Immunochemical analysis of membrane vesicles from Escherichia coli

Owen, Philip; Kaback, H. Ronald; Doherty, Helen

doi:10.1021/bi00575a004

Cited by 76 publications

(47 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As indicated, a perplexing problem was the continued acylation (even after addition of ample cold penicillin), before deacylation of some of the PBPs in only the osmotically fragile (3% [wt/vol] NaCl) membranes. It was suggested that membranes obtained by use of the X-press may lead to predominately "inside-out" (i.e., inverted) membrane vesicles, as opposed to sonication, which results in scrambled vesicles (11)(12)(13)16). Under these conditions, the possibility remained that cold penicillin might be greatly retarded in its penetration of these inside-out vesicles to terminate binding of labeled antibiotic (Dougherty, personal communication).…”

Section: Methodsmentioning

confidence: 99%

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms

Leon

Panos

1988

J Bacteriol

View full text Add to dashboard Cite

The penicillin-binding proteins (PBPs) of Streptococcus pyogenes and two of its derived, stabilized (i.e., nonreverting) L forms, an osmotically fragile L form and a physiologic isotonic L form, were compared. The numbers of PBPs in the membranes of these organisms were 6, 4, and 2 for the coccus and the osmotically fragile and physiologic isotonic L forms, respectively. Likewise, the relative amounts of total PBPs were 1.00: 1.48:0.32 for this coccus and the osmotically fragile and physiologic isotonic L forms, respectively. The two largest PBPs (PBPs 1 and 2) of the coccus were absent in both L forms, while the smallest PBPs (PBPs 5 and 6) were found in all three membranes. Deacylation (half-life) of three of the four PBPs in the osmotically fragile L form membrane required a significantly longer time than did deacylation of these presumed identical enzymes in the parental coccal membrane. Conversely, there was no such difference between the only two PBPs of the physiologic isotonic L form and the same coccal membrane proteins. Intact cells of all three organisms secreted PBPs and what appeared to be penicilloic acid and a minimal amount of free penicillin. A greater amount of these PBPs was secreted by both L forms than by the coccus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and ratios of secreted PBPs were identical to those from labeled membrane preparations. These differences are correlated with some of our previous findings and are discussed in terms of inhibition of cell wall synthesis and resulting membrane changes in these two derived, stabilized coccal L forms.A considerable body of information has been gathered on the inability of stabilized L forms of Streptococcus pyogenes to synthesize a rigid bacterial cell wall. These studies have included morphological, biochemical, and enzymological pursuits using L forms requiring a hypertonic environment for growth as well as those able to grow on all physiologic isotonic media which support growth of the parental coccus. Some of these studies have been recently reviewed (14). An earlier study had focused on the defective synthesis of lipid intermediates for peptidoglycan in an L form of this coccal pathogen. It concluded that the inability of this L form to synthesize a cell wall in vivo was, in part, due to its failure to form significant quantities of the lipid substrates needed for peptidoglycan formation. But evidence indicated that this lesion might also be at the enzymological level (15).Penicillin-binding proteins (PBPs) are considered penicillin-sensitive bacterial enzymes involved in terminal stages of cell wall formation (1,5,17,18,21). To our knowledge, a detailed study aimed at documenting differences in these particular enzymes in a coccal pathogen and its resulting L forms had not been documented previously. Therefore, this study compares changes in these PBPs within a nephritogenic S. pyogenes, type 12, and its stabilized osmotically fragile and physiologic isotonic L forms. These findings substantiate an earlier bel...

show abstract

Section: Methodsmentioning

confidence: 99%

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms

Leon

Panos

1988

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The E. coli NDH-2A was further characterized and showed to contain 1 mol of noncovalently bound FAD per mol of purified enzyme; moreover, it consists of a single polypeptide with M r 47,200, which in the mature form has an apparent molecular mass of 45 kDa (30). The NDH-2A from the E. coli respiratory chain faces the cytosol (62).…”

Section: Biochemical and Genomic Aspects Of Nad(p)h Dehydrogenasesmentioning

confidence: 99%

New Insights into Type II NAD(P)H:Quinone Oxidoreductases

Melo

Bandeiras

Teixeira

2004

Microbiol Mol Biol Rev

238

212

View full text Add to dashboard Cite

Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)+ pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of ∼50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the [NADH]/[NAD+] balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2

show abstract

“…Cytoplasmic membrane vesicles from Escherichia coli that have the same (right-side-out; refs. [6][7][8][9][10][11] or the opposite (inverted; refs. 12-15) orientation as the membrane in the intact cell retain the capacity to convert respiratory energy into a IATH+, and studies with these preparations have provided virtually unequivocal support for the central obligatory role ofchemosmotic phenomena in active transport (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

“…pBR322 DNA (10 Ag) was linearized in 300 Al of 20 mM Tris HCl, pH 7.0/100 mM NaCl/7 mM MgCl2/2 mM 2-mer--captoethanol containing 10 units of BamHI at 37"C for 15 …”

mentioning

confidence: 99%

ATP-driven active transport in right-side-out bacterial membrane vesicles.

Hugenholtz¹,

Hong²,

Kaback³

1981

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Membrane vesicles from Salmonella typhimurium induced for phosphoglycerate transport, were loaded with pyruvate kinase and ADP by lysing spheroplasts under appropriate conditions. Vesicles so prepared catalyze active transport of proline and serine in the presence of phosphoenolpyruvate; this activity is abolished by the protonophore carbonyl cyanide-m-chlorophenylhydrazone and by the HW-ATPase inhibitor NN' dicyclohexylcarbodiimide but not by anoxia or cyanide. In contrast, D-lactate-driven active transport is abolished by the hydrazone and by anoxia or cyanide but not by the carbodiimide. Moreover, phosphoenolpyruvate does not drive transport effectively in vesicles that lack the phosphoglycerate transport system. The results are consistent with an overall mechanism in which phosphoenolpyruvate gains access to the interior of the vesicles by means of the phosphoglycerate transporter and is then acted on by pyruvate kinase to phosphorylate ADP. ATP formed inside of the vesicles is then hydrolyzed by the H+-ATPase, leading to the generation of a proton electrochemical gradient that drives H+/ solute symport. By using pBR322 as vector and Escherichia coli. as host, a fragment of S. typhimurium DNA coding for the phosphoglycerate transport system has been cloned. E. coli membrane vesicles containing the phosphoglycerate transport system also catalyze transport in the presence of phosphoenolpyruvate when they are loaded with pyruvate kinase and ADP.According to the chemosmotic hypothesis of Mitchell (1-5), energy derived from respiration, light, or ATP hydrolysis can be transformed into a transmembrane electrochemical gradient of protons (IAAH+) that represents the immediate driving force for active transport and various other energy-dependent processes. Cytoplasmic membrane vesicles from Escherichia coli that have the same (right-side-out; refs. 6-11) (33). These workers also suggested the possibility of using the phosphoglycerate transport system to affect the intravesicular generation ofATP from phosphoenolpyruvate added to the medium (32).The experiments presented here demonstrate that external phosphoenolpyruvate drives active transport in right-side-out vesicles containing the phosphoglycerate transport system and an ATP-generating system consisting of pyruvate kinase and ADP. They also provide evidence suggesting that transport activity under these conditions is due to the intravesicular formation of ATP that is subsequently hydrolyzed by the H+-ATPase with generation of a &AfH+. In addition, the S. typhimurium gene encoding for phosphoglycerate transport activity has been cloned by transformation into E. coli, in which it is expressed functionally.MATERIALS AND METHODS Growth of Cells and Preparation of Membrane Vesicles. S. typhimurium LT-2 was grown on medium A (34) with either 0.5% sodium DL-lactate (uninduced) or 0.5% sodium 3-phosphoglycerate (induced) as indicated. Cells were harvested in the middle of the logarithmic growth phase (-=180 Klett units). E. coli/pBR322-pgt2 (see below) was gr...

show abstract

Immunochemical analysis of membrane vesicles from Escherichia coli

Cited by 76 publications

References 68 publications

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms

New Insights into Type II NAD(P)H:Quinone Oxidoreductases

ATP-driven active transport in right-side-out bacterial membrane vesicles.

Contact Info

Product

Resources

About