Regulation of Metabolite Flux through Voltage-Gating of VDAC Channels

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, ``closed' state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5–11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically. Received: 4 November 1996/Revised: 8 January 1997     

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13

Abstract The pCloDF1S encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murein lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF13.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

Dielectric analysis of mitochondria isolated from rat liver II. Intact mitochondria as simulated by a double-shell model

The dielectric dispersion of isolated intact mitochondria in suspension has been measured between 10 kHz and 500 MHz. In isotonic KCI media at 4°C, the mitochondria maintained their characteristic ‘double membrane’ structure as examined by electron microscopy, and the observed dispersion curves were successfully simulated in terms of a superposition of two sub-dispersions having different characteristic frequencies and different permittivity magnitudes. Taking these observations into account we analyzed the dispersion data on the basis of a ‘double-shell’ model in which two concentric shells are meant to represent the mitochondrial outer and inner membranes. The analyses by a computerized curve-fitting method revealed that: (i) electric capacities for the outer and the inner membrane are 1.7 and 0.5 μF/cm², respectively, (ii) relative permittivity for the inner compartment (or the equivalent homogeneous matrical space) = 50–60, (iii) outer compartment-to-external conductivity ratio = 0.4–0.6, and (iv) inner compartment-to-external conductivity ratio = 0.14. The implications of these parameter values are discussed with due attention paid to the limitations inherent in our ‘double-shell’ model approach.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

Studies on the cell cycle of Myxobacter AL-1

The properties of seven enzymes were studied in extracts from Myxobacter AL-1. The enzymes were isocitrate dehydrogenase (E.C. 1.1.1.42), succinate dehydrogenase (E.C. 1.3.99.1), alkaline phosphatase (E.C. 3.1.3.1), -glucosidase (E.C. 3.2.1.20), -glucosidase (E.C. 3.2.1.21), -galactosidase (E.C. 3.2.1.23), and N-acetyl-glucosaminidase (E.C. 3.2.1.30). Four of these enzymes: isocitrate dehydrogenase, -glucosidase, -glucosidase, and -galactosidase are cytosolic enzymes. Succinate dehydrogenase was found to be located on the cytoplasmic membrane system, whereas alkaline phosphatase and N-acetyl-glucosaminidase were considered as enzymes which bind the outer membranes resp. the cell wall. During the cell cycle, all enzymes have a pattern of discontinuous activity increase. Succinate dehydrogenase and isocitrate dehydrogenase exhibit a stepwise increase of activity, whereas the other enzymes follow the pattern of a peak enzyme.     

Molecular considerations in the evolution of bacterial genes

Summary Synonymous and nonsynonymous substitution rates at the loci encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and outer membrane protein 3A (ompA) were examined in 12 species of enteric bacteria. By examining homologous sequences in species of varying degrees of relatedness and of known phylogenetic relationships, we analyzed the patterns of synonymous and nonsynonymous substitutions within and among these genes. Although both loci accumulate synonymous substitutions at reduced rates due to codon usage bias, portions of thegap andompA reading frames show significant deviation in synonymous substitution rates not attributable to local codon bias. A paucity of synonymous substitutions in portions of theompA gene may reflect selection for a novel mRNA secondary structure. In addition, these studies allow comparisons of homologous protein-coding sequences (gap) in plants, animals, and bacteria, revealing differences in evolutionary constraints on this glycolytic enzyme in these lineages.     

Sites of ecdysteroid biosynthesis in female adults of Gryllus bimaculatus (Ensifera,Gryllidae)

Summary Ovaries from 4-day-old female adults of Gryllus bimaculatus produce about 5 ng of free and conjugated ecdysteroids per hour during a 16-h incubation in Grace's medium. During incubation of pieces of the abdominal integument together with the adjacent segmental fat body, a net synthesis of moulting hormones is observed (2.3 ng per hour per animal), similar to that in the ovary. Separate incubations of disunited abdominal epidermis and segmental fat body tissue result in much lower rates of ecdysteroid synthesis. Ecdysteroid synthesis in ovarian homogenates is about one-third of that in intact organs. This reduction is due to a lack of conjugate formation in homogenates. Homogenates of the abdominal integument complex are no longer capable of synthesizing ecdysteroids. For both tissues, a de novo synthesis of ecdysteroids is corroborated by following the in vitro incorporation of [¹⁴C]-label from cholesterol and [³H]-label from 2,22,25-trideoxyecdysone (5-ketodiol), respectively, into free ecdysone. The rate of incorporation into ecdysone is only 0.0014% for cholesterol but 0.48% for 5-ketodiol. Both tissues represent primary sources of ecdysteroids in female adult crickets.Abbreviations HPLC high performance liquid chromatography - IU international units - NP normal phase - RIA radioimmunoassay - RP reversed phase - SEM standard error of mean