B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

Stereospecificity of NADH-ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells

The stereospecificity of NADH-ferricyanide reductase and NADH-cytochrome c reductase in the endoplasmic reticulum (ER) for the α-hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α-specific NADH-acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time-course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH-ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β-specific and always showed a latency (increase upon addition of Triton X-100), whereas the α-specificity in the ER was non-latent. This is consistent with the presence of mainly right-side-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right-side-out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α-specific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α-specificity (in addition to β-specificity) which was probably due to ER contamination since the activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH-acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.     

Studies on plasma membranes XVII. On the chemical composition of plasma membranes prepared from rat and mouse liver and hepatomas

Summary Plasma membranes were isolated under hypotonic conditions from rat and mouse livers and five hepatomas, i.e. one rather anaplastic rat hepatoma (and its subline) and three well-differentiated mouse hepatomas. All these membranes contained some 25% protein soluble in 0.15m NaCl. Evidence is presented that this protein is mainly, if not exclusively of nonmembranous origin. Protein/phospholipid P (P=phosphorus) ratios did not differ significantly for the various plasma membrane species except the rat-hepatoma subline, which showed a markedly lower ratio and was thus identified. Hepatoma membranes contained more P of a nonphospholipid nature than did liver membranes and to this increase contributed in all instances an increased RNA content and in some cases also an increased DNA content. The presence of DNA in these plasma membranes is artefactual, but that of RNA is more complicated. Artefactually, Ca²⁺-associated RNA of low mol wt and soluble in 0.15m NaCl, and residual RNA (genuine?, in liver membranes less than 1% in respect of protein) have been demonstrated. The increase in hepatoma-membrane RNA is attributed to the ribosomal RNA of the few microsomal vesicles which are structurally connected with these plasma membranes. The sialic acid content and the percentage of neuraminidase-resistant sialic acid of hepatoma as compared with liver membranes was either similar or changed, depending on the hepatoma strain. Gelfiltration of trypsin-released peptides of liver plasma membranes showed hexosamine and hexose to be confined to the sialic acidcontaining fractions. In spite of quantitative differences among fractions, the relative contents of the three carbohydrates in the combined fractions were (about) similar to those in intact liver membranes. Similar experiments with the rat-hepatoma membranes showed a changed carbohydrate expression.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.     

Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction

A structural and biochemical study is presented concerning the agglutination of gametic flagella, the initial step in the mating reaction of Chlamydomonas reinhardtii. An alteration in the distribution of the intramembranous particles revealed by freeze-fracturing of flagella membranes is shown to accompany gametic differentiation in both mating types. The isolation and electrophoretic analysis of flagellar membranes and mastigonemes are reported; no electrophoretic differences can be detected when the membrane or mastigoneme glycoproteins from vegative and gametic cells are compared, nor when glycoproteins from the two mating types are compared, and no novel polypeptides are present in gametic preparations. The membrane vesicles, after they are freed of mastigonemes by sedimentation through a discontinuous sucrose gradient, are extremely active as an isoagglutinin, indicating a direct involvement of the membrane in the mating reaction.     

Purification of plasma membranes from leaves of conifer and deciduous tree species by phase partitioning and free-flow electrophoresis

Purified plasma membrane fractions were obtained from leaves of Picea abies L., Pinus sylvestris L., Fagus sylvatica L. and Quercus robur L., whereas plasma membranes from Pinus halepensis Mill, proved to be more difficult to obtain, perhaps due to the higher content of volatile substances in this plant species. Plasma membranes were purified by both phase partitioning and free-flow electrophoresis from microsomal fractions and identified on the basis of biochemical and in some cases morphological and cytochemical markers. Electron micrographs revealed that membrane vesicles from Pinus sylvestris exhibited a very clear dark-light-dark pattern and measurements of membrane thickness showed that it ranged from 6 to 10 nm. Most membranes were 8 nm thick and stained with phosphotungstic acid at low pH, both typical characteristics of the plasma membrane. Enzymatic identification of plasma membranes consisted in the determination of the vanadate-sensitive ATPase (EC 3.6.1.3) activity. The specific activity in the upper phase (U₂) fraction was 10–25 times higher than those in the lower phase and microsomal fractions, depending on plant species. 1,3-β-glucan synthase II (EC 2.4.1.3), another putative plasma membrane marker, was not detected in the plasma membrane-enriched fractions of conifer needles and showed a very low specific activity in membranes of deciduous trees. Contamination by membranes of other origin was determined by analysis of membrane markers: cytochrome c oxidase (EC 1.9.3.1) for mitochondria, inosine diphosphatase (EC 3.6.1.6) for Golgi apparatus, cytochrome c reductase (EC 1.6.2.4) for endoplasmic reticulum, and pyrophosphatase (EC 3.6.1.1) for tonoplasts. The main, but relatively low contamination, was due to tonoplasts, as determined by the activity of pyrophosphatase. Plasma membrane characteristics were quite different depending on the season during which needles were taken. Membrane preparations of better quality were more easily obtained from samples taken during winter.     

Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.     

Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [¹⁴C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [¹⁴C]SCN⁻ uptake and ³⁶Cl⁻ uptake activities coincided with [¹⁴C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.     

Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types

1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3–2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca²⁺-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/μm² for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/μm². 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca²⁺-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na⁺+K⁺)-dependent ATPase activity and of low amounts of Na⁺-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca²⁺-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg²⁺-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca²⁺-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotėin NADH:cytochrome b₅ reductase (mol.wt. 32000), cytochrome b₅ (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.     

Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.     

Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes

A procedure is described for the isolation of sub-cellular fractions from bloodstream forms of Trypanosoma brucei. The method leaves intact most of the nuclei, mitochondria and microbodies. All the fractions have been chemically characterized and tested for 10 enzymatic markers. About 5% of total cell protein was isolated as a microsomal fraction containing mostly plasma membranes and endoplasmic reticulum vesicles. Plasma membranes were purified by high-speed centrifugation on magnesium-containing Dextran, and on linear sucrose-density gradients. The yield of membranes was approximately 0.3% of the total cell protein. The purified material had a sucrose density of 1.14 g/cm3 and consisted of smooth vesicles. Specific activity of the membrane markers Na+, K+, ouabain-sensitive ATPase and adenylate cyclase were 26- and 20-fold higher, respectively, than in total cells. Neither DNA nor RNA was detected. The sum of the cholesterol and phospholipid content was 0.99 mg/mg protein. The cholesterol/phospholipid molar ratio was 1:2.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Phospholipid asymmetry in rough- and smooth-endoplasmic-reticulum membranes of untreated and phenobarbital-treated rat liver.

Phospholipase C was used as a probe for the distribution of phospholipids about the membrane of rough and smooth microsomal fractions from normal and phenobarbital-treated rat liver. All membranes exhibited an asymmetric distribution, with phosphatidylethanolamine and phosphatidylserine concentrated in the inner leaflet of the bilayer and phosphatidylcholine and sphingomyelin concentrated in the outer leaflet. The only phospholipid showing a significant difference in distribution between fractions was phosphatidylcholine, which was shifted towards the outer leaflet in the smooth microsomal fraction compared with the rough microsomal fraction, and towards the outer leaflet in both rough and smooth microsomal fractions from phenobarbital-treated liver compared with the same preparations from untreated rat liver. Apart from this small change, the asymmetric distribution of phospholipids was conserved in microsomal fractions which had proliferated in response to phenobarbital and in which the protein composition had changed.     

Fractionation of the thylakoid membranes from tobacco. A tentative isolation of 'end membrane' and purified 'stroma lamellae' membranes

Thylakoids isolated from tobacco were fragmented by sonication and the vesicles so obtained were separated by partitioning in aqueous polymer two-phase systems. By this procedure, grana vesicles were separated from stroma exposed membrane vesicles. The latter vesicles could be further fractionated by countercurrent distribution, with dextran-polyethylene glycol phase systems, and divided into two main populations, tentatively named 'stroma lamellae' and 'end membrane'. Both these vesicle preparations have high chlorophyll a/b ratio, high photosystem (PS) I and low PS II content, suggesting their origin from stroma exposed regions of the thylakoid. The two vesicle populations have been compared with respect to biochemical composition and photosynthetic activity. The 'end membrane' has a higher chlorophyll a/b ratio (5.7 vs. 4.7), higher P700 content (4.7 vs. 3.3 mmol/mol of chlorophyll). The 'end membrane' has the lowest PS II content, the ratio PS I/PS II being more than 10, as shown by EPR measurements. The PS II in both fractions is of the beta-type. The decay of fluorescence is different for the two populations, the 'stroma lamellae' showing a very slow decay even in the presence of K3Fe(CN)6 as an acceptor. The two vesicle populations have very different surface properties: the end membranes prefer the upper phase much more than the stroma lamellae, a fact which was utilized for their separation. Arguments are presented which support the suggestion that the two vesicle populations originate from the grana end membranes and the stroma lamellae, respectively.     

Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.     

Isolation of envelope membranes from bundle sheath chloroplasts of maize

Bundle sheath strands were isolated from maize (Zea mays L.) leaves treated with preparations of cellulase, hemicellulase, and pectinase. A three-phase discontinuous gradient yielded two fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm⁻³. The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of electron microscopy. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic, and electron microscopic examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes, and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56-g leaf sections. The Mg²⁺-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 μmoles Pi released hr⁻¹ mg protein⁻¹, a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants.