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 The Biological Significance of Haemoglobin in Nematode Parasites I. The Characteristics of the Purified Pigments

1949 ◽  
Vol 2 (3) ◽  
pp. 287 ◽  
Author(s):  
WP Rogers
Keyword(s):  
Carbon Monoxide ◽  
Linear Relationship ◽  
Equilibrium Constant ◽  
Haemonchus Contortus ◽  
Biological Significance ◽  
High Affinity ◽  
Nematode Parasites ◽  
A Value ◽  
Ammonium Sulphate Fractionation ◽  
Very High

Haemoglobins from Nippostrongylus muris, Nematodirus�spp., and Haemonchus contortus were purified by ammonium sulphate fractionation and their properties examined. All the haemoglobins showed a very high affinity for oxygen; the tension of half saturation (P50) for Nematodirus haemoglobin of concentrations about 1 x 10-4 g.-atoms of iron per 1. at pH 7.4 was in the region of 0.04 mm. of mercury. The P50 for H. contortus haemoglobin was similar to that of Nematodirus .spp.; N. muris haemoglobin had a somewhat higher p:;o' The. parasite haemoglobins all showed an unusually low affinity for carbon monoxide, the equilibrium constant, K = [HbCO] X p02/[Hb02] X pCO, having a value of about 1. The "span," the distance between the a-bands of oxyhaemoglobin and carboxyhaemoglobin, varied from 60 to 65 A. for the three parasites. None of the haemoglobins obtained from the parasites showed properties supporting the view that there is a linear relationship between log K and the "span."

scholarly journals Calcium-binding constants of trypsin and trypsinogen. A reassessment

1981 ◽  
Vol 193 (2) ◽  
pp. 655-658 ◽  
Author(s):  
S G R Cliffe ◽  
D A W Grant
Keyword(s):  
Affinity Chromatography ◽  
Calcium Binding ◽  
Cation Exchanger ◽  
Binding Constants ◽  
High Affinity ◽  
High Affinity Site ◽  
Logarithmic Unit

The Ca2+-binding constants for trypsin and trypsinogen have been reassessed by using enzyme that has been purified by affinity chromatography and measuring the distribution of 45Ca2+ between the protein and a cation exchanger. The pKCa2+ value of 4.5 for the high-affinity site on trypsin was 1 logarithmic unit greater than that previously reported.

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.

Glycerol Inhibits or Uncouples the Plasma Membrane (Ca2++Mg2+)ATPase of Kidney Proximal Tubules Depending on the Ca2+ Concentration

1995 ◽  
Vol 50 (11-12) ◽  
pp. 845-853 ◽  
Author(s):  
Mauro Sola-Penna ◽  
Adriana dos Passos Lemos ◽  
Adalberto Vieyra
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Proximal Tubules ◽  
Maximal Velocity ◽  
Soluble Enzyme ◽  
Inhibitory Component ◽  
High Affinity ◽  
Low Concentrations ◽  
High Affinity Site ◽  
Very High

In this report it is shown that glycerol (0.5-10% v/v) stimulate the C12-E9-solubilized renal (Ca2++Mg2+)ATPase in the presence of low concentrations of free Ca2+ (< 10-6ᴍ). At 4% (v/v), the polyol decreases the K0.5 for Ca2+ from 1.15 to 0.22 μᴍ at the high-affinity site, and a very-high-affinity Ca2+ component appears. This component has a K0.5 ≤10-9 ᴍ and its maximal velocity is about one-third that of the fully activated enzyme (at 10-20 μᴍ Ca2+), which is not affected by glycerol (21.1 and 20.2 nmol·mg-1 ·min-1 in the absence and presence of the polyol, respectively). The low-affinity, inhibitory component of the Ca2+ curve (50-1000 μᴍ ) is also unaffected by glycerol. With 0.07 μᴍ free Ca2+ and soluble enzyme, the stimulatory effect of glycerol saturates at ≈10% (v/v). In contrast, with 17 μᴍ free Ca2+, glycerol has little effect up to 10% (v/v), and then progressively inhibits ATPase activity. These data indicate that the effect of the polyol is modulated by the occupancy of the high-affinity Ca2+ sites. In native vesicles, the stimulation promoted by low concentrations of glycerol at low concentrations of Ca2+ is accompanied by inhibition of active Ca2+ transport, indicating that, in these conditions, the polyol uncouples ATPase activity and ATP-driven Ca2+ influx.

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

1980 ◽  
Vol 58 (10) ◽  
pp. 969-977 ◽  
Author(s):  
P. Nicholls ◽  
V. Hildebrandt ◽  
B. C. Hill ◽  
F. Nicholls ◽  
J. M. Wrigglesworth
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
The Other ◽  
Enzyme Preparations ◽  
Apparent Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Membrane Bound ◽  
Enzyme Molecules ◽  
Low Ionic Strength

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 μM > Kd′ > 0.2 μM) and readily reversible and the other of high affinity (0.01 μM > Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s−1 (30 °C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites are occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c – cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.

scholarly journals Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking

1996 ◽  
Vol 315 (3) ◽  
pp. 909-916 ◽  
Author(s):  
Francesco MALATESTA ◽  
Giovanni ANTONINI ◽  
Flavia NICOLETTI ◽  
Alessandro GIUFFRÈ ◽  
Emilio D'ITRI ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Beef Heart ◽  
State Reduction ◽  
High Affinity ◽  
Disulphide Bond Formation ◽  
High Affinity Site ◽  
Low Ionic Strength ◽  
Covalent Complex

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046–7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) Km and Vmax values obtained from the non-linear Eadie–Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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