Mg2+-activated ATP hydrolysis and sulfhydryl groups in membranes from human erythrocytes

1970 ◽  
Vol 48 (5) ◽  
pp. 604-612 ◽  
Author(s):  
Frances M. Smith ◽  
Jacob A. Verpoorte
Keyword(s):  
Atp Hydrolysis ◽  
Enzymatic Reaction ◽  
Maximal Activity ◽  
Maximum Activity ◽  
Human Erythrocytes ◽  
Sulfhydryl Groups ◽  
Sulfhydryl Reagents ◽  
Rate Of Reaction ◽  
Rate Profile ◽  
Sigmoid Shape

The ATP-hydrolyzing activity of membranes prepared from human erythrocytes depends on the presence of Mg2+ ions. Maximum activity is observed when the concentrations of Mg2+ and ATP are equal. The pH–rate profile of the enzymatic reaction shows a maximum at pH 7.8. The ATP-hydrolyzing activity has decreased to half the maximal activity at pH 6.3 and 9.8, respectively. The enzyme is inhibited by sulfhydryl reagents like p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide. Membranes that are titrated with NaOH or treated with p-CMB lose up to 30% of their protein content. This loss is reversed when Mg2+ or Mg2+ plus ATP is added. However, neither Mg2+ nor Mg2+ plus ATP detectably alters the rate of reaction between the sulfhydryl groups and specific reagents, nor protects the ATP-hydrolyzing activity from inhibition by p-CMB. The amount of protein which dissociates from the membrane increases with pH. The curve describing this extraction has a sigmoid shape with an inflection point at pH 10.2. Maximum solubilization is obtained at pH 11.2. Thus the results indicate that sulfhydryl groups and also the structure of the membrane are important for the ATP-hydrolyzing activity.

scholarly journals ON THE ROLE OF VIRUS SULFHYDRYL GROUPS IN THE ATTACHMENT OF ENTEROVIRUSES TO ERYTHROCYTES

1960 ◽  
Vol 112 (3) ◽  
pp. 455-478 ◽  
Author(s):  
Lennart Philipson ◽  
Purnell W. Choppin
Keyword(s):  
Virus Particle ◽  
Human Erythrocytes ◽  
Hemagglutinating Activity ◽  
Sulfhydryl Groups ◽  
Present Communication ◽  
Sulfhydryl Reagents ◽  
Thiol Compounds ◽  
Virus Particles ◽  
Receptor Sites

Many animal viruses possess the ability to agglutinate erythrocytes. In most but not all cases hemagglutination is due to the virus particle itself, and appears to result from the mechanical bridging of two or more erythrocytes by virus particles which attach to receptor sites on each erythrocyte (1, 2). Thus, attachment of virus particles to erythrocytes is a prerequisite for hemagglutination, and prevention of absorption of virus prevents hemagglutination. Among the enteroviruses, many ECHO viruses and some strains of Coxsackie B3 virus agglutinate human erythrocytes (3-7), and the evidence indicates that hemagglutination is due to the virus particle itself (3, 5, 6). The precise mechanism of attachment of enteroviruses to cells is unknown. Chymotrypsin treatment of erythrocytes prevents the absorption of some ECHO viruses (8). This suggests that the receptor sites on the erythrocyte may be at least in part protein in nature. The present communication is concerned with the mechanism of attachment of enteroviruses to cells. It is shown that sulfhydryl reagents block the hemagglutinating activity of enteroviruses. Treated virus fails to absorb to erythrocytes. Thiol compounds restore the hemagglutinating activity of enteroviruses treated with mercaptide-forming sulfhydryl reagents. The effect of sulfhydryl reagents on the infectivity of some enteroviruses is also described.

scholarly journals Sulfhydryl reagents induce altered spectrin self-association, skeletal instability, and increased thermal sensitivity of red cells

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1190-1196
Author(s):  
DK Smith ◽  
J Palek
Keyword(s):  
Thermal Stability ◽  
Reduced Glutathione ◽  
Thermal Sensitivity ◽  
Red Cells ◽  
Sulfhydryl Groups ◽  
Sulfhydryl Reagents ◽  
Sulfhydryl Reagent ◽  
Self Association ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Membrane Skeletons

Incubation of erythrocytes with the sulfhydryl reagent N-ethyl- maleimide (NEM) results in altered spectrin self-association and formation of dimers on the membrane. Skeletons isolated from these cells exhibit marked skeletal instability. In addition, NEM treatment induces increased thermal sensitivity of both cells and purified spectrin. These effects were not produced in aerobically incubated glucose-6-phosphate dehydrogenase deficient cells and were therefore presumably not due to depletion of intracellular reduced glutathione. These effects were produced by another permeant sulfhydryl reagent, monobromobimane, but not by its membrane-impermeant derivative. We conclude that spectrin sulfhydryl groups play an important role in spectrin self-association and thermal stability.

scholarly journals A nucleoside triphosphate-dependent deoxyribonucleic acid-breakdown system in Mycobacterium smegmatis, and the effect of iron limitation on the activity of this system

1969 ◽  
Vol 111 (5) ◽  
pp. 679-687 ◽  
Author(s):  
F. G. Winder ◽  
M. P. Coughlan
Keyword(s):  
Dna Content ◽  
Mycobacterium Smegmatis ◽  
Deoxyribonucleic Acid ◽  
Maximal Activity ◽  
Enzyme Concentration ◽  
Iron Limitation ◽  
Nucleoside Triphosphate ◽  
Primary Products ◽  
Rate Of Reaction ◽  
Optimum Ph

1. The presence of a nucleoside triphosphate-dependent DNA-breakdown system was demonstrated in extracts of Mycobacterium smegmatis. Its activity was increased substantially by iron limitation, apparently after the fall in DNA content that took place under these conditions. A maximal activity of about 0·2μmole of deoxyribonucleotide/30min./mg. of protein was found in crude extracts. 2. After slight purification by streptomycin treatment, the enzyme showed maximal activity with undenatured DNA (Km≃200μg./ml.), ATP (Km≃1·2mm) or UTP, CTP and GTP giving lower activity and pyrophosphate giving none, and Mg2+ ions (optimum concn. 12mm). The optimum pH was 8·5. 3. In the assay system there was proportionality between enzyme concentration and rate of reaction, but the rate fell off with time. 4. ATP was broken down in the reaction and monodeoxy-ribonucleotides were among the products, but the presence of some oligodeoxy-ribonucleotides was not excluded and the degree of phosphorylation of the primary products was uncertain.

Potassium-chloride cotransport in resealed human red cell ghosts

1986 ◽  
Vol 250 (4) ◽  
pp. C578-C583 ◽  
Author(s):  
P. B. Dunham ◽  
P. J. Logue
Keyword(s):  
Membrane Protein ◽  
Potassium Chloride ◽  
Cell Volume ◽  
Human Erythrocytes ◽  
Sulfhydryl Groups ◽  
Red Cell ◽  
Intact Cells ◽  
Protein Sulfhydryl ◽  
Protein Sulfhydryl Groups ◽  
Resealed Ghosts

Furosemide-inhibitable K influx is threefold higher in resealed ghosts of human erythrocytes than in intact cells. The enhancement is specific for K in that furosemide-inhibitable Na influx is the same in resealed ghosts and intact cells. The enhanced K influx resembles K-Cl cotransport in intact cells in that it requires Cl but not Na. N-ethylmaleimide (NEM), which stimulates furosemide-inhibitable K influx in intact cells, is without effect (or slightly inhibitory) in resealed ghosts. The failure of NEM to enhance the flux was not due to low ATP in the ghosts. These findings suggest that enhancement of the K flux in ghosts occurs by oxidation of membrane protein sulfhydryl groups, known to occur with lysis, the same sulfhydryl groups at which NEM acts by alkylation. This conclusion is supported by two observations: dithiothreitol completely prevents the increase in K influx in ghosts; this agent inhibits both oxidation of sulfhydryl groups and alkylation of them by NEM; and K influx in resealed ghosts is sensitive to changes in cell volume, just as it is in NEM-treated intact cells.

Pyruvate kinase from the moderate halophile, Vibrio costicola

1977 ◽  
Vol 55 (8) ◽  
pp. 825-833 ◽  
Author(s):  
Eveline de Médicis ◽  
Bertrand Rossignol
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Chloride Concentration ◽  
Maximum Velocity ◽  
Maximal Activity ◽  
Maximum Activity ◽  
Moderate Halophile ◽  
Exclusion Chromatography

Pyruvate kinase (EC 2.7.1.40)from Vibrio costicola was purified to homogeneity. The tetramer had a molecular weight of 270 000 measured by exclusion chromatography on Sepharose 4B and was dissociated into apparently identical subunits with a molecular weight of 65 000 measured by SDS–polyacrylamide gel.The kinetic parameters Vm, K0.5, and n were measured at variable KCl concentrations. Both affinity and activity were inhibited when potassium chloride concentration was at 0.025 M, a fact which indicates that low potassium concentration is required for activity. Maximal activity and affinity were obtained at 0.125 M KCl. Potassium chloride appeared as an allosteric inhibitor in the range of 0.125 M to 1.025 M. Above 1.025 M KCl, both maximum velocity and affinity were decreased. NaCl had the same effect as KCl. The pH profiles of K0.5 and Vm of the phosphoenolpyruvate dependence revealed two main pK values: at 6.4 which controls affinity, and at 8.6, which controls activity.The activity of the enzyme showed a hysteretic response to salt. The specific activity measured under fixed standard conditions of temperature and salt concentrations showed a reversible variation which depended upon the preincubation conditions of temperature and salt concentration. The maximum activity tested was reached after low (0.1 M) or high (2 M) KCl preincubation at room temperature.

The influence of haptoglobin on the reactivity of the –SH groups of hemoglobin

1969 ◽  
Vol 47 (12) ◽  
pp. 1205-1208 ◽  
Author(s):  
B. Malchy ◽  
G. H. Dixon
Keyword(s):  
Conformation Change ◽  
Sulfhydryl Reagents ◽  
Sh Groups ◽  
Rate Of Reaction

The rate of reaction of the β93 sulfhydryl of hemoglobin with iodoacetamide and 2,2′-, and 4,4′- dithiodipyridine is much slower when the hemoglobin is bound by haptoglobin than when it is free. However, these sulfhydryl reagents will react with the haemoglobin–haptoglobin complex, and the reaction of the complex with iodoacetamide is still at position β93. The results suggest that haptoglobin might induce a conformation change on oxyhemoglobin which is similar to that occurring upon deoxygenation.

scholarly journals MYROSINASE ACTIVITY IN ARMORACIA RUSTICANA

1970 ◽  
Vol 62 ◽  
Author(s):  
Daniela Mucete ◽  
Mariana Poiana ◽  
I. Jianu ◽  
Florina Radu
Keyword(s):  
Reaction Time ◽  
Hplc Analysis ◽  
Enzymatic Reaction ◽  
Maximum Activity ◽  
Spectrophotometric Analysis ◽  
Armoracia Rusticana ◽  
Different Temperatures ◽  
Myrosinase Activity ◽  
Best Parameters

This work is part of a study, which is more complex regarding the activity of sinigrin-myrosinase complex. With the help of this study we are following the optimization and the characterization of myrosinase activity with the help of HPLC chromatography and the comparison of the obtained results by spectrophotometric analysis. At different temperatures conditions, (25°C ÷ 75°C), and reaction time (30 ÷ 390 minutes) were determined sinigrin concentration in extract (CSinExtr) and sinigrin concentration consumed in enzymatic reaction (CSinCons) by HPLC analysis. The appreciation of sinigrin-myrosinase system activity was done by transforming CSinExtr and CSinCons in μg/glucose/g sample*hour. The best parameters, adequate to myrosinase maximum activity, in Armoracia rusticana extracts were the following: pH = 7, temperature of 55°C, and reaction time was of 210 minutes for rubbed out horseradish samples and of 240 minutes for unrubbed horseradish samples.

Studies on phosphoglyceromutase from chicken breast muscle: number and reactivity of sulfhydryl groups

1976 ◽  
Vol 54 (4) ◽  
pp. 307-320 ◽  
Author(s):  
T. J. Carne ◽  
D. J. McKay ◽  
T. G. Flynn
Keyword(s):  
Enzymatic Activity ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Marked Change ◽  
Sulfhydryl Groups ◽  
Breast Muscle ◽  
Performic Acid ◽  
Chicken Breast ◽  
Fluorescence Emission Spectrum ◽  
Sulfhydryl Reagents

Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5′-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB. The total number of sulfhydryl groups determined by amino acid analysis of the performic acid oxidized and carboxymethylated enzyme was also found to be six. Disc gel electrophoresis in sodium dodecyl sulfate indicated that the enzyme is composed of subunits having the same molecular weights which suggests there are three sulfhydryl groups per subunit. Reaction with Nbs2 iodoacetate or iodoacetamide resulted in the modification of only two sulfhydryl groups. In each of the modifications, except that due to iodoacetamide both substrate and cofactor reduced the rate, but not the extent, of the reaction. Substrate was more effective than cofactor in reducing the rate of reaction with sulfhydryl reagents suggesting that a cysteine residue is involved in the binding of substrate. That the involvement is indirect is shown by the lack of protection offered by substrate and cofactor to reaction with iodoacetamide. Inactivation of PGM following titration with sulfhydryl reagents is not likely to be due to a conformational change since none of any significance was indicated by circular dichroism of the treated enzyme. This result was substantiated by the lack of a marked change in the fluorescence emission spectrum of the PMB treated enzyme.

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