Sulfhydryl Oxidation Research Articles

Effects of hyperoxia on composition and rate of synthesis of fatty acids in Escherichia coli

Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.

The Binding of Reduced Nicotinamide Adenine Dinucleotide Phosphate to Mammalian and Avian Fatty Acid Synthetases: NUMBER OF BINDING SITES AND THE EFFECT OF REAGENTS AND CONDITIONS ON THE BINDING OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE TO ENZYME

Abstract The binding of NADPH to pigeon and rat liver fatty acid synthetases has been examined by the techniques of fluorescence enhancement, fluorescence polarization, and equilibrium dialysis. Pigeon and rat liver fatty acid synthetases increase the fluorescent yield from NADPH 4.6- and 3.8-fold, respectively. Fluorescent yield appears to be independent of the buffer concentration and the amount of coenzyme bound to enzyme. The number of binding sites on each synthetase is independent of the protein concentration. The pigeon liver enzyme binds 2 moles of NADPH per mole of enzyme in 0.2 m phosphate and 3 moles of coenzyme at lower buffer concentrations. The binding affinity for NADPH is independent of the quantity of coenzyme bound; however, binding of coenzyme is competitive with charged species supplied by the buffer. The Kd value in 0.2 m phosphate is 8 µm, and in 0.1 m phosphate it is 3.5 µm. The rat enzyme binds maximally 4 moles of NADPH per mole of enzyme in 0.2 m and in lower buffer concentrations. This enzyme has either two pairs of binding sites or it exhibits negative cooperativity. The affinity of binding of NADPH to each of these pairs of sites is dependent upon the buffer concentration. The dissociation constant for the stronger pair of binding sites is 1 µm in 0.2 m phosphate and less than 1 µm at lower buffer concentrations. Neither the binding of NADPH to protein nor enzyme activity for fatty acid synthesis requires phosphate. Fatty acid synthesis proceeds most rapidly at ionic strengths higher than those yielding the strongest NADPH binding. The binding affinity of the pigeon liver enzyme for NADPH is not changed by the binding of either acetate or malonate (from their respective CoA esters) to protein, thereby indicating a separate binding site for NADPH. It is not affected either by a pH change in the region where the rate of fatty acid synthesis changes rapidly. It is also not changed appreciably by dissociation (with Tris-glycine buffer) of the enzyme complex into half-molecular weight units, a condition that completely eliminates fatty acid synthesis. The binding capacity of the enzyme for NADPH is destroyed by extensive sulfhydryl oxidation and by alkylation with iodoacetamide. It is suggested that the binding of NADPH to enzyme is primarily electrostatic and that it is dependent on one or more sulfhydryl groups. It is not, however, dependent on the noncovalent binding forces responsible for maintaining the integrity of the enzyme complex.

Senile cataract: The influence of blood factors on lens and serum sulfhydryl oxidation

When dialyzed lens extract, pH 7·8, is incubated in the presence of serum dialysate made from patients with senile cataract, a significantly higher sulfhydryl oxidation of lens proteins than that produced by serum dialysate from normal humans of the same age, is observed. The effects of the hydrogen ion concentration, ionic strength, temperature and serum dialysate concentration on the reaction between lens proteins and serum dialysate were studied. The incubation of a lens extract, previously incubated in oxygen at pH 7·8 or pH 9 for 40 hr, in the presence of “cataract” serum dialysate, produces further decrease of protein —SH groups; under the same conditions, “normal” serum dialysate produces no effect at all. To the same extent, “normal” and “cataract” serum dialysates stimulate sulfhydryl oxidation in a lens protein fraction obtained by chromatography on DEAE Sephadex of nuclear γ-crystallin from calf lenses, which appears susceptible to autoxidation as evidenced by a high —S—S— bond levle. On the other hand, a different protein fraction, which also belongs to the γ-crystallin group but is quite resistant to autoxidation, is found to be oxidized only by “cataract” serum dialysate, accounting for the higher oxidation rate of the protein —SH groups of a lens extract produced by “cataract” serum dialysate. Human serum dialysate is also able to oxidize the —SH groups of serum albumin, provided the incubation is carried out in oxygen or in the presence of CuSO4. As with the lens extract, “cataract” serum dialysate oxidizes the —SH groups of bovine and buman serum albumin to a greater extent than “normal” serum dialysate. Various methods for the estimation of protein —SH groups were compared and all gave similar results.

Reduction and reoxidation of turkey ovomucoid—a protein with dual and independent inhibitory activity against trypsin and α-chymotrypsin

1. 1.|The reduction and reoxidation of the disulfide bonds of turkey ovomucoid were investigated in relationship to its independent inhibitory activities against bovine trypsin and α-chymotrypsin. When all, or nearly all, of the 8 disulfide bonds were reduced by 2-mercaptoethanol, inhibitory activities against both enzymes were lost. On air reoxidation, the inhibitory activity against trypsin was regained faster than was the activity against α-chymotrypsin. It appeared that only 5 or 6 of the disulfides were required for activity against trypsin and 7 for activity against α-chymotrypsin. 2. 2.|On reduction of all the disulfides, absorbance at 280 mμ decreased by 20 to 25%. The extent of reoxidation could be determined by measuring the absorbance. 3. 3.|Neither trypsin nor α-chymotrypsin could serve as a template for reactivation of inhibitory capacity or oxidation of sulfhydryl. However, either enzyme stopped the reactivation of both inhibitory activities, apparently by extensive proteolysis of the inactive inhibitor. 4. 4.|The reoxidized inhibitor had a faster rate of interaction with the enzymes.

Aggregation of an immunoglobulin fragment by sulfhydryl oxidation.

An immunoglobulin of the IgG family has been isolated from the serum of a patient with multiple myeloma. The IgG was subjected to plasmin digestion and the resulting Fab fragment was isolated and characterized. Incubation of this fragment in denaturing solvents, such as aqueous 8 M urea at either pH 3.4 or pH 8.2, resulted in the formation of a dimer as the main product. In some cases more complex products were formed in addition to the dimer. Free sulfhydryl titrations and blocking experiments indicated that the transformation reaction consisted of cross-linking of two Fab fragments by oxidation of sulfhydryl groups. The original immunoglobulin proved to have two sulfhydryl groups per mole, which are unreactive except in the presence of denaturing solvents.

Phosphofructokinase: CORRELATION OF PHYSICAL AND ENZYMATIC PROPERTIES

Abstract Purified rabbit muscle phosphofructokinase required K+ ion for maximal activity and was inactive at pH 7 unless the Mg++ concentration exceeded the total adenosine triphosphate concentration. Enzymatic activity was proportional to sulfhydryl reduction, and was reversibly lost by sulfhydryl oxidation with oxidized glutathione. The molecular weight of enzyme crystallized in the presence of adenosine triphosphate was determined. The smallest fully active form had a molecular weight of 3.8 x 105 and could be reversibly dissociated into units of either one-half or one-quarter this size; 95 to 98% of the enzymatic activity was lost by dilution at pH 6.7, by urea treatment at pH 5.8, or by lowering the pH to 5.0, all of which decrease the molecular weight to 1.92 x 105. Protein of this size had an intrinsic activity 0.02 to 0.05 times that of the higher molecular weight form and was completely reactivated by reaggregation.

Studies on the Specificity and Mechanism of Action of Hepatic Glutathione-Insulin Transhydrogenase

Studies on the specificity and mechanism of action of hepatic glutathione-insulin transhydrogenase, an enzyme capable of catalyzing the reductive cleavage of the disulfide bonds of insulin in the presence of excess reduced glutathione, have been carried out. By coupling reactions catalyzed by glutathione-insulin transhydrogenase to that of glutathione reductase, it was indicated that during the course of enzyme action, transhydrogenase undergoes reduction by GSH and that the reduced enzyme is autoxidizable; it is inferred that this reduced form is an active intermediate during transhydrogenase-catalyzed reductions of disulfide-substrates. Under conditions of limited spontaneous air oxidation of protein sulfhydryls, transhydrogenase was shown to promote also the net oxidation of such thiols as the reduced forms of insulin and RNase. During the process of reoxidation of reduced forms of insulin and RNase, glutathione-insulin transhydrogenase was capable of enhancing the rates of regeneration of the native proteins from their reduced precursors as determined by their immunologic and enzymic properties, respectively. Furthermore, this transhydrogenase was able to promote the reconstitution of the native activity from a reduced form of RNase already extensively oxidized by dehydroascorbate. Contrary to previous indications, glutathione-insulin transhydrogenase was found to have no observable activity in specifically directing the preferential re-establishment of the native configuration of insulin. On the basis of evidence presented in this study, a mechanism involving a series of disulfide-interchange reactions has been proposed to explain the aforementioned reactions catalyzed by glutathione-insulin transhydrogenase. The possible relationship or identity of the transhydrogenase to other insulin-reducing and RNase-reactivating enzymes is pointed out. In substrate specificity studies on a number of chemically modified and proteolytically degraded forms of insulin, it was found that neither biological activity nor native structure is prerequisite for activity as substrate for glutathione-insulin transhydrogenase-catalyzed reduction.

The Effects of Iodination and Related Modifications on the Supercontraction of Wool Fibers

Iodination of wool fibers in ethanol or n-propanol accelerates the first stage and retards the second stage of supercontraction. Iodination in aqueous KI3 has little effect on the rate of the first stage but retards the second stage of supercontraction. Evidence is presented that the rate of supercontraction is affected by at least two chemical processes which occur during iodination in ethanol : oxidation of sulfhydryl and disulfide groups, and the reaction of ethanol with the fiber in the presence of HI with consequent esterifica tion of carboxyl groups. Oxidative treatments, such as reaction with peracetic acid, aqueous KI3, or ethanolic I2 are considered to retard sulfhydryl-disulfide interchange reactions which determine the rate of the second stage of supercontraction.

Increased Urinary Excretion of Taurine and Urea by Rats after X-Irradiation

An excessive urinary excretion of taurine and urea has been observed in the rat after whole-body X-irradiation (1-3). Since taurine is one of the major end products of sulfhydryl oxidation (4-7) its excessive excretion suggests an increase in the rate of oxidation of this radical. Barron et al. (8-11) have shown sulfhydryl-containing enzymes to be especially sensitive to inactivation by X-irradiation. In addition, when given prior to irradiation, cysteine (12), glutathione (13), and cysteamine (14, 15) protect against radiation death through a mechanism not due to the redox potential of these substances (16). All these observations indicate a fundamental importance of sulfhydryl groups in radiation damage. Therefore, it appeared that a study of the excretion of the end products of sulfhydryl oxidation would be of value. In addition, since sulfhydryl groups are components of most proteins, the simultaneous investigation of the excretion of the end products of protein degradation might provide additional useful information. Excretion of the sulfur products of metabolism is confined almost entirely to the urine. Therefore, the extent of sulfhydryl oxidation can be estimated by measuring urinary taurine and sulfate, and the extent of protein degradation evaluated by determining urinary urea. This paper will present and attempt to interpret data on the urinary excretion of these compounds by the X-irradiated rat.

The effect of ultraviolet radiation on sulhydryl and disulfide containing amino acids.

Abstract : According to Rothman and his associates pigmentogenic stimuli, such as ultraviolet radiation, cause pigmentation by oxidizing or destroying inhibitory sulfhydryl compounds, thus enabling an enzyme to act on the pigment precursor. It was therefore of interest to study the effect of ultraviolet irradiation on the possible reduction of the -S-S- (disulfide) linkage of cystine and homocystine to -SH and possible oxidation of the -SH (sulfhydryl) group of cysteine to -S-S-. Methionine was also studied.

Synergistic action of cortisone and total-body irradiation in mice.

Laboratory evidence has indicated that at least part of the biological action of ionizing radiations is due to inhibition of sulfhydryl obligate intracellular enzyme systems by oxidation of the sulfhydryl radical, e.g., cysteine to cystine (1, 2). The protective action against lethal total-body irradiation offered by sulfhydryl donors such as cysteine and glutathione is further evidence of the importance of this mechanism (3, 4). Similarly, substances which temporarily inhibit oxidation of sulfhydryl (—SH) provide significant protection against lethal irradiation. Cyanide, azide, and malononitrile (5), and anoxic anoxia (6) are examples. Cortisone and ACTH have recently been shown also to inhibit the sulfhydryl obligate systems as part of their fundamental biological action (7, 8). Therefore, some synergistic action of these drugs and irradiation could logically be expected. On the other hand, attempts have been made to demonstrate their value as protective agents against whole-body irradiation— one being reported as successful (9) and another as unsuccessful (10). The present report concerns an investigation into what seemed to us to be a more logical possibility, namely, a synergistic action between cortisone and whole-body irradiation. Method As test animals white male mice of a single inbred strain were used. To provide further uniformity they were fed and observed for ten days, reaching a fairly uniform weight of 25 gm. They were then divided at random into four groups of 12 mice each for the initial experiment. One group received a single dose of 220-kv. roentgen irradiation amounting to 750 r, including back-scatter, estimated to be at least and probably more than an LD 50 dose. A second group was irradiated in the same way after being given 1 mg. of cortisone subcutaneously twenty-four and twelve hours previously. The third group also received 750 r, but the cortisone was given in daily injections of 1.0 mg. following the irradiation. The animals of the fourth group served as controls, given cortisone daily only as long as there were survivors in the third group. This initial experiment was more or less exploratory, to determine gross effects and investigate protection or synergism with different timing. The results are summarized in Figure 2. On the basis of these observations, discussed below, another experiment was set up with dosages and timing selected with a view to proving more conclusively the apparent synergism initially observed. For the second experiment the same kind of mice and the same treatment conditions were used. Three groups of 12, 13, and 13 mice, respectively, were again selected at random after an initial period of feeding and observation.