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The phospholipid/protein ratios of rat liver peroxisomes, mitochondria and microsomes were determined and found to be 257 +/- 26, 232 +/- 20 and 575 +/- 20 nmol.mg-1, respectively. After correction for the loss of soluble protein, a peroxisomal ratio of 153 nmol.mg-1 was calculated. Organelle fractions were treated with sodium carbonate, whereafter membrane fragments containing integral membrane proteins were pelleted. For the membrane fractions of peroxisomes, mitochondria and microsomes phospholipid/protein ratios of 1054 +/- 103, 1180 +/- 90 and 1050 +/- 50 nmol.mg-1 were found, whereas 26 +/- 2, 20 +/- 2 and 49 +/- 2% of the organelle protein was recovered in these membrane fractions, respectively. The phospholipid composition of the different organelle fractions were determined, but no large differences were obtained, except for cardiolipin that was found only in the mitochondrial fraction. After sodium carbonate treatment virtually all enzymatic activity of the enzymes tested was lost. Therefore Triton X-114 phase separation was used to obtain the peroxisomal membrane components. In this fraction 42.9 +/- 3.5% of the protein and 90.2 +/- 3.7% of the phospholipid was found. Enzymatic activity of two integral membrane proteins was recovered for over 90% in the membrane fraction, whereas activity of two matrix proteins was mainly found in the soluble fraction. Urate oxidase, the peroxisomal core protein, behaved differently and was recovered mainly with the membrane components. Recoveries of enzymatic activities after the Triton X-114 phase separation varied from 45 to 116%, and together with the good separation that was obtained between soluble proteins and integral membrane proteins this method provides a useful alternative for the isolation of membrane components.

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.

By immunolabeling of cryosections, we have characterized in rat cardiac myocytes the cation-independent mannose-6-phosphate receptor (MPR), a lysosomal membrane glycoprotein, lgp120, and a lysosomal enzyme, MEP (homologous to cathepsin L). Most of the MPR label was located in large membrane-filled structures (MPR structures) in large clusters of mitochondria adjacent to but distinct from the Golgi complex. Lpg120 and MEP showed typical lysosomal localization throughout the cell, often associated with regions that appeared to contain autophagosome-like structures. In addition, MEP and lgp120 co-localized within MPR structures. MEP and MPR were localized inside the lumen of MPR structures. MPR was associated mostly with inner membranes, whereas lgp120 was predominantly bound to the outer limiting membrane. MPR, lgp120, and MEP were not detected in Golgi stacks, but some labeling was seen in the putative TGN. Our data suggest that the MPR structures are prelysosomes involved in lysosomal enzyme targeting in rat cardiac myocytes.

The ordering state and changes in fatty acid composition of microsomal (MS) and mitochondrial (MC) membranes of two dominant temperature-sensitive (DTS) lethal mutations and the wild-type Oregon-R strain larvae of Drosophila melanogaster have been studied at 18 and 29 degrees C and after temperature-shift experiments. The membranes of wild-type larvae have a stable ordering state, with "S" values between 0.6 (18 degrees C) and 0.5 (29 degrees C) in both membranes which remained unchanged in shift experiments, although the ratios of saturated/unsaturated fatty acids were changed as expected. The strongly DTS mutation 1(2) 10DTS forms very rigid membranes at the restrictive temperature (29 degrees C) which cannot be normalized after shift down, while shift up or development at the permissive temperature results in normal ordering state. This mutant is less able to adjust MS and MC fatty acid composition in response to the growth temperature than the wild type. The less temperature-sensitive 1(2)2DTS allele occupies an intermediate state between Oregon-R and 1(2)10DTS in both respects. We assume and the genetical data suggest that the DTS mutant gene product is in competition with the wild-type product, resulting in a membrane structure which is not able to accommodate to the restrictive temperature.

The receptor protein for the mitochondrial protein precursor synthesized in the cytosol was extensively purified from the mitochondrial membrane fraction by affinity column chromatography using a synthetic peptide containing the extrapeptide of ornithine aminotransferase as a ligand. The purified fraction contained two major proteins with molecular masses of 52 and 29 kDa. Of these proteins, only the 29 kDa protein bound to the extrapeptide of ornithine aminotransferase. Furthermore, anti-29 kDa protein Fab fragments inhibited the import of pre-ornithine aminotransferase into mitochondria, suggesting that the 29 kDa protein plays an essential role in the process of import of the mitochondrial protein precursor.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

Ultrastructural localization of InsP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. We noticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. We suggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like InsP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.

Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes was determined from the increase of extracellular Mg2+ concentration or decrease of intracellular Mg2+ content, as measured by means of atomic absorption spectrophotometry. Mg2+ efflux was specifically combined with the uptake of Na+ at a stoichiometric ratio of 2Na+:1Mg2+, indicating electroneutral Na+/Mg2+ antiport. Na+/Mg2+ antiport depended on intracellular ATP and was inhibited by amiloride and quinidine, but was insensitive to strophanthin. Net Mg2+ efflux was only occurring at increased concentration of intracellular Mg2+ ([Mg2+]i), and stopped when the physiological Mg2+ content was reached. Intracellular Mg2+ acted cooperatively with a Hill coefficient of 2.4, which may indicate gating of Na+/Mg2+ antiport at increased [Mg2+]i. At increased intracellular Na+ concentration, Na+ competed with intracellular Mg2+ for Mg2+ efflux and Na+ could leave the rat erythrocyte via this transport system. Na+/Mg2+ antiport was working asymmetrically with respect to extra- and intracellular Na+ and Mg2+, and did not perform net Mg2+ uptake.

Recently, analysis of protein distribution in rat brain mitochondria suggested the existence of distinct cholesterol domains in the outer membrane (Dorbani et al., 1987, Arch. Biochem. Biophys. 252, 188-196) while such domains were not detected in rat liver mitochondria (Jancsik et al., 1988, Arch. Biochem. Biophys. 264, 295-301). We studied cholesterol distribution in both types of mitochondria by analyzing the kinetics of filipin-cholesterol complex formation, using the stopped-flow technique. In liver mitochondria, the kinetics are characterized by a biphasic curve which presumably corresponds to the two membranes. This was confirmed by the finding that pretreatment with digitonin abolished one of the kinetic components. Sonication of the mitochondria increased the rate of the filipin-cholesterol complex formation and also abolished one of the two components. In the case of brain mitochondria, several distinct cholesterol domains could be revealed: one of them was cholesterol-free and it was directly accessible to filipin. Two other domains were revealed by differences found in the rate of the cholesterol-filipin complex formation. It is noteworthy that only a part of the cholesterol is accessible to filipin. Sonication of mitochondria decreased the proportion of cholesterol molecules accessible to filipin. This suggests specific interactions of cholesterol with other mitochondrial components, which occur only in brain mitochondria.

The isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes were characterized by lectin cytochemistry using concanavalin A (ConA), Ricinus communis agglutinin 120 (RCA-120), and wheat germ agglutinin (WGA). We found that RCA-120, ConA, and WGA bind to these membranes. The distribution of the lectins on the isolation membranes was heterogeneous, mainly found on the rims, which we referred to as the peripheral dilated portion. When the rims fused and thus formed autophagosomes the apparent sites of fusion were strongly labeled by the lectins. After autophagosomes were transformed to autolysosomes by fusion with lysosomes, the limiting membranes became more densely and homogeneously labeled with the lectins. We previously reported that cytochrome P-450 does not exist on the limiting membranes of the autophagosomes. Taken together, these results suggest that the isolation membranes may originate not from endoplasmic reticulum membranes but from some post-Golgi membranes that contain complex type N and/or O-linked oligosaccharide chains.

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class.

A method for fractionation of membrane structures of Yersinia pestis is developed. It involves the following basic stages: the cultivation of bacteria in a liquid nutrient medium, mechanical destruction from the solid state in the X-press or ultrasound treatment of the suspension, subsequent two-stage centrifugation in the step (70-15%) and linear (70-45%) gradients of the sucrose density, collection of fractions and their storage. The method makes it possible to separate rapidly and efficiently the outer and cytoplasmic membranes which preserve biochemical and morphological integrity. This is confirmed by the distribution pattern of marker enzyme activities, by the electron microscopic control as well as by other modern sediment tests. High heterogeneity of the polypeptide composition of the isolated membrane preparations has been shown by electrophoresis in PAAG in the presence of sodium dodecyl sulphate as well as definite sensitivity of certain protein subunits to variations of the temperature (28 or 37 degrees C) during cultivation of a plague agent.

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.

When many ligands, polypeptidic hormones, growth factors, metabolic carriers, plasma glycoproteins, etc., bind to cell-surface receptors, ligand-receptor complexes are internalized by a process called receptor-mediated endocytosis towards the endocytic compartment. The endocytic compartment is an extensive network of anastomosing vesiculo-tubular membranes that differs biochemical and functionally from other intracellular organelles. Endosome fractions were prepared and antibodies raised against endosome membrane proteins. In addition to a detailed biochemical study of proteins and glycoproteins the antibodies were used to immunolocalize the endocytic structures in the hepatic cell. These studies aided to demonstrate the involvement of endocytic compartment not only in the sorting of proteins to specific domains of the plasma membrane but in the identification of "resident" endosome components.

The use of the adsorption chromatography on the hydroxyl apatite makes it possible to yield and partly purify the triton X-100-solubilized mitochondrial protein fraction of the rat liver able to bind specifically [3H]-alpha-tocopherol. The method permits removing simultaneously both free detergent and [3H]-alpha-tocopherol from the protein mixture without disturbance of the established equilibrium. When compared with methods used for the removal of free hydrophobic ligands in the in vitro binding experiments, the applied method is the most effective.

We report a methodology for the isolation of peroxisome membranes from the yeast Candida tropicalis pK233 grown on oleic acid, and the characterization of the polypeptide and lipid compositions of these membranes. Peroxisomes purified in either sucrose or Nycodenz gradients are treated with Tris-HCl (pH 8.5) and then with sodium carbonate (pH 11.5) to yield a final peroxisome membrane preparation (hereafter called 'peroxisome membranes'). Electron microscopy revealed peroxisome membranes that are approximately 8.1 nm thick, have a typical trilaminar appearance, and form either flattened sheets or whorled structures. Peroxisome membranes contain 3.1% and 2.2% of the total protein of sucrose- and Nycodenz-gradient-purified peroxisomes, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed three predominant polypeptide bands of 34 (PMP 34), 29 (PMP 29), and 24 (PMP 24) x 10(3) Mr in peroxisome membranes. Immunoblotting with an antiserum to PMP 24 showed that PMP 24 segregates with the peroxisome membrane fractions and is induced by growth of Candida tropicalis on oleic acid. Peroxisome membranes contain neutral lipids and phospholipids. The principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine. The phospholipid/protein ratio of peroxisome membranes is approximately 430 nmol mg-1.

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.

Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome-c oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.

Ascorbic acid uptake was investigated in isolated, plated human neutrophils using high-performance liquid chromatography with coulometric electrochemical detection. Freshly isolated neutrophils contained 1.3 mM ascorbic acid and accumulated significantly greater amounts when physiologic concentrations of the vitamin were present in the extracellular buffer. In several different buffers uptake was dependent on the presence of calcium and magnesium. Under these conditions, scintillation spectrometry of [14C]ascorbic acid in conjunction with high-performance liquid chromatography was suited for measuring ascorbic acid transport.

By using extraction in the presence of Ca2+ and Triton X-100 and then in the presence of EGTA without detergent, a set of Ca2+-dependent phospholipid binding proteins has been identified in the membranes of transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), isolated from rabbit skeletal muscles. Longitudinal SR, junctional SR and T-tubule membranes yielded about 9, 14 and 3.3 micrograms of EGTA-soluble proteins per 1 mg of membrane protein, respectively. In the presence of 1 mM CaCl2, 68 and 33 kDa proteins of T-tubules and junctional SR as well as 30 kDa protein of T-tubules were shown to bind to liposomes made of 1:1 w/w mixtures of (i) phosphatidylcholine and (ii) phosphatidylserine, phosphatidic acid, or phosphatidyl ethanolamine. In the presence of EGTA, the above-mentioned proteins were mostly found in the supernatants. Binding of the proteins with liposomes consisting of pure phosphatidylcholine was negligible.

The effect of chronic dietary iron overload on the lipid composition and physical state of rat liver mitochondria, microsomes and plasma membranes was investigated. After 9 weeks of iron treatment, a significant decrease of polyunsaturated and a parallel increase of saturated fatty acids was observed in mitochondrial and plasma membrane phospholipids. By contrast, no appreciable modification of the fatty acid composition of microsomal membranes was detected. The cholesterol/phospholipid molar ratio as well as the lipid/protein ratio, did not reveal any significant difference in any of the fractions studies. Finally, no change in the molecular order of the various membranes, as assessed by electron spin resonance spectrometry, was observed following iron intoxication. These data indicate that, although in vivo chronic hepatic iron overload induces a modification of fatty acid profile in cellular structures consistent with the in vivo occurrence of lipid peroxidation, these changes do not bring about appreciable modifications of other physico-chemical parameters relevant to membrane integrity and cell viability.

The changes in synaptosomal membranes in comparison with those of macrophages and mitochondria during repeated psychogenic stress were evaluated using a specially designed multiprobe procedure. Activity of Na,K-ATPase activity was measured as a functional marker for synaptosomal membranes. The interaction of peritoneal macrophages with nitrobluetetrazole (NBT-test) was also evaluated. Some bioenergetic parameters were measured in mitochondria using spectrophotometric techniques. The data revealed profound structural and functional changes in synaptosomal membranes on 15th day of stress. Those in macrophage membranes, present on 3rd and 6th day were not accompanied by functional ones. In conclusion the present data suggest that stress repeated for 15 days leads to a modelled psychopathology in rats and induce selective structural and functional changes in synaptosomal membranes from the cerebral cortex. This model may be used for investigations of the effects of psychotropic drugs.

We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.

The smooth endoplasmic reticulum (SER) has been implicated in glycogen deposition and depletion. It has been suggested that SER proliferation plays a role in elevated glucose release during rapid glycogenolysis (Striffler et al., Am. J. Anat. 160: 363, 1981). In these studies, the role of SER in glucagon-stimulated hepatic glucose release was examined by assessing changes in microsomal glucose-6-phosphatase (G-6-Pase) and membrane cholesterol to phospholipid ratios. In control fed rats given 6 i.p. injections of glucagon 350 micrograms/injection) at hourly intervals, percentage hepatic glycogen decreased nearly 30 fold, with liver homogenate G-6-Pase (U/mg protein) increasing 50% (p less than .02 relative to vehicle-injected controls) from .055 +/- .003 at 0h (n = 12) to .081 +/- .004 at 6h (n = 11). The increase in homogenate G-6-Pase was reflected in the isolated SER fraction by a 48% rise (p less than .01 relative to controls) in activity from a 0h value of .210 +/- .010 (n = 10) to a peak activity of .310 +/- .027 U/mg microsomal protein at 5 h (n = 12). G-6-Pase also increased in the rough endoplasmic reticulum (RER) reaching .242 +/- .023 U/mg protein at 4h (n = 14), but then declining to .209 +/- .019 U/mg protein at 6 h (n = 11). The changes in G-6-Pase were accompanied by alterations in the ratio of microsomal cholesterol to phospholipid, which decreased by 50% (p less than .002) in both RER and SER fractions signifying changes in membrane lipid environment. Ultrastructural analysis revealed a marked reduction in hepatic glycogen and conspicuous presence of elements of the SER in regions of the cytoplasm where glycogen was or presumably had been located.

Milk lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk-secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.

Zymogen granules (ZG) of rat pancreas have been isolated by the procedure of Pâquet et al. The granules lysed when exposed to alkaline pH (pH 8.2), and their membranes could be subfractionated by centrifugation on a sucrose gradient. Four discrete types of membranes corresponding to densities of 1.105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively and characterized both by morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands but in different proportions. Type A membranes are comprised of four major bands corresponding to molecular weights of 80, 69, 54, and 20 kDa, being in higher concentration than the others. Types B and C contain three major bands at 80, 54 and 20 kDa whereas type D is comprised of only two major bands at 69 and 54 kDa, the latter polypeptide corresponding to ATP-diphosphohydrolase activity which is present in all four membrane types. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM) showed that type A are large superimposed sheets of membranes with amorphous material between sheets. The surface area of these sheets corresponds grossly to the surface of an intact ZG with a few intramembrane particles (IMP) distributed at random or in small aggregates on large smooth fracture planes. Types B and C exhibit a totally different aspect, forming closed vesicles about the size of a small ZG with few IMP distributed at random or in small aggregates on smooth fracture planes. Type D membranes are very small vesicles with no detectable IMP on relatively smooth fracture planes.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional gel electrophoresis is used to analyse the polypeptide composition of thylakoid membranes during chloroplast development in Euglena gracilis. The number of polypeptide spots resolved on the two-dimensional gels is 3-4 times the number of bands previously resolved on one-dimensional gels. Groups of multiple polypeptides with the same Mr but with differing isoelectric points are detected throughout the period of chloroplast development. The pattern of polypeptide synthesis and insertion into the forming thylakoid membranes of dark-grown Euglena exposed to light conforms with previous biochemical measurements on the assembly of the electron transport chain located in these membranes.

Model bilayer systems from individual purified chloroplast thylakoid membrane lipids, from reconstituted mixtures of these purified lipids, and from leaf total polar lipid extracts have been prepared in water, and the longitudinal relaxation times (T1's) of the individual carbon atoms of the fatty acyl chains measured by 13C-NMR spectroscopy. The T1's increase with increasing distance of the carbon atoms from the polar headgroups in all cases, and as the results from each of the preparations are similar, all can be used as models of chloroplast membrane bilayers. Relaxation time measurements on intact chloroplast thylakoid membranes indicate the presence of chlorophyll resonances in the 13C-NMR spectrum of the membrane.

A sorbent specific for cholesterol enabled to isolate effectively the substance from blood plasma and membranes of erythrocyte shadows. Cholesterol was not isolated from microsomal membranes neither with the sorbent MGU-I nor by means of phospholipid liposomes. The phenomenon was apparently related to the localization of cholesterol on the inner surface of lipid bilayer of microsomal membranes.

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.

Intracellular element concentrations were measured in rat sympathetic neurones using energy dispersive electron microprobe analysis. The resting intracellular concentrations of sodium potassium and chloride measured in ganglia maintained for about 90 min in vitro at 25 degrees C were 3, 155 and 25 mmol/kg total tissue wet weight respectively. Recalculated in mmol/l cell water, these values are 5, 196 and 32 respectively. There were no significant differences between the nuclear and cytoplasmic values of these ions. Incubation in either carbachol (180 mumol/l, 4 min) or ouabain (1 mmol/1, 60 min) significantly increased the intracellular sodium and decreased the intracellular potassium concentrations. Neither substance materially altered the intracellular chloride concentration. The data obtained are compared and contrasted to those obtained in mammalian sympathetic neurones using chemical analysis and ion-sensitive microelectrodes.

A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones.

Experimental details of a new method for the cytochemical characterization of the membrane faces and cytoplasm produced by freeze-fracture of isolated cells and tissues are presented. This new method-"fracture-label"-involves grinding of frozen samples immersed in liquid nitrogen, thawing, cytochemical labeling of the fractured faces, and processing for thin section electron microscopy. Cationized ferritin (at pH. 7.5 and 4.0), colloidal iron, as well as concanavalin A are used to label the fractures faces of leukocytes and Hela cells embedded in a cross-linked matrix of bovine serum albumin and of liver and spleen tissues. Our results show the presence of numerous anionic binding sites on the fracture faces of all plasma and cytoplasmic membranes, and of concanavalin A binding sites preferentially associated to the exoplasmic fracture faces of plasma and nuclear envelope membranes. A proportion of the anionic sites appears to be revealed by, or during, the freeze-fracture process. Colloidal iron labeling also shows preferential association with the chromatin areas of cross-fractured nuclei. The results show that "fracture-label", i.e., the combined application of freeze-fracture and cytochemical labeling techniques, can be used to study the surface chemistry of the fractures faces of biological membranes as well as of cross-fractured cytoplasm.

Sodium dodecyl sulfate/polyacrylamide gels of coated vesicles from porcine brain (mean 76% coated vesicles) show three major proteins (180,000, 125,000, and 55,000 daltons) that account for 73% of the total protein. Preparations consisting predominantly of coats (65%) have less of the 55,000-dalton protein. Clathrin (180,000 daltons) comprises 40% of the protein of a coated vesicle. Conditions of 2 M urea, 0.25 M MgCl2, or pH 7.5 disrupt the coat and solubilize clathrin. Solubilized clathrin reforms coat structures after dilution of urea or MgCl2. High-pH-solubilized clathrin reassembles after dialysis against buffer at pH 6.5 containing dithiothreitol (5 mM). Reassembled coats are predominantly clathrin.

A simple method is proposed for the analysis of the distribution and changes in membrane lipids subjected to different treatments (lipolytic, aging, etc.). This technique involves only one thin-layer chromatographic step followed by a scanning of the photographic negative of the charred thin layer. This method is time saving, inexpensive and does not require the technical skill usually demanded in lipidology. The precision of this method is compared with that obtained with the classical TLC-GC method: its variability is roughly twice that of the TLC-GC method.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.

Applications of lanthanum as an electron microscopic tracer have been reviewed. This electron-dense trivalent cation, which binds avidly to calcium binding sites, can be used as tracer for delineating extracellular spaces and intercellular junctions. It has served as a basis for classification of junctional structures. It can also be used as a calcium probe, a tracer in studying the permeability of barriers, as an intracellular marker and as an electron microscopic stain for such membrane components as surface glycoprotein. Each of these applications may require a different methodology. Thus methodological considerations in the use of this tracer have also been reviewed. The recent recognition that lanthanum is more than a passive tracer and that by reacting with different cell components may serve as a true stain, will extend the use of lanthanum in electron microscope histochemistry.

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.

Extracts of detergent-treated thylakoids, when reconstituted into azolectin/cholesterol/dicetyl phosphate vesicles, stimulate chloride ion efflux as measured with a Cl--sensitive electrode. This stimulation is inhibited by piretanide, an active chloride ion transport inhibitor in fish intestinal epithelia. This stimulation is also abolished by pretreatment of extracted proteins with trypsin. Antiserum raised to efflux-active proteins inhibits a cation-driven Cl- influx in nonenergized thylakoids, as measured by a flow dialysis technique.

The glycolipids of the green alga Chlamydomonas reinhardtii 137+ have been quantitated in the phototrophically-cultured cell and in its thylakoid membrane. Three lipids, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the sulfated glycolipid sulfoquinovosyldiacylglycerol (SL), constitute the total glycolipid complement and some 70-80% of the total polar lipid at both levels. About 70% of the alga's sulfolipid, but only about half of its galactolipid, is localized in the thylakoid. The three cellular and thylakoid glycolipids all contain prominent hexadecanoic and octadecanoic fatty acids. Quantitatively, each lipid has a distinctive acyl profile, making SL the most highly saturated and MGDG the least saturated glycolipid. Differences between the fatty acid profiles of each corresponding cellular and thylakoid glycolipid indicate that discrimination of the glycolipid species assembled into photosynthetic membrane takes place during thylakoid membrane biogenesis.

We have been developing a procedure for localizing intracellular antigens in cultured cells, by using peroxidase-labeled antibodies, that allows good morphologic preservation. Although useful, our previous technique did not preserve the morphology of membranes, and the location of the peroxidase reaction product was difficult to establish. In this paper, we report major improvements on the basic technique that markedly enhance the quality of localization and of morphology. Saponin is used to permeabilize membranes without destroying their morphology. The amount of reaction product is enhanced with a peroxidase-antiperoxidase label. The clarity of morphologic detail and contrast of reaction product density are increased by using postsectioning staining with the osmium/thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures. We have applied this technique to the ultrastructural localization of alpha2-macroglobulin and demonstrated that it is localized in membrane-limited vesicles. We have also used this method to improve the preservation of structures for localization by fluorescence microscopy.

The recently developed capability to separate and quantify each of several proteins concurrently in single red cells presents an opportunity to test for biological variations in intercellular distribution of a protein as well as the extent of correlation between quantities of gene products derived from a single cell genome. In this preliminary study, erythrocytes from 30 sickle trait subjects were subjected to single cell electrophoresis and the resulting hemoglobin electropherograms were scanned by a recording densitometer. There was found to be heterogeneity among subjects in the form of the intercellular distribution of Hb S fraction, as tested by g statistics for skewness and kurtosis. Additionally, in all subjects there was statistically significant correlation between relative quantities of cellular Hb A and Hb S as measured concurrently in the same cell. These observations provide a basis for future research on the hypothesis that the form of the distribution of hemoglobin among erythrocytes is a heritable variable.

The phospholipids of phototrophically-cultured Chlamydomonas reinhardtii 137+ have been quantitated at the cellular and thylakoid membrane levels. The alga contains three major phospholipids, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), which together constitute about 14% of the cellular polar lipid complement. PG is the only phospholipid in an analytical fraction of thylakoid membrane isolated from the alga, and represents about 9% of the membrane's total polar lipid. Slightly more than half the cellular PG is localized in the photosynthetic lamellae. Hexadecanoic and octadecanoic fatty acids make up over 70% of the acyl groups esterified to these phospholipids, PG being the most highly unsaturated of the three. Differences in fatty acid profile between cellular and thylakoid PG, as well as the presence of PE and PC in the alga but not in its thylakoid, are indicative of a strict biogenetic regulation of the specific types and species of phospholipid associated with the photosynthetic membrane.

A membrane-DNA complex was isolated by centrifugation of sheared lysate of isolated mitochondria in 20-60% sucrose step solution. Analyses using Hoechst 33258/CsCl density gradient centrifugation and restriction endonuclease treatment showed that DNA in the membrane-DNA complex was AT-rich compared with total mitochondrial DNA (mt DNA) and contained Eco RI fragments of E-4, 5 and 8, which were localized on the right hand of Physarum mitochondrial genome. Phenethyl alcohol (PEA) and ethidium bromide (EB) could disrupt the membrane-DNA complex to release DNA fragments from their complex in vitro. Addition of 0.5% or more PEA, which released 80-90% of the DNA from the membrane-DNA complex in vitro, inhibited not only mitochondrial nuclear division but also mitochondrial division in vivo. EB treatment at more than 1 mg/ml disrupted the membrane-DNA complex in vitro to release 77% of the total DNA in the complex. Addition of 10 micrograms/ml EB induced unequal mitochondrial nuclear division in the microplasmodia, e.g., a dividing dumbbell-shaped mitochondrion had the mt-nucleus in one side and as a result formed then one nucleated and one enucleated mitochondrion. From the EB-pretreated mitochondria, a lesser amount of the membrane-DNA complex was isolated than from the control. These findings mean than the unequal mt-nuclear division is due to dissociation of DNA and the membrane system in the membrane-DNA complex. They strongly suggested that the DNA region (E-4, 5 and 8), where the mitochondrial nucleus is associated with the mitochondrial membrane system plays an important role in mitochondrial nuclear division.