scholarly journals Loss of protection by nucleotides against proteolysis and thiol modification in the isolated α-subunit from F1 ATPase of Escherichia coli mutant uncA401

1984 ◽  
Vol 224 (1) ◽  
pp. 145-151 ◽  
Author(s):  
H Stan-Lotter ◽  
P D Bragg
Keyword(s):  
Escherichia Coli ◽  
Adenosine Triphosphatase ◽  
Alpha Subunit ◽  
Thiol Groups ◽  
Α Subunit ◽  
Partial Protection ◽  
E Coli ◽  
F1 Atpase ◽  
High Affinity ◽  
High Affinity Site

Binding of nucleotides to the high-affinity site of the isolated alpha subunit of normal Escherichia coli F1 adenosine triphosphatase (ATPase) results in partial protection against digestion by trypsin [Senda, Kanazawa, Tsuchiya & Futai (1983) Arch. Biochem. Biophys. 220, 398-440]. In contrast, the isolated alpha subunit from the defective ATPase of the E. coli uncA401 mutant (strain AN120) is cleaved by trypsin to peptides of less than 8000 Da in the presence of ADP or ATP (2.5 microM-110 mM). The nucleotide-dependent accessibility of thiol groups of the isolated alpha subunit was also studied. Two out of four thiol groups of the alpha subunit from normal ATPase are labelled by fluorescent maleimides or iodoacetates, but in the presence of ADP or ATP (0.14-1.2 mM), reaction of thiol groups with these labels is almost absent. Mutant alpha subunit, however, is labelled by these reagents at all four thiol groups in the presence or absence of ADP or ATP (1 mM). These results suggest that the mutation in the ATPase of strain AN120 leads either to the loss of the high-affinity nucleotide-binding site or affects transmission of allosteric changes that occur on binding of nucleotide to the isolated alpha subunit.

scholarly journals The uncA gene codes for the α-subunit of the adenosine triphosphatase of Escherichia coli. Electrophoretic analysis of uncA mutant strains

1979 ◽  
Vol 180 (1) ◽  
pp. 103-109 ◽  
Author(s):  
A E Senior ◽  
J A Downie ◽  
G B Cox ◽  
F Gibson ◽  
L Langman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Adenosine Triphosphatase ◽  
Alpha Subunit ◽  
Subunit Structure ◽  
Net Charge ◽  
Α Subunit ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Low Ionic Strength

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.

Binding of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli to phospholipid vesicles

1978 ◽  
Vol 56 (6) ◽  
pp. 559-564 ◽  
Author(s):  
P. D. Bragg ◽  
C. Hou
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Adenosine Triphosphatase ◽  
Lipid Binding ◽  
Phospholipid Vesicles ◽  
Α Subunit ◽  
E Coli ◽  
Soybean Phospholipid

Incubation of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli with phospholipid vesicles resulted in binding of the enzyme to the lipid. Binding was observed with vesicles of soybean phospholipid (asolectin), phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and cardiolipin. Binding was not affected by alterations in pH in the range of pH 6.5 to 8.5, by ionic strength, or by the presence of Mg2+. Loss of the δ subunit from the enzyme had no effect on binding. However, removal of the δ and ε subunits by treatment of the enzyme with trypsin prevented binding to phospholipid. This treatment also removed a small portion (<2000 daltons) of the α subunit. It is concluded that the ATPase of E. coli binds to phospholipid vesicles mainly by nonpolar interactions through the α and (or) ε subunits of the enzyme.

Nanomolar Levels of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Glycine Betaine Are Sufficient To Confer Osmoprotection to Escherichia coli

1999 ◽  
Vol 65 (8) ◽  
pp. 3304-3311 ◽  
Author(s):  
Anne Cosquer ◽  
Vianney Pichereau ◽  
Jean-Alain Pocard ◽  
Jacques Minet ◽  
Michel Cormier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salinity Tolerance ◽  
Glycine Betaine ◽  
Standard Laboratory ◽  
E Coli ◽  
High Affinity ◽  
Bacterial Cultures ◽  
Transport Studies ◽  
Ecological Implications ◽  
Substrate Concentrations

ABSTRACT We combined the use of low inoculation titers (300 ± 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently,proU + strains of E. coli, but not the proP + strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.

scholarly journals The interaction of cations with lipopolysaccharide from Escherichia coli C as shown by measurement of binding constants and aggregation reactions

1989 ◽  
Vol 263 (3) ◽  
pp. 695-702 ◽  
Author(s):  
A M Field ◽  
E Rowatt ◽  
R J P Williams
Keyword(s):  
Escherichia Coli ◽  
Sodium Salt ◽  
Binding Constants ◽  
Assay Method ◽  
Aggregation State ◽  
High Affinity ◽  
A Value ◽  
High Affinity Site ◽  
Low Ionic Strength ◽  
Very High

Lipopolysaccharide from Escherichia coli C interacts with polyvalent cations at low ionic strength at more than one site. The first site has high affinity with a KD value of 10(-8) M for Ca2+ and even stronger binding for [(NH3)5CoNH2Co(NH3)5]5+ and La3+. The high-affinity site for the latter cations is beyond the sensitivity of the assay method. The second, low-affinity, site for bivalent cations has a Km of 10(-3) M, whereas, for tervalent and quinquevalent metal cations and spermine and hexacyclen (1,4,7,10,13,16-hexa-azacyclo-octadecane), this constant has a value of 10(-5) M. Binding of cations to the high-affinity site does not alter the aggregation state of the lipopolysaccharide, but combination with the low-affinity site gives particles twice the size of those of the sodium salt. Very high Ca2+ concentrations (30 mM) give particles eight times the size of those of the sodium salt.

scholarly journals Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions

1991 ◽  
Vol 275 (1) ◽  
pp. 151-158 ◽  
Author(s):  
M Kusche ◽  
H H Hannesson ◽  
U Lindahl
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Binding Sites ◽  
Proteinase Inhibitor ◽  
Capsular Polysaccharide ◽  
Polymer Modification ◽  
Sulphated Polysaccharide ◽  
E Coli ◽  
High Affinity ◽  
K5 Polysaccharide

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)→(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3′-phosphate 5′-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

scholarly journals Affinity of Tomopenem (CS-023) for Penicillin-Binding Proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa

2008 ◽  
Vol 53 (3) ◽  
pp. 1238-1241 ◽  
Author(s):  
Tetsufumi Koga ◽  
Chika Sugihara ◽  
Masayo Kakuta ◽  
Nobuhisa Masuda ◽  
Eiko Namba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Binding Proteins ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
High Affinity

ABSTRACT Tomopenem (formerly CS-023), a novel 1β-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drug's PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

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