scholarly journals Thiol-dependent changes in the properties of rat liver sulphotransferases

1971 ◽  
Vol 123 (3) ◽  
pp. 427-434 ◽  
Author(s):  
D. J. Barford ◽  
J. G. Jones
Keyword(s):  
Ionic Strength ◽  
Methyl Ester ◽  
Molecular Size ◽  
Degree Of Polymerization ◽  
Prolonged Storage ◽  
Kinetic Properties ◽  
Female Rat ◽  
Km Value ◽  
Low Ionic Strength ◽  
Rat Livers

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The Km values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5μm and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0°C when the Km and Vmax. values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0°C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The Km value for adenosine 3′-phosphate 5′[35S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.

scholarly journals Contraction transients of skinned muscle fibers: effects of calcium and ionic strength.

1978 ◽  
Vol 72 (5) ◽  
pp. 701-715 ◽  
Author(s):  
J Gulati ◽  
R J Podolsky
Keyword(s):  
Ionic Strength ◽  
Muscle Fibers ◽  
Kinetic Properties ◽  
Cross Bridge ◽  
Skinned Muscle ◽  
The Cross ◽  
Cross Bridge Mechanism ◽  
Low Ionic Strength ◽  
Bridge Number ◽  
Steady Force

Calcium and ionic strength are both known to modify the force developed by skinned frog muscle fibers. To determine how these parameters affect the cross-bridge contraction mechanism, the isotonic velocity transients following step changes in load were studied in solutions in which calcium concentration and ionic strength were varied. Analysis of the motion showed that calcium has no effect on either the null time or the amplitude of the transients. In contrast, the transient amplitude was increased in high ionic strength and was suppressed in low ionic strength. These results are consistent with the idea that calcium affects force in skeletal muscle by modulating the number of force generators in a simple switchlike "on-off" manner and that the steady force at a given calcium level is proportional to cross-bridge number. On the other hand, the effect of ionic strength on force is associated with changes in the kinetic properties of the cross-bridge mechanism.

scholarly journals Purification from rat liver of an enzyme that catalyses the sulphurylation of phenols

1971 ◽  
Vol 123 (5) ◽  
pp. 901-906 ◽  
Author(s):  
F. A. McEvoy ◽  
J. Carroll
Keyword(s):  
Enzyme Activity ◽  
Methyl Ester ◽  
Rat Liver ◽  
The Other ◽  
Male Rat ◽  
Thiol Compounds ◽  
Km Value ◽  
Rat Livers

1. An enzyme (EC 2.8.2.1) that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to phenols was purified approx. 2000-fold from male rat livers. 2. The purified preparation did not catalyse the sulphurylation of dehydroepiandrosterone, butan-1-ol, l-tyrosine methyl ester, 1-naphthylamine or serotonin. 3. At pH8.0 and 37°C the Km values of the enzyme for p-nitrophenol and adenosine 3′-phosphate 5′-sulphatophosphate are 51 and 14μm respectively. The Km value for either substrate is independent of the concentration of the other. 4. The sulphurylation of phenol is inhibited by thiol compounds and glutathione at a concentration of 3mm caused an approx. 50% decrease in enzyme activity. 5. The Km of the enzyme for adenosine 3′-phosphate 5′-sulphatophosphate is unaffected by the presence of added glutathione but at a concentration of 5mm-glutathione the Km of the enzyme for its phenolic substrate is decreased.

scholarly journals Partial purification and properties of an enzyme from rat liver that catalyses the sulphation of l-tyrosyl derivatives

1970 ◽  
Vol 116 (5) ◽  
pp. 797-803 ◽  
Author(s):  
P. Mattock ◽  
J. G. Jones
Keyword(s):  
Methyl Ester ◽  
Rat Liver ◽  
Potent Inhibitor ◽  
Quantitative Difference ◽  
Partial Purification ◽  
Female Rat ◽  
Mixed Substrate ◽  
Partial Separation ◽  
Purification And Properties ◽  
Rat Livers

1. An enzyme that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′[35S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3′-phosphate 5′-sulphatophosphate–phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or -10°C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.

scholarly journals Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles

1976 ◽  
Vol 159 (3) ◽  
pp. 627-631 ◽  
Author(s):  
M S Wooster ◽  
J M Wrigglesworth
Keyword(s):  
Ionic Strength ◽  
Dehydrogenase Activity ◽  
Association Constant ◽  
Phospholipid Vesicles ◽  
Kinetic Properties ◽  
Km Value ◽  
Triton X ◽  
Positively Charged ◽  
Apparent Association ◽  
Apparent Association Constant

1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 × 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.

scholarly journals Interaction of macrophage-migration-inhibitory factor with haematin

1998 ◽  
Vol 331 (3) ◽  
pp. 905-908 ◽  
Author(s):  
Joanne L. PENNOCK ◽  
Jiraprapa WIPASA ◽  
Michael P. GORDGE ◽  
David J. MEYER
Keyword(s):  
Ionic Strength ◽  
Methyl Ester ◽  
Immune Responses ◽  
Migration Inhibitory Factor ◽  
Human Liver ◽  
Macrophage Migration Inhibitory Factor ◽  
Inhibitory Factor ◽  
High Yield ◽  
Macrophage Migration ◽  
Low Ionic Strength

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione–agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Åman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2, 143–149]. The l-dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC50 100–200 nM). The inhibition was non-competitive and showed positive co-operativity (h = 5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.

scholarly journals Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH.

1990 ◽  
Vol 96 (1) ◽  
pp. 1-22 ◽  
Author(s):  
P A Pappone ◽  
G L Barchfeld
Keyword(s):  
Ionic Strength ◽  
Single Channel ◽  
Ph Dependence ◽  
Kinetic Properties ◽  
Magnesium Ions ◽  
Channel Kinetics ◽  
Open Time ◽  
Calcium And Magnesium ◽  
Low Ionic Strength ◽  
Calcium And Magnesium Ions

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

An Evaluation of a Plasma Fractionation System

1960 ◽  
Vol 4 (01) ◽  
pp. 031-044
Author(s):  
George Y. Shinowara ◽  
E. Mary Ruth
Keyword(s):  
Biological Activity ◽  
Ionic Strength ◽  
Plasma Proteins ◽  
High Concentration ◽  
Significant Evidence ◽  
Native Proteins ◽  
Mobility Data ◽  
Fractionation Procedure ◽  
The Individual ◽  
Low Ionic Strength

SummaryFour primary fractions comprising at least 97 per cent of the plasma proteins have been critically appraised for evidence of denaturation arising from a low temperature—low ionic strength fractionation system. The results in addition to those referable to the recovery of mass and biological activity include the following: The high solubilities of these fractions at pH 7.3 and low ionic strengths; the compatibility of the electrophoretic and ultracentrifugal data of the individual fractions with those of the original plasma; and the recovery of hemoglobin, not hematin, in fraction III obtained from specimens contaminated with this pigment. However, the most significant evidence for minimum alterations of native proteins was that the S20, w and the electrophoretic mobility data on the physically recombined fractions were identical to those found on whole plasma.The fractionation procedure examined here quantitatively isolates fibrinogen, prothrombin and antithrombin in primary fractions. Results have been obtained demonstrating its significance in other biological systems. These include the following: The finding of 5 S20, w classes in the 4 primary fractions; the occurrence of more than 90 per cent of the plasma gamma globulins in fraction III; the 98 per cent pure albumin in fraction IV; and, finally, the high concentration of beta lipoproteins in fraction II.

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