DIFFERENCES IN THE SUBCELLULAR AND SUBSYNAPTOSOMAL DISTRIBUTION OF THE PUTATIVE ENDOPLASMIC RETICULUM MARKERS, NADPH-CYTOCHROME c REDUCTASE, ESTRONE SULFATE SULFOHYDROLASE AND CDP-CHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE IN RAT BRAIN

Abstract— A comprehensive study has been undertaken on the subcellular and subsynaptosomal distribution of a number of markers for subcellular organelles in preparations from rat brain. Although the activity of most enzymatic markers was decreased by freezing and storage at - 70^oC, no significant changes were noted in the distribution of these activities. This demonstrates that contamination of brain fractions by subcellular organelles can be accurately assessed after freezing and thawing. A marked discrepancy was noted between the distribution of three putative markers for endoplasmic reticulum. CDP-choline-diacylglycerol cholinephosphotransferase (EC 2.7.8.1) activity was mainly limited to the microsomal fraction and was present to a lesser extent in the synaptosomal fraction than the other putative markers for endoplasmic reticulum. Estrone sulfate sulfohydrolase (EC 3.1.6.2) activity demonstrated a bimodal distribution between the crude nuclear and microsomal fractions. However, considerable activity was associated with the synaptosomal fraction. NADPH-cytochrome c reductase (EC 2.3.1.15) activity sedimented in the microsomal and the synaptosomal fractions. Calculations based on the relative specific activities of the microsomal and synaptic plasma membrane fraction indicated that the contamination of the synaptic plasma membranes by endoplasmic reticulum was 44.5% (NADPH-cytochrome c reductase), 38.0% (estrone sulfatase) and 9.0% (cholinephosphotransferase). Since it is believed that virtually all of the synthesis of phosphatidylcholine by cholinephosphotransferase occurs in the neuronal and glial cell bodies, it was concluded that cholinephosphotransferase is a satisfactory marker for the endoplasmic reticulum derived from these sources. The results suggest that NADPH-cytochrome c reductase and estrone sulfatase may be present in the smooth endoplasmic reticulum system responsible for the fast transport of macromolecules along the axon to the nerve endings as well as in the endoplasmic reticulum of the cell bodies. The possible relation between that portion of the smooth endoplasmic reticulum involved in fast axonal transport and the GERL (Golgi, Endoplasmic Reticulum, Lysosomes) complex discovered by Novikoff and his coworkers (Novikoff , 1976) is discussed.     

Labeling and isolation of plasma membranes from corn leaf protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [¹²⁵I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg²⁺-dependent, K⁺-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Prostaglandins E₁ and E₂ are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E₂ binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine β-hydroxylase). The only other fractions enriched in prostaglandin E₂ binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E₂ binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.     

Subcellular localization of nuclease in barley aleurone

The intracellular localization of an endonuclease (nuclease I) in barley aleurone responding to gibberellic acid was investigated by subcellular fractionation and immunocytochemistry with monoclonal and polyclonal antibodies. Organelle separations were performed with aleurone layers and protoplasts; immunefixations were carried out on protoplasts only. Nuclease was detected in fractions from isopycnic sucrose density gradients which were enriched in either endoplasmic reticulum or Golgi apparatus membranes. These two organelles were also labelled by the indirect immunogold method on thin sections. Intensive labelling of protein and developing vacuoles was observed. Therefore, as noted in other plants nuclease in barley is essentially a vacuolar enzyme.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Identification of secretory vesicles in homogenates of pea stem segments

In homogenates of stem sections from etiolated pea (Pisum sativum L.) seedlings, secretory vesicles can be separated from Golgi-apparatus cisternae by rate-zonal centrifugation in renografin gradients. Optically, two bands of turbidity are observed, the uppermost containing the secretory vesicles and the lower one the Golgi-apparatus cisternae. The absence of glutaraldehyde in the homogenizing medium has allowed the effective characterization of marker-enzyme activities. Golgi-apparatus cisternae have been recognized by the presence of inosine-diphosphatase and glucan-synthase I activities as well as by electron microscopy. In contrast, although secretory vesicles also bear inosine diphosphatase they do not appear to possess glucan-synthase activity. Three plasma-membrane markers, NPA-binding, glucan synthase II, and KCl,Mg²⁺-adenosine triphosphatase (pH 6.5), were not detected in secretory vesicles. Pulse-chase experiments with [³H]glucose support our designation of secretory vesicles and Golgi-cisternal fractions.Abbreviations ER endoplasmic reticulum - GSI, GSII glucan, synthase I, II, respectively - IDPase inosine diphosphatase - PM plasma membrane - SV(s) secretory vesicle(s)     

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     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

The ultrastructure of cellular division (autosporogenesis) in the coccoid green alga,Trebouxia aggregata,revealed by rapid freeze fixation and freeze substitution

Summary Adequate ultrastructural preservation of cells of the green algaTrebouxia aggregata is achieved by immersion freeze fixation using liquid propane followed by freeze substitution and resin embedding at ambient temperature. Despite differential staining of membranes, using this method we have been able to study plasma membrane biogenesis during cellular division. Daughter protoplasts are separated by an ingrowing septum of plasma membrane that extends into the cell from a particular site at the peripheral plasma membrane marked by centrioles. Septum development involves tip growth followed by lateral growth. This growth seems to involve transfer of membrane from an adjacent partially coated reticulum to the septum plasma membrane. The reticulum which extends from nearby Golgi stacks to the area of septum growth is associated with an extensive array of microtubules. After daughter protoplasts are completely separated, each one becomes surrounded by a cell wall which is distinct from the persisting mother wall. The ultrastructural evidence suggests that cells ofT. aggregata are autospores rather than vegetative cells.Abbreviations C centriole - ER endoplasmic reticulum - G Golgi body - MTOC microtubule organizing center - Mt(s) microtubule(s) - N nucleus - P primary septum - PCR partially coated reticulum - PM plasma membrane - Py pyrenoid - S septum     

Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of a K ATP + channel

A valuable method to isolate and purify mitochondria from embryonal masses of two coniferous species (Picea abies [L.] Karst. and Abies cephalonica Loud.) is described. Crude mitochondria from both species were shown to be intact, oxygen consuming (with malate plus glutammate, succinate and NADH as substrates) and well coupled (respiratory control ratio ca. 4). The oxidation of the substrates was only partially KCN-insensitive (alternative oxidase) in some cases. However, these fractions were contaminated by membranes (e.g. plasmalemma, tonoplast, Golgi and endoplasmic reticulum). After purification by a discontinuous Percoll gradient (18, 23, 40%, v/v), three mitochondrial populations were separated. The 0/18 interface fraction was composed mainly of broken and uncoupled mitochondria, while the other two (18/23 and 23/40 interface fractions) contained intact and coupled mitochondria, but only 23/40 interface fraction revealed to be better purified starting from both coniferous embryonal masses. In the latter purified fraction, the presence of a cyclosporin A-sensitive K_ATP⁺ channel was demonstrated. These findings were discussed in the light of the potential use of these mitochondrial fractions in bioenergetic studies, or in the involvement of these organelles to stress response in conifers.     

Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg²⁺ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[¹⁴C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[¹⁴C]mannose, [¹⁴C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [¹⁴C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [¹⁴C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [¹⁴C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER endoplasmic reticulum - TCA trichloroacetic acid - SDS sodium dodecylsulphate - GDP guanosine diphosphate     

Role of the cell wall in the ability of tobacco protoplasts to form callus

Cellular membranes from dark grown hypocotyls of Phaseolus aureus Roxb. were separated by centrifugation on a continuous sucrose gradient. Each gradient fraction was monitored for activity of inosine diphosphatase (EC 3.6.1.6) and the ability to transfer glucose from UDP-[¹⁴C]glucose to endogenous lipids in vitro. The highest incorporation of radioactivity into lipids occurred in a particulate fraction correlated with the Golgi apparatus, sedimenting at sucrose densities of 31.5–33% w/w. Three endogenous lipids were glucosylated in vitro. The two main lipids were characterized as steryl glucoside and acylated steryl glucoside; data from chromatography and hydrolysis of the third lipid suggests that it is dolichyl-monophosphate-glucoside. Steryl glucoside was found to be the main glucoside synthesized, but the proportion of the acylated form increased with time. The results are discussed in the context of the role of the Golgi apparatus as a centre of membrane modification within the plant cell.Abbreviations DMP-mannose dolichyl monophosphate mannose - ER endoplasmic reticulum - GA Golgi apparatus - ID-Pase inosine diphosphatase     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-³H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.     

Highly enriched,minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture

A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5′-nucleotidase and ouabain-sensitive (Na⁺ + K⁺)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.     

The Glucan Synthases from Suspension-Cultured Sugar Cane Cells

Organelles from 10 g phase suspension-cultured sugar cane cellshave been analysed by isopyenic sucrose density gradient centrifugation.The distribution profiles for marker enzymes have allowed therecognition of tonoplast, endoplasmic reticulum, Golgi apparatus,plasma membrane, mitochondrial and microbody fractions. In thissystem the glucan synthases I and II, which have previouslybeen regarded as specific marker enzymes for the Golgi apparatusand plasma membrane respectively, show a two-peak profile inthe gradient. For each glucan synthase the peaks correspondroughly with the positions of the Golgi apparatus and plasmamembrane. Analysis of the in vitro synthesized polymers fromthe glucan synthase assay indicates that a mixed-linked (ß,l 3; ß l 4) glucan is produced by both organelle fractions.Supported by individual observations from other authors we suggestthat, in the case of members of the Gramineae, the allocationof the two glucan synthases to two different membrane fractionsis not possible. Key words: Golgi apparatus, Glucan synthases, Plasma membrane, Sugar cane cells     

Transition vesicle formationin vitro

Summary Isolated fractions enriched in transition elements derived from part rough—part smooth regions of endoplasmic reticulum of rat liver respondin vitro to ATP plus a concentrated fraction of cytoplasmic proteins by formation of ca. 60 nm vesicles with nap-like coats resembling those of transition vesicles of the intact cell. Similar vesicles are normally considered to function in the transfer of materials from endoplasmic reticulum to cis elements of the Golgi apparatus.