Immunogold localization of acyl carrier protein in plants and Escherichia coli: Evidence for membrane association in plants

Immunogold labelling was used to study the distribution of acyl carrier protein (ACP) in Escherichia coli and a variety of plant tissues. In E. coli, ACP is distributed throughout the cytoplasm, confirming the observation of S. Jackowski et al. (1985, J. Bacteriol., 162, 5–8_. In the mesocarp of Avocado (Persea americana) and maturing seeds of oil-seed rape (Brassica napus cv. Jet Neuf), over 95% of the ACP is localised to plastids. The protein is almost exclusively located in the chloroplasts of leaf material from oil-seed rape. Approximately 80% of the gold particles associated with the ACP were further localized to the thylakoid membrane of the chloroplast. Since acetyl-CoA carboxylase has been reported to be localized to the thylakoid membrane (C.G. Kannangara and C.J. Jensen, 1975, Eur. J. Biochem., 54, 25–30), these results are consistent with the view that the two sequential enzymes in fatty-acid synthesis are in close spacial proximity.Abbreviations ACC acetyl CoA carboxylase - ACP acyl carrier protein - FAS fatty-acid synthetase     

Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.     

Changes in Insulin Binding to Developing Embryonic Chick Neural Retina Cells

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.