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.

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 Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal β-subunits

1983 ◽  
Vol 210 (2) ◽  
pp. 395-403 ◽  
Author(s):  
A E Senior ◽  
L Langman ◽  
G B Cox ◽  
F Gibson
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Atpase Activity ◽  
Atp Synthesis ◽  
Beta Subunit ◽  
F1 Atpase ◽  
Dependent Atpase ◽  
Mutant Strains ◽  
Synthesis Activity ◽  
Low Ionic Strength

To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

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.

scholarly journals Properties of membranes from mutant strains of Escherichia coli in which the β-subunit of the adenosine triphosphatase is abnormal

1979 ◽  
Vol 180 (1) ◽  
pp. 111-118 ◽  
Author(s):  
A E Senior ◽  
D R Fayle ◽  
J A Downie ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Beta Subunit ◽  
Beta Subunits ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Membrane Bound ◽  
Phenotypic Variations

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

scholarly journals Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

1972 ◽  
Vol 59 (4) ◽  
pp. 375-387 ◽  
Author(s):  
William Lehman ◽  
Andrew G. Szent-Györgyi
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Limulus Polyphemus ◽  
Muscle Tropomyosin ◽  
Low Ionic Strength ◽  
Full Activation

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits

1993 ◽  
Vol 1144 (3) ◽  
pp. 278-284 ◽  
Author(s):  
Holger Lill ◽  
Andreas Burkovski ◽  
Karlheinz Altendorf ◽  
Wolfgang Junge ◽  
Siegfried Engelbrecht
Keyword(s):  
Escherichia Coli ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Atpase Subunits

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

Sign in / Sign up

Export Citation Format

Share Document