Identification of an anion channel protein from electric organ of Narke japonica

The anion permeability of membrane vesicles prepared from the electric organ of $\text{Narke japonica}$ was inhibited by the addition of 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid (DIDS). The permeability was measured by measuring changes in the scattered-light intensity caused by the osmotic volume change of vesicles; and also by the efflux measurement of ions from the vesicles using radioisotopes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane vesicles treated with dihydro analog of DIDS ([³H]H₂DIDS) showed that the H₂DIDS binding protein has a molecular weight of 180,000, and exists in membrane vesicles as a dimer formed by a disulfide bond between monomers of molecular weight 90,000.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and $\text{p-}\text{nitrophenylphosphate}$ activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ or $\text{p-}\text{nitrophenylphosphatase}$ activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Translocation and assembly of outer membrance proteins of Escherichia coli. Selective accumulation of precursors and novel assembly intermediates caused by phenethyl alcohol.

Selective and step-wise inhibition of bioysnthesis and assembly of three major outer membrane proteins of Escherichia coli (matrix protein, tolG protein (DiRienzo et al., 1978), and lipoprotein) was achieved in the presence of phenethyl alcohol. At a lower concentration (0·3% or higher) PEA4 specifically inhibited the processing and assembly of matrix protein, resulting in the accumulation of promatrix protein. The promatrix protein thus synthesized in the presence of PEA was chased into matrix protein and properly assembled into the outer membrane upon the removal of PEA, demonstrating a direct precursor-product relationship between the two proteins. Promatrix protein was sensitive to trypsin and was also solubilized from the membrane fraction by sodium sarcosinate. However, promatrix protein was also found to be loosely associated with the outer membrane fraction. These data indicate that promatrix protein was translocated across the cytoplasmic membrane and localized external to the cytoplasmic membrane, although it was not yet properly inserted into the outer membrane structure.The inhibition of processing of protolG (DiRienzo et al., 1978) protein was observed at higher levels of PEA (0·4% or higher). However, at all concentrations of PEA tested, the accumulation of prolipoprotein was not detected. On the other hand, when PEA was added at concentrations lower than the above critical concentrations for each protein, the precursor was properly processed but the processed proteins (tolG protein, and lipoprotein) were accumulated in the periplasmic space, since they were released by osmotic shock. tolG protein of the soluble cell fraction was chased into the outer membrane after removal of PEA and regrowth of the cells in culture. The processed lipoprotein of the soluble fraction was trypsin-sensitive in contrast to mature lipoprotein. These results indicate that the precursor protein with the peptide extension is transformed into a new assembly intermediate after the extended peptide is cleaved off. This intermediate may be released into the periplasmic space in the presence of PEA before it can be assembled into the outer membrane. These data indicate that the peptide extension is not essential for the insertion of the outer membrane protein into the outer membrane.When PEA (0·3%) was added to a growing culture, the production of not only matrix protein but also promatrix protein was completely inhibited. However, synthesis of promatrix protein was restored when rifampicin was added before the PEA treatment. These results are discussed in terms of control of gene expression for matrix protein. PEA was found to increase the membrane fluidity.     

Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA

The integration of DNA of highly oncogenic simian adenovirus type 7 (SA7) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonuclease SalI (SA7) and BglII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome. Additionally integrated fragments were stable and preserved during numerous passages of cells lines, although they were no expressed, at least in the case of the Ad6-transformed cell line. The integration of the fragments of SA7 DNA was accompanied by loss of 25-50% of the mass of each fragment. Adding the linear form of the pBR322 plasmid to the preparation of transforming Ad6 DNA also contributed to its cointegration into the genome of the transformed cell. This technique of cell cotransformation with any foreign DNAs together with the viral oncogens may be used as an equivalent of an integration vector for eukaryotic cells.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

Effects of amphotericin B on the electrical properties ofNecturus gallbladder: Intracellular microelectrode studies

Summary Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (V _ms) in the gallbladder ofNecturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B,P _Na/P _Cl across the paracellular pathway did not change significantly, whereasP _K/P _Na doubled. These results indicate that V _ms is due to an increase of g_Na across the luminal membranes of the epithelial cells (Cremaschiet al., 1977,J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976.J. Membrane Biol. 29:1) that V _ms results from a reduction in shuntP _Na/P _Cl acting in combination with a rheogenic basolateral Na pump.