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R D Simoni

Publications and source records attributed to R D Simoni.

At least 73 records · Page 4Linked to original sources
PubMed1984

In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli.

The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit. In addition, the epsilon-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0.

Antigen-Antibody Complex↗
PubMed1983

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit.

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.

ATP Synthetase Complexes↗
PubMed1983

Assembly of a functional F0 of the proton-translocating ATPase of Escherichia coli.

We have investigated both structural and functional assembly of the F0 portion of the Escherichia coli proton-translocating ATPase in vivo. Fractionation of E. coli minicells containing plasmids which code for parts of the unc operon shows that each of the F0 peptides a, b, and c insert into the cytoplasmic membrane independent of each other and without the polypeptides which form the F1 portion of the complex alpha, beta, gamma, delta, and epsilon. Assays of membrane energization indicate that, while formation of a functional proton channel requires the presence of all three F0 polypeptides a, b and c, they are not sufficient. Synthesis of both the alpha and beta subunits of the F1 are required for formation of a functional proton channel.

Adenosine Triphosphatases↗
PubMed1983

Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells.

We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP(+) oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [(35)S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band of M(r) 92,000 and a minor band of M(r) 63,000. We conclude that the M(r) 92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii) Isolation and solubilization of [(35)S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of the M(r) 92,000 protein and the appearance of two proteins of M(r) 52,000 and 38,000. (iv) Analysis of cells labeled for 30 min with [(35)S]methionine, well under the half-life of HMG-CoA reductase, revealed only the M(r) 92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of M(r) 62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982) Proc. Natl. Acad. Sci. USA 79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO(4) gel electrophoresis. Analysis of C100 cells labeled with [(35)S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is the M(r) 92,000, rather than the M(r) 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.

Animals↗
PubMed1983

Escherichia coli mutants defective in the uncH gene.

Plasmids carrying cloned segments of the unc operon of Escherichia coli have been used in genetic complementation analyses to identify three independent mutants defective in the uncH gene, which codes for the delta subunit of the ATP synthetase. Mutations in other unc genes have also been mapped by this technique. ATPase activity was present in extracts of the uncH mutants, but the enzyme was not as tightly bound to the membrane as it was in the parental strain. ATP-dependent membrane energization was absent in membranes isolated from the uncH mutants and could not be restored by adding normal F1 ATPase from the wild-type strain. F1 ATPase prepared from uncH mutants could not restore ATP-dependent membrane energization when added to wild-type membranes depleted of F1. Membranes of the uncH mutants were not rendered proton permeable as a result of washing with low-ionic-strength buffer.

ATP Synthetase Complexes↗
PubMed1983

Cloning and expression of uncI, the first gene of the unc operon of Escherichia coli.

The unc operon of Escherichia coli consists of eight genes coding for the eight subunits of the proton-translocating ATPase. In vitro transcription-translation of DNA cloned from the beginning of the operon onto plasmids reveals that the reading frame uncI, which precedes the other genes of the operon, codes for a protein with a molecular weight of 14,500, called i. In minicells, the i protein is synthesized in amounts comparable to the amounts of the ATPase subunits, suggesting that it may be part of the ATPase complex. The presence of the unc promoter and uncI on a plasmid containing the other eight genes of the unc operon has little effect on the differential expression of the unc genes or the partitioning of the newly synthesized subunits into soluble or sedimentable fractions in the in vitro system. The i protein partitions into the sedimentable fraction.

Adenosine Triphosphatases↗
PubMed1983

Promoter for the unc operon of Escherichia coli.

Fragments of DNA carrying possible promoters for the unc operon of Escherichia coli were cloned into a promoter detection plasmid (pRZ5255). Similar fragments were transcribed in vitro to produce transcripts whose sizes were used to determine the approximate start site for transcription. One strong promoter and at least two very much weaker ones were detected by these methods. The exact position of the strongest promoter, presumed to be the true unc promoter, was determined by S1 nuclease mapping and shown to lie 73 base pairs upstream from the open reading frame that precedes uncB. It therefore appears that this reading frame (uncI) is part of the unc operon. S1 mapping also revealed the presence of a third weak promoter 25 base pairs upstream of uncI. All of the weak promoters occur between the proposed unc promoter and uncB, but their role in vivo, if any, is unclear.

Adenosine Triphosphatases↗
PubMed1982

Intracellular transport of cholesterol to the plasma membrane.

We have modified a plasma membrane isolation procedure which utilizes DEAE-Sephadex beads (Gotlib, L. J., and Searls, D. B. (1980) Biochim. Biophys. Acta 602, 207-212) to rapidly measure intracellular transport of cholesterol from the site of synthesis in the endoplasmic reticulum to the plasma membrane. This transport process is rapid, with a half-time of about 10 min, has different kinetics from that of intracellular glycoprotein transport, and appears to be energy-dependent.

Acid Phosphatase↗
PubMed1982

Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli.

We have constructed an extensive set of plasmids that carry the genes specifying the eight polypeptides of the proton-translocating ATPase of Escherichia coli. Using detailed restriction analysis and in vitro protein synthesis directed by these plasmids, we have established the order of the eight unc genes to be BEFHAGDC and the corresponding polypeptides to be a, c, b, delta, alpha, gamma, beta, and epsilon. These analyses include determining the location of the gene coding for the delta subunit of the F1 portion of the complex. We call this gene uncH. We have now established the gene order and gene-polypeptide relationships of the unc operon. This approach should be of use for study of other multigene bacterial operons, especially those with genes coding for polypeptides with unknown or unmeasurable catalytic activity.

Adenosine Triphosphatases↗
PubMed1982

Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli.

We investigated the regulation of the synthesis of the eight polypeptides of the Escherichia coli proton-translocating ATPase. A plasmid carrying the eight genes of the unc operon was used to direct in vivo and in vitro protein synthesis of the eight polypeptides. Analysis of these data indicates that the ATPase polypeptides are synthesized in unequal amounts both in vitro and in vivo. We identified several regions within the unc operon at which expression of a gene is either increased or decreased from that of the preceding gene. Since genetic information indicates a single polycistronic mRNA for all eight genes of this operon, the observed differential synthesis of the polypeptides is most likely the result of translational regulation. The effect of varying the temperature suggests that the secondary structure in the mRNA may affect the rate of translation initiation in the region between uncE and uncF.

Adenosine Triphosphatases↗
PubMed1982

In vitro membrane association of the F0 polypeptides of the Escherichia coli proton translocating ATPase.

The F0 polypeptides a, b, and c of the H+-translocating ATPase associated with membranes when synthesized in vitro. This association occurred when the membranes were present either cotranslationally or post-translationally. In addition, the F0 polypeptides associated with liposomes. The membrane association seemed to be an insertion process since there was protection of polypeptides a and c from proteolysis. The in vitro insertion of the F0 polypeptides a, b, and c was independent of the synthesis of each polypeptide and of the F1 polypeptides.

Adenosine Triphosphatases↗
PubMed1981

Isolation and characterization of cells resistant to ML236B (compactin) with increased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

ML236B is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (EC 1.1.1.34), the major regulatory enzyme in cholesterol biosynthesis. This compound inhibits cell growth when present in the culture medium of CHO-K1 cells at a concentration as low as 0.1 micrograms/ml. Addition of the product of the HMG-CoA reductase reaction, mevalonate, to the culture medium prevents the cytotoxic effects of ML236B at a concentration of inhibitor as high as 50 micrograms/ml. Using a stepwise selection procedure, we have obtained two variant cell lines which are resistant to the presence of 8 micrograms/ml of ML236B in the culture medium. The rates of cholesterol synthesis and the cholesterol levels in the variant cell lines, grown in the presence of ML236B, are similar to those of the parental CHO-K1 cell line grown in the absence of inhibitor. Assays of HMG-CoA reductase activity from extracts of variant cells, grown in the presence of inhibitor, reveal that the variant cell lines have an approximately 40-fold higher HMG-CoA reductase activity than does the parental CHO-K1 cell line grown in the absence of inhibitor. However, when the variant cell lines are grown without ML236B in the culture medium, the HMG-CoA reductase activity returns to the parental CHO-K1 level within 5 days, but the resistant phenotype is stable for up to 9 months. We conclude that the variant cell lines are unable to overcome the cytotoxic effects of ML236B by a mechanism which leads to overaccumulation of HMG-CoA reductase which in turn permits normal mevalonate metabolism and cholesterol synthesis to take place.

Animals↗
PubMed1981

In vitro synthesis of the F0 and F1 components of the proton translocating ATPase of Escherichia coli.

Specialized lambda transducing phage DNA containing the unc region of the Escherichia coli chromosome was used as template to direct an in vitro transcription/translation system. The results demonstrated synthesis of seven of the eight polypeptides of the proton translocating ATPase of this organism. The three polypeptides a, b, and c, constituting the F0 portion of the complex, were resolved by sodium dodecyl sulfatepolyacrylamide gel analysis and have apparent molecular weights (Mr = 24,000, 18,000, and 8,000-9,000) similar to the corresponding proteins produced in vivo. In addition, the alpha, beta, delta, and epsilon polypeptides of the F1 portion of the ATPase were also detected and their molecular weights correspond to the in vivo peptides. A 4.3-kilobase HindIII-generated lambda unc DNA fragment was cloned onto plasmid vectors and was demonstrated to contain the genes for the three F0 and two of the F1 (alpha, delta) subunits. In addition, the polypeptides synthesized in vitro were precipitable with antibody prepared against purified F1.

Adenosine Triphosphatases↗
PubMed1980

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition.

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.

Adenosine Triphosphatases↗