stacks, the fractions contain numerous smooth vesicles. Swollen cisternae, which are also present, may account for these vesicles. Three dictyosome-enriched fractions are obtained by centrifugation in a discontinuous sucrose gradient. Fractions differ morphologically in the degree of stacking of cisternae. Further identification of the membrane fractions was accomplished by measuring IDPase activities in each of the fractions. Inosine diphosphatase activity is enriched 8–10-fold relative to the initial homogenate. The highest IDPase activity was present in the fraction containing the greatest number of stacked cisternae.     

Purification of liver membranes highly enriched in phosphatidylinositol kinase.

Membranes highly enriched in phosphatidylinositol (PtdIns) kinase were purified from rat liver by sucrose density gradient centrifugation of a plasma membrane-depleted microsomal fraction. PtdIns kinase-containing membranes had a lower density than membranes containing Golgi and plasma membrane markers, both in sucrose and Nycodenz gradients, without being completely resolved from these other membranes. They also had a lower density than an endosomal marker. Furthermore, lectin affinity partitioning showed that PtdIns kinase did not reside in plasma membranes. PtdIns kinase in different membrane fractions was of type II and had similar kinetic properties. We suggest that the isolated membranes are the major site for phosphatidylinositol 4-phosphate formation in the liver cell, and that these membranes are part of the exocytic pathway. Thus, PtdIns kinase might be a convenient marker for the exocytic process.     

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.     

Localization of Mannose, TV-Acetylglucosamine and Galactose in the Golgi Apparatus, Plasma Membranes and Cell Walls of Scenedesmus acuminatus

The localization of lectin binding sites in the Golgi apparatus,plasma membranes and cell walls of Scenedesmus acuminatus wasinvestigated by cytochemical electron microscopy. The lectinsused were concanavalin A (Con A), peanut agglutinin (PNA) andwheat germ agglutinin (WGA), all labeled with gold. Con A-goldparticles were deposited not on the Golgi apparatus, but onthe outer cell-wall layer. PNA-gold and WGA-gold particles weredeposited on distal Golgi cisternae and vesicles derived fromthe Golgi apparatus. Entire cell-wall layers were evenly labeledby PNA-gold. The plasma membrane and cytoplasmic regions closeto the plasma membrane were labeled with WGA-gold. The processingof oligosaccharide in the Golgi apparatus, plasma membranesand cell walls of Scenedesmus acuminatus is discussed in referenceto that reported for animal cells. (Received March 5, 1987; Accepted July 18, 1987)     

Structure and Function of the Golgi Complex in Rice Cells: Characterization of Golgi Membrane Glycoproteins

The structure and synthesis of the saccharide chains of Golgimembrane glycoproteins in suspension-cultured rice (Oryza sativaL.) cells were studied. Peanut lectin (PNA) and Ulex europaeuslectin-I (UEA-I) have high affinity for typical O-linked saccharidechains and both recognized the saccharide chains of rice Golgimembrane glycoproteins. These glycoproteins were also sensitiveto alkali and to O-glycanase. These results indicate that theGolgi membrane glycoproteins have O-linked saccharide chains.Brefeldin A, a specific inhibitor of Golgi-mediated secretion,induced morphological changes in Golgi complexes and preventedthe synthesis of the saccharide chains of the membrane glycoproteinsthat could be recognized by PNA and UEA-I. These glycoproteinswere typically localized in all compartments of the Golgi complex.Monensin can arrest the transport of secretory proteins frommedial to trans Golgi compartments but did not affect the formationand localization of the Golgi membrane glycoproteins. Tunicamycin,an inhibitor of the synthesis of N-linked saccharide chains,did not inhibit the synthesis of the saccharide chains of theseGolgi membrane glycoproteins. These results strongly suggestthat the synthesis of O-linked saccharide chains of Golgi membraneglycoproteins is initiated in the cis Golgi compartment. (Received September 24, 1992; Accepted June 4, 1993)     

Separation of K+-and Cl --selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K⁺channels, and a 148-pS Cl^– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K ⁺-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K ⁺ channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K⁺ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl^–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K⁺ channel, an 8-pS K⁺channel and a 4-pS K⁺ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl^–) channel, potassium (K⁺) channel, planar lipid bilayer, root, rye, Secale cerealeL.     

Synaptic junctional glycoconjugates from chick brain

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins.     

Combined biochemical and cytological analysis of membrane trafficking using lectins

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz–sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein β-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER–Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.     

Properties of Membranes from the Halophyte Suaeda maritima: II. DISTRIBUTION AND PROPERTIES OF ENZYMES IN ISOLATED MEMBRANE FRACTIONS

Membrane fractions from the shoots of Suaeda maritima have beenseparated by sucrose gradient centrifugation. Certain stainingand enzymatic activities were studied with the aim of identifyingspecific membrane types, particularly the plasma membrane. Chloroplastand mitochondrial membranes were largely distributed in thedenser regions of the gradient ( 1.16). A plasma membrane fractionwas not easily identifiable and the problems of separating thesemembranes from plant cells are discussed.     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Separation and localization of two classes of auxin binding sites in corn coleoptile membranes

Summary Further evidence is presented for the discrete nature of the two classes of high affinity auxin binding sites in corn (Zea mays L.) coleoptile membranes, site 1 and site 2. Fractions can be obtained by differential centrifugation that exhibit binding kinetics characteristic of site 2, but not site 1. Membrane preparations containing both binding sites may be resolved on sucrose gradients into a light and a heavy band, whose binding kinetics and analogue binding specificities correspond to those deduced for site 1 and site 2 respectively in unfractionated membranes. Evidence from enzymic and chemical assays and from electron microscopy suggests that site 2, the auxin-specific binding site, is located in fractions enriched in plasma membrane, whereas site 1 is associated with Golgi membranes and/or endoplasmic reticulum.Abbreviations NAA 1-naphthylacetic acid - IAA 3-indolylacetic acid - TIBA 2,3,5-triiodobenzoic acid - SDH succinic dehydrogenase - IDPase inosine diphosphatase