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Soluble cytochrome c-553 and membrane-bound cytochrome f-553 from the alga Scenedesmus acutus were purified to apparent homogeneity. The properties of cytochrome c-553 are comparable to preparations obtained from other eukaryotic algae, whereas the thylakoid-bound species resembles higher plant cytochrome f. Common characteristics are: 1. An asymmetrical alpha-band at 553 nm. 2. A midpoint redox potential of +38 MV (pH 7.0), with a pH dependency above pH 8.0 of -60mV/pH unit. 3. Formation of a pyridine hemochromogen with a maximum at 550 nm; no adducts with CN- or CO are observed. Distinguishing features are: 1. Cytochrome f-553 has a more complicated beta-band, with maxima at 531.5 and 524 nm, and hence a more complex low-temperature spectrum. Also the positions of the gamma- and delta-bank at 421.5 and 331 nm, respectively, distinguish cytochrome f-553 from cytochrome c-553, with gamma- and delta-bands at 416 and 318 nm. 2. The ferricytochrome c-553 spectrum exhibits a weak band at 692 nm, which is not observed with cytochrome f.

An entire organelle, the peroxisome, appears to be missing in Zellweger syndrome, causing profound neurological problems and neonatal death. One hypothesis for the molecular cause of this defect is a failure in the assembly of the peroxisomal membrane. An alternative is that the peroxisomal membrane is assembled, but the post-translational import of the matrix proteins is defective. We have investigated these possibilities by analytical cell fractionation, immunoblotting, and immunoelectron microscopy of fibroblasts. We identified four integral membrane proteins that can serve as markers for the human peroxisomal membrane. In Zellweger fibroblasts, peroxisomal membranes were found but they were abnormal; they had an equilibrium density of 1.10 g/cm3 instead of the normal density of 1.17 g/cm3, their diameters were generally 2-4 times greater than normal, and they lacked most content. The existence of these peroxisomal ghosts in Zellweger syndrome fibroblasts supports the hypothesis that the defect in this disease is in the protein import machinery.

An antiserum which recognizes high molecular mass enkephalin-containing proteins was used to compare proenkephalin intermediates in both the soluble and membrane components of bovine adrenal chromaffin granules by immunoblotting. While a range of molecular mass forms were identified in the soluble lysate the major form in the membranes corresponded to a 27-kDa enkephalin-containing protein. Enzymic digestion of bands of 27-kDa material and quantitation of the enkephalin released showed that 22% of this material was membrane-associated. High concentrations of chaotropic agents were required to extract this material from the membranes. Association of hormone and neuropeptide precursors with membrane components may be important for targeting of precursors to secretory granules or correct processing.

Glycosomal membranes of bloodstream form Trypanosoma brucei were purified to apparent homogeneity by sodium carbonate treatment and found to contain two major integral membrane proteins of 26 kDa (band 10) and 24 kDa (band 11). The procyclic-form glycosomal membranes isolated by the same procedure also resulted in a homogeneous preparation, but each piece of membrane thus purified was associated with an electron-dense granule. Procyclic membranes also contained primarily bands 10 and 11. These two proteins were selectively reduced by protease treatment of intact glycosomes, suggesting surface exposed domain(s) of the two proteins accessible to proteolytic digestion. They and band 8 protein also vanished in glycosomal lysates by apparent proteolysis, implying the presence of a specific protease which degrades the integral membrane proteins upon the loss of membrane integrity. The two proteins, hydrophobic in nature and apparently unglycosylated, have no known biological functions, but their possible involvement in translocating precursor proteins from the cytoplasm into the glycosome of T. brucei is postulated.

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.

The X-ray diffraction pattern from chromatophore membranes of a photosynthetic bacterium, Rhodospirillum rubrum, indicates that a highly organized protein assembly exists in the membrane. The X-ray scatterer was solubilized from chromatophores by a mixture of cholate and deoxycholate. The basic component was identified as the photoreaction unit, which consists of light-harvesting bacteriochlorophyll proteins and a reaction center. The radial autocorrelation function, calculated directly from the X-ray intensity dats, made it possible to deduce certain structural features of the X-ray scatterer. 1. The maximum dimension of the X-ray scatterer is estimated to be 110-130 A. 2. The arrangement of the units in the chromatophore membrane is random. 3. Protein molecules in the unit form a rigid structure, being arranged mutually in fixed positions to give a distinct X-ray diffraction pattern. 4. The most probable structure is one which has rotational symmetry.

A simple method for the preparation of lysosomes from livers of rats injected with Triton WR 1339 was developed. Enzymic characterization showed that the Triton WR 1339-filled lysosome (tritosome) preparation isolated by this procedure was almost completely free from mitochondria, peroxisomes, and microsomes. With this method, tritosomes were purified about 50 times with a yield of 8%. The tritosomal membrane fraction was prepared by osmotic disruption of the purified tritosomes followed by washing with 1 M NaCl. Analysis by SDS-polyacrylamide gel electrophoresis showed that tritosomal membrane proteins were distinct from those of plasma membranes. The major glycoproteins found in tritosomal membranes in the higher molecular weight region were not detected in plasma membranes. When livers were labeled with L-[3H]leucine or D-[3H]glucosamine, the incorporation of both isotopes into tritosomes attained the maximum value at around 3 h after the isotope injection. Radioactivities associated with the tritosomal membranes decayed slower than those of the tritosomal contents. SDS-polyacrylamide gel electrophoresis of the membranes labeled with the isotopes for various times demonstrated the distribution and variation with time of radioactivity in the protein and glycoprotein components. The results indicate that the turnover rate of the protein and glycoprotein components in the tritosomal membrane is heterogeneous.

Recently we described a protein component, from the inner mitochondrial membrane, which binds thyroid hormone with high affinity, low capacity (saturable) characteristics. This partially purified rat liver mitochondrial membrane component appears to be a 150,000 daltons lipoprotein complex. Phospholipids, tentatively identified as lecithin, phosphatidyl ethanoamine, and cardiolipin, appear to constitute 50% of this complex. A similar hormone binding marcomolecule was also found in mitochondria from rabbit kidney, as well as human liver and kidney. In the rat this saturable thyroid hormone binding component was found in mitochondria from liver, kidney, myocardium, skeletal muscle, intestinal mucosa, whole small intestine, adipose tissue, and lung. It was absent from the mitochondria of adult brain, spleen and testis, organs known to be calorigenically unresponsive to thyroid hormones injected in vivo. In contrast, neonatal rat brains contain the protein with binding constants similar to those of neonatal or adult rat liver mitochondria, but in older rat brains (14 and 17 days) the saturable binding was no longer present, as in adult brain. These data provide strong support for the biological relevance of the mitochondrial component as a thyroid hormone receptor.

The congenitally jaundiced Gunn rat does not conjugate bilirubin but does conjugate bilirubin dimethyl diester. Partial defects in conjugating p-nitrophenol and demethylating aminopyrine are also evident. A proposed mechanism to explain this combination of findings is a defective microsomal membrane. To examine the 'matrix' of Gunn microsomal membranes, hepatic microsomes were isolated from Gunn (jj) and outbred Wistar (JJ) rats and were studied by electron paramagnetic resonance spectroscopy of 7-doxylstearic and 12-doxylstearic acid probes, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, glucose-6-phosphatase activity vs. temperature, and lipid analysis. The data indicate several factors related to lipid bilayer order do not differ in microsomes from jj and JJ.

We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commercial compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3-12, the concentration of urea compatible with 2% detergent increases from 4.5 M for the parent molecule up to 7 M. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl-N,N-dimethylammonio-3-propanesulfonate of a major protein (pI = ca. 5.5, Mr = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a pK of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

A method has been developed for the functional reconstitution of membrane proteins in phospholipid vesicles. This method is an extension of a previously published procedure (Ueno, M., Tanford, C. and Reynolds, A. (1984) Biochemistry 23, 3070-3076) for the formation of unilamellar vesicles from mixed micelles of egg phosphatidylcholine and dodecyl octaoxyethylene ether. Mixed micelles are formed from detergent-solubilized protein and egg-yolk phospholipid vesicles. These micelles are subjected to repeated passage through small columns filled with Amberlite XAD-2 beads. Several carrier proteins from the inner mitochondrial membrane have been reconstituted in this way; experimental data are shown for the aspartate/glutamate carrier and the ADP/ATP carrier. Certain parameters proved to be important for optimal efficiency of reconstitution: the ratio of detergent/phospholipid in the mixed micelles, the concentration of phospholipid during the hydrophobic chromatography, the ratio of phospholipid/protein, (d) the ratio of detergent/Amberlite XAD 2 beads, the number of column passages, and the type of detergent. After optimization of these parameters, phospholipid vesicles with a diameter of about 150 nm were obtained. The main advantage of this procedure, however, lies in the fact that high amounts of membrane protein can be incorporated into the phospholipid vesicles, i.e. up to 15% (w/w).

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.

The major neutral, glycolipids and phospholipids from envelopes of spinach chloroplasts were analyzed with respect to proportions, positional distribution and pairing of fatty acids. All specificities in the diacylglycerol portions of lipids known from previous analyses of lipids from whole leaves were also found in envelope lipids. Diacylglycerols and galactolipids share a common diacylglycerol portion. The only exception is digalactosyl diacylglycerol, which contains 18:3/16:0 but lacks 18:3/16:3 species reverting the distribution in other galactolipids. Phosphatidylcholine, phosphatidylglycerol and sulfoquinovosyl diacylglycerol are distinct from the galactolipids, because each one has a unique diacylglycerol profile. The diacylglycerol species present in phosphatidylcholine and galactolipids or free diacylglycerols do not provide evidence for a biogenetic relation between phosphatidylcholine and galactolipids at the level of envelopes.

Microsomal membranes from rat liver were treated with the cross-linking reagent 1,5-difluoro-2,4-dinitrobenzene (DFDNB). Experimental work showed that at a probe concentration of 0.75 mM all free phosphatidylethanolamine (PE) and phosphatidylserine (PS) were found as dinitrophenyl derivatives: 29% of PE was in monomeric form, 9% dimeric, 2% interacted with PS, and 63% cross-linked to protein. PS showed a greater percent in monomeric and dimeric form and only 31% was cross-linked to protein. The cross-linking pattern of PE was clearly different from that pattern which is present in the inner mitochondrial and erythrocyte membranes. In vivo labeling of PE with [(3)H]glycerol and [(3)H]ethanolamine followed by phospholipase A(2) treatment of isolated microsomes established a heterogeneous labeling pattern during the first 2 hours. During this period, the specific activity of the phospholipase A(2)-sensitive compartment was considerably higher. The differential distribution of radioactivity after in vivo labeling in the part of the PE which reacted with increasing concentrations of DFDNB also indicated compartmentalization. After in vivo labeling with the precursors, the time course of the specific radioactivity demonstrated an initial high labeling, almost exclusively in the monomeric form, followed by a later appearance of the label in the protein-bound PE. The experiments indicate that the biosynthesis of PE takes place in a compartment that is more accessible to surface probes and that the labeled molecules are transferred in a time-dependent process to a second compartment where the lipid is not available for phospholipase A(2) action but is available for cross-linking to protein.-Valtersson, C., and G. Dallner. Compartmentalization of phosphatidylethanolamine in microsomal membranes from rat liver.

Membrane distribution in human milk was investigated. Milk samples from six women taken at intervals throughout 9 months of lactation were fractionated into fat globules, skim milk, fluff, and cells. These fractions and the intact milks were analyzed for protein, total lipid, and membrane material as revealed by phospholipid and cholesterol contents. All fractions showed initial levels of membrane that dropped by 1 month and then remained relatively unchanged thereafter. Total lipid (fat globules) in the milks was the primary factor determining membrane content. Distribution of membrane in mature milks was: fat globules, 80%; skim milk, 20% (including fluff, 5%); and cells, less than 1%. Mature milk assures the infants a relatively steady supply of membrane material. However, there appears to be approximately twice as high a concentration of it in milk during the first several weeks of lactation. At that time, the skim milk phase accounts for approximately 30% of milk membrane as compared to 20% during subsequent lactation.

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar head-group detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.

Paired lines of metastasizing and non-metastasizing transplantable rat mammary tumors were studied for their sensitivity to hyperthermia. The metastasizing TMT-081 and SMT-2A tumors were markedly more sensitive to heat injury as measured by tumor growth delay than were their non-metastatic counterparts MT-100 and MT-W9B. The metastasizing MT-081 tumor was also significantly more sensitive than the SMT-2A tumor. The differences in heat sensitivity were not the result of differences in intra-tumor temperatures attained during water bath heating. A more likely explanation for the variable response obtained with these tumors after heat treatment may be the inherent differences in stability and composition of their cellular membranes, particularly the plasma membrane.

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.

Secretory and endocytic vesicles have analogous functions as cyclic carriers between specific cellular compartments. The centrifugally functioning secretory system operates from the Golgi complex, whereas the centripetally functioning endocytic system operates from the cell surface. Further, within polarized epithelial cells the export traffic can be directed to a distinct plasmalemmal domain which distinguishes exocrine from endocrine secretion and import traffic can be directed transcellularly. These shuttle operations involve a special class of lipid-rich, protein-poor membranes that appear to use an inwardly directed H+-translocase activity to varying extents for pH-dependent sorting and for accumulation and concentration of transported molecules. Comparative analyses of purified membrane preparations from exocrine and endocrine sources identify compositional overlap between different types of shuttle membrane. However, the structural elements that specify a particular origin or destination for a given carrier or determine function in storage and stimulus-dependent shuttling remain unknown.

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.

The cytoskeleton of human ventricular myocardium in normal and pathological hearts has been analysed with immunocytochemical techniques. Specific antibodies against the intermediate filament proteins desmin (Mr 55 000) and vimentin (Mr 58 000) and antibodies against two cytoskeleton-associated proteins, a spectrin-like protein (Mr 230 000) and vinculin (Mr 130 000), have been used. We show that desmin is localized in the myocytes as an intermyofibrillar lattice at the Z disk level of the myofibrils, and at the intercalated disks. The spectrin-like protein is localized as a transverse striated pattern interlinked with fine longitudinal strands in the subplasmalemmal region of the myocytes. Vinculin is abundant in the intercalated disks and in myotendinous junctions but occurs also at the peripheral sarcolemma in the form of a regular repeat of dots and of fine bar-like extensions into the cytoplasm from the dots. These patterns were observed both in normal and in abnormal hearts, but a number of altered patterns in pathological myocytes were also seen. It is concluded that the intermediate filament system has important implications in the structural function of normal and abnormal hearts but that further studies are needed to elucidate how the different components are related to each other and how they are influenced by different disease processes.

A method has been designed to prepare inside-out thylakoid vesicles from a cyanobacterial species (Phormidium laminosum). Everted thylakoid vesicles could be generated by Yeda press treatment after induced membrane pairing. Membrane pairing was induced either by addition of high concentrations of Mg2+ ions or by lowering the pH of the fragmentation media. The inside-out vesicles were isolated by aqueous polymer two-phase partition. The membrane orientation was determined by proton translocation studies and freeze-fracture electron microscopy.

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.

A glycoprotein was isolated from detergent solubilized membranes of bovine chromaffin granules by high-performance liquid chromatography. Specific antisera raised against this glycoprotein reacted in one- and two-dimensional immunoblots with a heterogeneous component with a pI of 4.2-4.7 and Mr 100,000. The antiserum against bovine glycoprotein II cross-reacted with an analogous component in several species. The specific localization of glycoprotein II in chromaffin granules was established by density gradient centrifugation followed by immunoblotting. The antiserum, as shown by one- and two-dimensional immunoblotting, reacted with an analogous antigen in the posterior pituitary, in endocrine (anterior pituitary, parathyroid gland) and exocrine (parotid gland, pancreas) organs. In the pancreas the protein reacting with the antiserum was found in the membranes of zymogen granules. The results demonstrate for the first time that secretory vesicles of endocrine and exocrine tissues have at least one common antigen, i.e. the glycoprotein II. It seems likely that this protein is involved in a basic function common to all secretory vesicles.