scholarly journals The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule

1977 ◽  
Vol 167 (3) ◽  
pp. 739-748 ◽  
Author(s):  
David A. Thorley-Lawson ◽  
N. Michael Green
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Rate Constant ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Reaction Rates ◽  
Cysteic Acid ◽  
Thiol Groups ◽  
Low Concentrations

The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[14C]maleimide or 5,5′-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[14C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A1 and A2). The kinetics of the reaction of the ATPase with 5,5′-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca2+ the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca2+ the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca2+ of Mg2+ had very little effect on the reactivity.

Effect of thyroid hormone on carbohydrate content of Na+-K+-adenosine triphosphatase

1984 ◽  
Vol 247 (3) ◽  
pp. C282-C287 ◽  
Author(s):  
C. S. Lo ◽  
L. E. Klein ◽  
T. N. Lo
Keyword(s):  
Thyroid Hormone ◽  
Sodium Dodecyl Sulfate ◽  
Rate Constant ◽  
Gel Electrophoresis ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Carbohydrate Content ◽  
Beta Subunits ◽  
Reverse T3 ◽  
Dodecyl Sulfate

The effect of L-3,5,3'-triiodothyronine (T3) (50 micrograms/100 body wt) on the incorporation of labeled glucosamine and fucose into the subunits of Na+-K+-ATPase was examined by gel electrophoresis in sodium dodecyl sulfate. T3 augmented the incorporation of glucosamine into the alpha- and beta-subunits by 51 and 58%, respectively, in the 22-h chase experiments. Similarly T3 augmented the incorporation of fucose into the alpha- and beta-subunits by 58 and 43%, respectively. Reverse T3 did not alter the incorporation of labeled fucose in either subunit. The effect of T3 on the rate constant of degradation of renal cortical Na+-K+-ATPase was assessed. The rate constant of degradation (Kd) of the [3H]fucose labeled alpha- and beta-subunits for the hypothyroid rats were both 0.20, and for T3-treated rats, the Kd of the alpha- and beta-subunits were 0.23 and 0.18, respectively, suggesting that T3 enhanced fucose incorporation into the subunits of Na+-K+-ATPase rather than retarding the degradation of this enzyme.

scholarly journals Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence

1991 ◽  
Vol 275 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Y K Li ◽  
J Boggaram ◽  
L D Byers
Keyword(s):  
Isotope Effect ◽  
Rate Constant ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Acidic Group ◽  
Irreversible Inhibitors ◽  
Effects Of Ph ◽  
Solvent Isotope ◽  
Bell Shaped Curve

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

scholarly journals Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

1977 ◽  
Vol 75 (1) ◽  
pp. 135-147 ◽  
Author(s):  
A L Blitz ◽  
R E Fine ◽  
P A Toselli
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Coated Vesicles ◽  
Potassium Oxalate ◽  
Triton X ◽  
Trapping Agent ◽  
The Brain

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

scholarly journals The location of the thiol groups of myosin that are protected by pyrophosphate against reaction with 2,4-dinitrophenyl β-hydroxyethyl disulphide

1971 ◽  
Vol 125 (1) ◽  
pp. 261-266 ◽  
Author(s):  
Irena Kakol
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Adenosine Triphosphatase ◽  
Thiol Groups ◽  
Low Concentrations ◽  
Light Components

Myosin modified in the presence or in the absence of pyrophosphate by 2,4-dinitrophenyl β-hydroxyethyl disulphide was treated with iodo[1-14C]acetamide. The residual Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the modified myosin was different depending on the presence or absence of PPi during modification and the number of 2,4-dinitrophenyl β-hydroxyethyl disulphide-modified thiol groups. The radioactivity incorporated into the light components of myosin correlated with the Ca2+-stimulated ATPase activity of the modified myosin and decreased with decreasing residual Ca2+-stimulated ATPase activity of the modified myosin. When native myosin was treated with low concentrations of iodo[1-14C]acetamide the residual Ca2+-stimulated ATPase activity of carboxyamidomethylated myosin was high and the radioactivity incorporated into the light components of myosin was negligible. The thiol groups of the light components of myosin are essential to preserve the ATPase activity of the protein and are close to the pyrophosphate-binding sites.

scholarly journals Organization of thiol groups of electric-eel electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents

1980 ◽  
Vol 185 (3) ◽  
pp. 787-790 ◽  
Author(s):  
W E Harris ◽  
W L Stahl
Keyword(s):  
Adenosine Triphosphatase ◽  
Polar Solvent ◽  
Electric Organ ◽  
Thiol Group ◽  
Cross Linking ◽  
Thiol Groups ◽  
High Solubility ◽  
Efficient Inhibitor ◽  
Polar Environment ◽  
Electric Eel

The reactions of three bifunctional thiol-blocking reagents of differing cross-linking spans and two monofunctional thiol-blocking reagents with the Na+ + K+-stimulated ATPase of the electric-eel electric organ were examined. 1,5-Difluoro-2,4-dinitrobenzene with a cross-linking span of 0.3-0.5 nm (3-5 A) and high solubility in non-polar solvent was the most efficient inhibitor of enzyme activity; thus essential thiol groups exist in a non-polar environment and are approx. 0.3-0.5 nm (3-5 A) from their nearest thiol-group neighbours. Ligands promoting phosphorylation of the Na+ + K+-stimulated ATPase decreased the number of thiol groups bridged by 1,5-difluoro-2,4-dinitrobenzene and by 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone [0.7-1.0 nm (7-10 A) span]. Phosphorylation is associated with a conformational change in the enzyme.

scholarly journals Complex-formation and inhibition of urokinase by blood plasma proteins

1983 ◽  
Vol 215 (1) ◽  
pp. 123-131 ◽  
Author(s):  
E K Waller ◽  
W D Schleuning ◽  
E Reich
Keyword(s):  
Complex Formation ◽  
Rate Constant ◽  
Plasma Proteins ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Order Rate Constant ◽  
Reducing Conditions ◽  
Blood Plasma Proteins

We have studied the formation of covalent complexes between 125I-urokinase (125I-UK) and proteins in human plasma. Although 125I-UK reacts with many proteinase inhibitors in purified systems, the predominant complexes formed in plasma are with antithrombin III (ATIII) and alpha 2-macroglobulin (alpha 2M). 125I-UK interacts with purified alpha 2M or alpha 2M in plasma to form a characteristic pattern of multiple complexes whose Mr values by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis are in the range of 380 000-720 000, under non-reducing conditions, and 180 000-430 000 after reduction. We also examined the inhibition of UK amidolytic activity by plasma and by purified ATIII. In the presence of saturating concentrations of ATIII and heparin, an apparent first-order rate constant of 6.8 X 10(-1) s-1 was calculated for the inhibition of urokinase. In contrast, the rate constant for the formation of covalent ATIII-UK complexes was lower, suggesting the inhibition of UK proceeds first via the formation of transient non-covalent intermediates that are then transformed more slowly into covalent end products. The observed rate constants for enzyme inhibition or complex-formation with plasma or purified inhibitors are insufficient to account for the reported clearance rate of injected UK in vivo.

Activation of Factor VIII by Thrombin as Measured by Competition Kinetics

1979 ◽  
Author(s):  
H. Sandberg ◽  
K. Brodén ◽  
L.-O. Andersson
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Blood Coagulation ◽  
Rate Constant ◽  
Order Rate Constant ◽  
The Other ◽  
Peptide Substrate ◽  
Physiological Importance ◽  
Low Concentrations ◽  
One Step

It is known that the procoagulant activity of purified Factor VIII is strongly increased when incubated with concentrations of thrombin below 0.1 unit/ml. It has been assumed that this also is a mechanism of physiological importance thus that activation of Factor VIII by low concentrations of thrombin is one step in normal blood coagulation. The rate constant tor the reaction between Factor VIII and thrombin was estimated by two different methods. In one method formation of “activated” Factor VIII in a system containing thrombin, purified Factor VIII and albumin added as stabilizer was measured. In the other method the competition of purified Factor VIII as substrate for thrombin was compared with fibrinogen and a chromogenic peptide substrate. As expected it was found that thrombin had higher affinity for Factor VIII than for fibrinogen. The values calculated for the second order rate constant, using 240,000 as the molecular weight of the reactive unit in Factor VIII, were similar for the various systems, around 106s-1M-1.

The Influence of Detergents on the Ca2+-and Mg2+-Dependent Adenosine Triphosphatase of the Sarcoplasmic Reticulum

1982 ◽  
Vol 37 (3-4) ◽  
pp. 299-307 ◽  
Author(s):  
Hans Lüdi ◽  
Bernhard Rauch ◽  
Wilhelm Hasselbach
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Tryptophan Fluorescence ◽  
The Other ◽  
Phosphorylated Protein ◽  
Intrinsic Tryptophan Fluorescence ◽  
Low Concentrations ◽  
Calcium Affinity

Abstract During the stepwise solubilization of sarcoplasmic reticulum vesicles with detergents, the following changes in the structural and enzymatic properties of the preparation are observed: 1. The viscosity of the vesicular suspension initially rises. This change is accompanied by the formation of elongated tubules. Subsequently the membranes are completely desintegrated, resulting in a considerable reduction of the viscosity. 2. A decrease in the activity of the Ca2+-dependent ATPase, which is restored after complete solubilization. 3. A decrease in the change of intrinsic tryptophan-fluorescence on removal of calcium ions, which is also restored after complete solubilization. 4. A decrease of the calcium affinity of the ATPase. 5. A decrease in the amount of phosphorylated protein formed by the incorporation of inorganic phosphate. On the other hand, the amount of phosphoprotein formed from ATP is not affected during solubilization. 6. The dependence of the initial rates of phosphoprotein formation from inorganic phosphate on either magnesium or inorganic phosphate at low concentrations of the respective ligand changes from an S-shape profile to a normal hyperbolic profile after solubilization.

scholarly journals Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism

1989 ◽  
Vol 263 (3) ◽  
pp. 875-881 ◽  
Author(s):  
J M May
Keyword(s):  
Thiol Group ◽  
Thiol Groups ◽  
Intact Cells ◽  
Nitrobenzoic Acid ◽  
Low Concentrations ◽  
Transport Inhibitors ◽  
Membrane Bound ◽  
And Function ◽  
Maleimide Derivative ◽  
Glucose Carrier

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5′-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.

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