Protein Sulfhydryl Content Research Articles

Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by ∼30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.

Thiol-Disulfide Exchange Systems in the Liver of Aging Fischer 344 Rats

The purpose of this study was to determine whether the major thiol-disulfide oxidoreductase activities of the rat liver were altered as a consequence of aging, and whether the alterations had any consequences in terms of hepatic thiol concentrations. Liver fractions were prepared from male and female Fischer 344 rats at ages representing young adulthood (5 months), middle age (15 months) and old age (24–29 months), and the activities of the major thiol-disulfide exchange enzymes, together with protein and nonprotein sulfhydryl contents, were measured using spectrophotometric procedures. Thioltransferase, protein disulfide isomerase and thioredoxin reductase activities in livers of male and female rats were unchanged with aging, while glutathione disulfide (GSSG) reductase activity remained the same (in male livers) or increased (in female livers) as a consequence of aging. Both protein and nonprotein sulfhydryl concentrations were well maintained in old age. The absence of age-dependent alterations in the thiol-protein disulfide exchange enzymes and the lack of compromise in the glutathione GSSG reductase system suggest that aged livers retain their capacity to regulate their thiol-disulfide redox balance under normal physiological conditions.

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.

Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes.

We have studied the effects of thimerosal, a mercurial compound extensively used as a preservative, as well as other sulfhydryl reagents (e.g. p-hydroxymercurybenzoate, hydrogen peroxide, bromophenacyl bromide, and mercuric chloride) on Ca2+ homeostasis and the redox status of sulfhydryl groups in thymus lymphocytes. They all induced an increase in [Ca2+]i which was blocked with dithiothreitol, suggesting that they act via the oxidation or blockade of sulfhydryl groups. [Ca2+]i increase could be directly related to the effect of the different reagents on cellular protein sulfhydryl content. Experiments with ethidium bromide indicate that the observed rise in [Ca2+]i was not due to a non-specific increase in membrane permeability. Thimerosal differs from the other agents studied in its oxidative properties, which is probably linked to the production of a potent reductor molecule, thiosalicylic acid, which may modulate its oxidative capacity.

Rapid neutrophil accumulation and protein oxidation in irradiated rat lungs

Exposure of lungs to high doses of ionizing radiation can initiate an injurious acute inflammatory response. We show that neutrophil content in the lungs of rats exposed to 10 Gy whole body gamma radiation increased threefold 4.5 h after irradiation and returned to normal by 24 h. Oxidized methionine in the proteins of the lungs, heart, liver, kidney, and jejunum increased significantly in 2 h. Treatment with the antioxidant dithiothreitol immediately after irradiation prevented methionine oxidation. Methionine oxidation was also observed after intrabronchial instillation of phorbol myristate acetate, a model of neutrophil oxidant-mediated pulmonary injury, as well as in isolated lungs perfused with hypochlorous acid, confirming the ability of neutrophil oxidants to cause protein oxidation in lungs. No change in glutathione or protein sulfhydryl content was detected in irradiated lungs 4.5 h after irradiation, possibly as a result of protection by the observed increase in pulmonary glutathione reductase. We therefore show that the acute pulmonary inflammatory response to radiation involves rapid neutrophil accumulation, oxidant production, and protein oxidation.

Degradation of native and oxidized beta- and gamma-crystallin using bovine lens epithelial cell and rabbit reticulocyte extracts.

In many types of cells, modified proteins are selectively and rapidly removed by various proteolytic systems. In eye lens, as in most cells, there appears to be a multiplicity of proteolytic pathways, including ubiquitin-dependent, ATP-dependent and ATP-independent pathways. Each of these appears to be involved in the degradation of alpha-crystallins. The objective of this study was to determine if oxidized beta- and gamma-crystallins would be selectively degraded and which proteolytic systems might be involved. beta- and gamma-crystallins were oxidized by exposure to 137Cs radiation under N2O. This system generates *OH primarily. Oxidation of beta- and gamma-crystallins was indicated by decreased protein sulfhydryl content and tryptophan fluorescence, as well as by increased levels of carbonyl and high molecular weight aggregates with increasing radiation dose. gamma-crystallin was more susceptible than beta-crystallin to oxidation based on loss of native crystallin, increase in aggregates and fragmentation products, and loss of tryptophan. Low molecular mass polymers (dimers) appear to be the precursors of high mass aggregates induced upon oxidation. At a specific level of oxidative insult interchain covalent bonds in addition to disulfides were more extensive in the polymers of gamma-crystallin as compared to beta-crystallin. Except for beta-crystallin irradiated with 1 krad, the degradation rate of crystallins using both reticulocyte and bovine lens epithelial cells (BLEC) proteolytic systems increased in proportion to the extent of oxidation. Proteolysis of oxidized gamma-crystallins increased 1775% and 900%, respectively, using reticulocytes and BLEC supernatants as the source of proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrolein-Induced Injury to Cultured Pulmonary Artery Endothelial Cells

We evaluated the specific effects of acrolein on sulfhydryl status and plasma membrane-dependent functions of cultured pulmonary artery endothelial cells. Acrolein exposure caused a dose-dependent increase in lactate dehydrogenase (LDH) release and decreases in reduced glutathione (GSH) and protein sulfhydryl (P-SH) content, whereas oxidized glutathione (GSSG) content was not altered. Exposure to 4.5 μM, but not 1.5 or 3.0 μM, of acrolein caused significant (p < 0.05) LDH release. With increasing concentrations (25 μM) of acrolein, LDH release was increased to 66% (p < 0.001). Acrolein (3.0-25 μM) resulted in 36 to 100% reductions in GSH content, whereas reductions in P-SH content at these concentrations of acrolein ranged from 11 to 37%. Uptake of amino acids (cystine, glycine, and glutamic acid) and incorporation of valine into the protein fraction were significantly reduced in a dose-dependent fashion in acrolein (1.5-4.5 μM)-exposed cells. Reductions in cystine, glycine, and glutamic acid uptakes were maximal in cells exposed to 3 and 4.5 μM acrolein (p < 0.001). Similarly, maximum reductions (p < 0.001) in both uptake and incorporation of valine into the protein fraction were observed at 3.0 and 4.5 μM acrolein. Acrolein (1.5 μM) also resulted in significant loss of plasma membrane-specific Na+/K+-ATPase as well as plasma membrane P-SH content (p < 0.05 for both). When cells were treated with ouabain, reductions in amino acid uptake were observed, and this appeared to mimic the effect of acrolein exposure. When isolated plasma membranes were exposed to a known SH-alkylating agent, N-ethylmaleimide, losses of Na+/ K+-ATPase and P-SH content were observed and were similar to the effects following exposure to acrolein. These results demonstrate that acrolein exposure results in alterations of plasma membrane-dependent transport in pulmonary artery endothelial cells, leading to reduced availability of precursor amino acids used in GSH and protein synthesis. This plasma membrane injury is accompanied by reductions in the GSH and PSH contents of these cells. Loss of the plasma membrane P-SH appears to be associated with specific inactivation of Na+/K+ATPase.

Intracellular calcium disruption as a secondary event in acetaminophen-induced hepatotoxicity.

To examine a role for disturbances in intracellular calcium homeostasis in acetaminophen-induced hepatotoxicity, freshly isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen for 1.5 h to allow for covalent binding and initiation of cell damage. The hepatocytes were then washed and the cells incubated in fresh medium containing either 2.0 mM N-acetylcysteine or 1.5 mM dithiothreitol for the duration of a 4-h incubation period. These agents were used as tools in the elucidation of the biochemical events responsible for acetaminophen-induced cell necrosis. The reduced protein sulfhydryl content, cytosolic [Ca2+], and plasma membrane integrity were quantitated. Acetaminophen produced protein sulfhydryl depletion, an increased cytosolic [Ca2+], and cell injury; however, cytotoxicity preceded the increase in [Ca2+]. Both N-acetylcysteine and dithiothreitol restored the acetaminophen-induced protein sulfhydryl loss. Dithiothreitol prevented both further cell injury and an increase in the cytosolic [Ca2+]. However, cell death and a subsequent increase in cytosolic [Ca2+] proceeded unabated following N-acetylcysteine addition. Although both agents restored protein sulfhydryl content, in view of their contrasting ultimate effects on cell viability the role of reduced protein sulfhydryl depletion in acetaminophen-induced hepatic injury requires further investigation. The increase in cytosolic [Ca2+] with acetaminophen alone and with subsequent N-acetylcysteine addition was determined to be a secondary event in cell injury because cytotoxicity occurred by 1.5 h; however, the increase in cytosolic [Ca2+] was not observed until 2.5 h. Additional evidence for changes in cytosolic [Ca2+] as a secondary event was obtained by incubating the hepatocytes with acetaminophen in the presence of fura 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Influences of retinal activity on choroid and sclera

It has recently been demonstrated that, with age, UV filters such as 3-hydroxykynurenine glucoside, bind to proteins in the human lens. This covalent interaction leads to colouration of the normal lens, and results from the instability of the kynurenine side chain. Other primate UV filters, in addition to containing the same side chain, can also be readily oxidized. One such compound is 3-hydroxykynurenine (3OHKyn). It has been proposed that oxidation of bound and/or free UV filters, such as 3OHKyn may give rise to the lens colouration associated with age-related nuclear cataract. Therefore it has become important to understand the oxidation of 3OHKyn within the lens. In this study, intact bovine lenses (which lack UV filters) were incubated with 3OHKyn and various lens parameters monitored. The effect of exposure to hyperbaric oxygen (HBO) was also assessed, both alone, and in combination with prior 3OHKyn incubation. Glutathione (GSH), protein sulfhydryl and protein-bound sulfhydryl levels, as well as soluble protein content and gel filtration profiles, were obtained for cortical and nuclear regions after defined periods of incubation. The presence of the primate UV filter, 3OHKyn, at concentrations similar to those present in the human lens, was shown to produce considerable oxidative stress within the lens, as judged by its effect on GSH. This effect was noted under normobaric conditions, but was exacerbated by increased oxygen. Exposure of lenses to HBO caused a marked fall in GSH in cortical and nuclear regions. This effect was exaggerated in the presence of 3OHKyn. HBO treatment also lead to a fall in protein sulfhydryl content, however, this was only partial (approximately 1 mol SH per mol protein) and changed only slowly, even with extended periods of exposure to HBO, suggesting that most crystallin sulfhydryl groups may be buried. 3OHKyn did not appreciably affect this oxidation although it did cause an increase in the level of protein-bound sulfhydryl. HBO treatment produced a more than two-fold increase in protein-bound sulfhydryl content in the cortex. There was little influence of 3OHKyn alone on protein solubility, even with extended periods of incubation, however, incubation for 72 hr in the presence of HBO caused a significant increase in insoluble protein particularly in the nucleus. This insolubilization was further increased in the presence of 3OHKyn. FPLC profiles showed that the proportion of gamma and beta crystallins in the soluble fraction decreased following HBO, suggesting that these may be involved in disulfide bond formation. This study demonstrates that a readily oxidized compound, such as the primate UV filter 3OHKyn, represents an oxidative stress within the lens and that such oxidative processes can be exacerbated if the concentration of oxygen within the lens is increased. We speculate that this factor may account for the evolution of unusually high levels of glutathione reductase in human lenses.

Characteristics of five human tumor cell lines and sublines resistant to cis‐diamminedichloroplatinum(II)

In order to study the mechanisms responsible for resistance to CDDP, 5 human tumor cell lines were made resistant to CDDP by repeated in vitro exposures. After cloning it was found that the cell lines developed were between 3.3-fold and 17-fold more resistant to CDDP than the parental cell lines at the IC90. These lines were also resistant to carboplatin and tetraplatin; however, resistance to tetraplatin was lower than to the other platinum complexes. Sensitivity was also assessed to Adria, MTX, 5-FU, chlorambucil, 4-HC, 4-HIF, BCNU, Thiotepa, HN2, Mito C and L-PAM, and no consistent cross-resistance was observed. As compared with the parental lines, non-protein sulfhydryl content was elevated in 3 resistant lines, and protein sulfhydryl was elevated in all 5 lines, as was glutathione-S-transferase activity. Measurements of platinum in whole cells and nuclei after exposure of the cultures to 25 microM CDDP for either 1 or 6 hr showed that nuclear levels reflected those in whole cells and that, per mg protein, platinum levels were lower in resistant cells at both time points. Formation of DNA cross-links, determined by alkaline elution, was lower in resistant cell lines than in parental cell lines, but did not correlate with the absolute cell kill observed. These results indicate that cellular resistance to CDDP often involves decreases in drug accumulation and increases in protein sulfhydryl content. Possible strategies for overcoming these mechanisms are discussed.

Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice

Microcystin-LR (MCLR), a cyclic heptapeptide produced by the blue-green algae Microcystis aeruginosa, produces death in female mice treated with 100 micrograms MCLR/kg. Kupffer-cell hyperplasia was observed histologically after treatment with 50 or 100 micrograms MCLR/kg. No other changes or lethality were observed with the 50 micrograms MCLR/kg, while 100% lethality occurred in less than 2 h in mice treated with 100 micrograms/kg. In these animals liver weights increased by 45% and hepatic hemoglobin content increased 106% at 60 min posttreatment. Liver histology showed loss of hepatic architecture and necrosis 30 min after treatment, and congestion with blood became evident at 45 min after treatment. Serum enzymes were significantly increased 45 min posttreatment. Hepatic nonprotein sulfhydryl content decreased 19% when calculated on the basis of cytosolic protein and 39% when based upon the total protein content, respectively. The sulfhydryl content of the liver cytoskeletal fraction decreased 26% by 30 min after treatment. Decreased enzyme-mediated and increased non-enzyme-mediated lipid peroxidation were observed in hepatic microsomes following both in vivo and in vitro exposure of hepatic microsomes to MCLR. The toxicity of MCLR may be related to alterations in the sulfhydryl content of the cytoskeletal protein. Furthermore, MCLR may either directly or indirectly affect microsomes, suggesting alterations in structure and function of smooth endoplasmic reticulum.

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P2 fraction) from Fischer 344 female rats were incubated in the presence of ADP-chelated Fe3 (0.5-50 microM) and ascorbate (250 microM). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by 14CO2 evolution from L-[1-14C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe3./ascorbate was found with 50% inhibition occurring at 2.5 microM Fe3 concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe3 in the absence of exogenous ascorbate was effective only above 25 microM. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P2 actions using sucrose gradients. Membrane microviscosity of the purified synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagnetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe3./ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulfhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulfhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulfhydryl content. It is concluded that iron-induced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.

Allopurinol does not enhance antioxidant properties of extracellular fluid.

Allopurinol has been shown to provide significant protection against ischemia/reperfusion-induced microvascular and parenchymal cell injury. It has been hypothesized that the protection seen with allopurinol after ischemia/reperfusion (I/R) is caused by inhibition of xanthine oxidase. However, recent reports suggest that the beneficial effects of allopurinol in I/R may be caused by direct free radical scavenging. The objective of this study was to determine whether the regimen of allopurinol administration used in most I/R studies leads to a significant modification of the free radical scavenging properties of extracellular fluid (ECF), i.e., plasma and lymph. Plasma and intestinal lymph samples obtained from both control and allopurinol-treated cats were used to assess the following: 1) allopurinol and oxypurinol concentrations, 2) xanthine oxidase inhibition, 3) myoglobin-catalyzed linolenic acid peroxidation, 4) hypochlorous acid scavenging, and 5) protein and nonprotein sulfhydryl content. ECF from allopurinol-treated animals contained approximately 10 microM each of allopurinol and oxypurinol. Ten percent ECF resulted in 80% inhibition of xanthine oxidase activity. Comparable volumes of control ECF did not inhibit xanthine oxidase. Furthermore, allopurinol treatment did not enhance the antioxidant properties of ECF. The results of this study do not support the contention that the beneficial effects of allopurinol in I/R injury are caused by the scavenging of oxidants produced in ECF by activated granulocytes.

Modification of cellular protein sulfhydryl groups by activated soluble immune response suppressor.

Soluble immune response suppressor (SIRS), a product of murine Ly-2+ T lymphocytes, is activated to SIRSox by H2O2 produced by macrophages: SIRSox directly inhibits cell division by normal and neoplastic cells and antibody secretion by B lymphocytes. To examine the mechanism of SIRSox-mediated inhibition, a variety of cellular functions were measured after treatment of cells with SIRSox. These included respiration, glucose transport, microtubule content, glutathione content, production of H2O2 or superoxide anion, and the activities of a variety of different enzymes. Several cellular activities or measurements were inhibited or lowered after SIRSox-treatment, including cell division, microtubule content, glutathione reductase activity, and thioredoxin reductase activity; inhibition was partially reversed by the sulfhydryl reducing agent dithiothreitol. Protein sulfhydryl content of P815 mastocytoma cells and several other cell types was lowered by 35 to 45% after exposure to SIRSox. Protein sulfhydryl loss was also partially restored after incubation with dithiothreitol. Sulfhydryl loss was not due to cell lysis. In addition, treatment of crude cellular particulate fractions with SIRSox resulted in protein sulfhydryl loss and formation of protein sulfenyl derivatives. A comparison of the amount of SIRS and H2O2 present to the number of protein sulfhydryls lost or sulfenyl derivatives formed suggests that SIRSox acts catalytically, serves as a co-factor in protein sulfhydryl oxidation, or that it activates a second pathway that is directly responsible for sulfhydryl oxidation.

The thiol-disulfide balance during maturation of Xenopus laevis oocytes.

We measured several of the components involved in the thiol-disulfide balance of immature and mature oocytes of Xenopus laevis: total soluble native sulfhydryl, reduced and oxidized glutathione, native sulfhydryl content of soluble protein, and disulfide content of soluble protein. None of these values changed during maturation. Both the immature and mature oocyte were characterized by a relatively high disulfide to sulfhydryl ratio. We also measured native sulfhydryl content of germinal vesicle proteins and enucleated oocyte proteins in immature oocytes. Contrary to early cytochemical findings, our measurements suggest that the germinal vesicle has a lower concentration of protein sulfhydryl than does the cytoplasm.

Dietary antioxidants and the biochemical response to oxidant inhalation: II. Influence of dietary selenium on the biochemical effects of ozone exposure in mouse lung

We examined the influence of dietary selenium (Se) on the pulmonary biochemical response to ozone (O 3) exposure. For 11 weeks, weanling female strain A St mice were fed a test diet containing Se either at 0 ppm (−Se) or 1 ppm (+Se). Each diet contained 55 ppm vitamin E (vit E). Mice from each dietary group were exposed to 0.8 ± 0.05 ppm (1568 ± 98 μg/m 3) O 3 continuously for 5 days. After O 3 exposure, they were killed along with a matched number of unexposed controls, and their lungs were analyzed for various biochemical parameters. The Se contents of lung tissue and whole blood were determined, and the levels were seven- to eightfold higher in +Se mice than in −Se mice, reflecting the Se intake of the animals. In unexposed control mice, Se deficiency caused a decline in glutathione peroxidase (GP) activity relative to +Se group. After O 3 exposure, the GP activity in the −Se group was associated with a lack of stimulation of glutathione reductase (GR) activity and the pentose phosphate cycle (PPC) as assessed by measuring glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. In contrast, the +Se group after O 3 exposure exhibited increases in all four enzyme activities. Other parameters, e.g., lung weight, total lung protein, DNA and nonprotein sulfhydryl contents, and O 2 consumption, were not affected by dietary Se in the presence or absence of O 3 exposure. The data indicate that dietary Se alters the GP activity, which in turn influences the GR and PPC activities in the lung evidently through a reduced demand for NADPH. The level of vit E in the lung was found to be twofold higher in the −Se group than in the +Se group, suggesting a compensatory relationship between Se and vit E in the lung. With O 3 exposure, both Se and vit E contents further increased in the lungs of each dietary group. It is plausible that Se and vit E under oxidant stress are “mobilized” to the lung from other body sites.

Glutathione metabolism in lenses of dogs and rabbits: Activities of five enzymes

Several biochemical parameters were examined in clear dog and rabbit lenses as functions of age, and in posterior subcapsular cararacts in the Alaskan malamute. Tabulated data include soluble protein, reduced sulfhydryl content of soluble protein, reduced glutathione, water, and activity of five enzymes of glutathione metabolism. The enzymes include the glutathione biosynthesis system consisting of γ-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione peroxidase, glutathione reductase and glutathione-S-transferase. Each enzyme, acting last in a sequential reaction of either two or three reactions, was in excess activity over the preceding enzyme(s) in every case but one. In the exception, the ratio of glutathione reductase to glutathione peroxidase activity was about 1 : 600 and 1 : 155 in the dog and rabbit lens, respectively.

Biochemical changes associated with selenite-induced cataract in the rat

Subcutaneous injection of 20 μmol sodium selenite/kg body weight into 9-day-old male or female Sprague-Dawley rats caused the appearance of large nuclear cataracts within 72hr. Lens glutathione was depressed to 40% of normal after 24 hr, 50% of normal at 48 hr, and was still depressed to 70% of normal through 12 days post-injection. The lens NADPH pool was only 70% of normal 24 hr after selenite administration but was normal at 72 hr. Lens protein sulfhydryl content was also diminished by 12% when measured at 6 days post-injection and a reduced rate of lens growth was apparent by 12 days after treatment. Maximum lens selenium accumulation was observed at 24 hr post-injection and amounted to a tripling of the normal level of this element. The results indicate a direct and sustained interference of selenium with glutathione metabolism. The model should be useful for the further study of the role of glutathione in normal and abnormal lens metabolism.

Sulfhydryl Groups in Individual Normal and Nuclear-Cataractous Human Eye Lenses

Evaluation of sulfhydryl data derived from the nucleus and cortex of individual human lenses, ranging in age from 40 to 90 years, revealed the following: (a) no significant correlation between the age and the sulfhydryl content of normal and nuclear-cataractous lenses; (b) a significant relationship between the distribution of non-protein-bound sulfhydryl compounds among the lens parts and both age and cataractous state; (c) no significant difference in sulfhydryl data from the cortex between groups of cataractous lenses with increasing nuclear color; and (d) a significant difference in nuclear protein sulfhydryl content between the groups of lenses, varying in cataractous state. If lens parts are separated on the basis of cataract localization, sulfhydryl oxidation in nuclear cataractogenesis seems to be confined to the colored nuclear region. It is suggested that nuclear cataract develops from the inner nucleus towards the cortex of the lens.

Structural and functional properties of myosin associated with the compensatory cardiac hypertrophy in the rabbit

Right ventricular hypertrophy was produced in rabbits by pulmonary artery constriction. Measurement of intraventricular pressure, ventricular wet and dry weights and histologic examination of the liver demonstrated substantial hypertrophy with no signs of congestive failure 3 to 6 weeks following constriction. Right ventricular papillary muscle from these hearts exhibited a depressed force-velocity relationship and an increased time to peak tension but otherwise were normal. Myosin prepared from control and hypertrophied hearts was of comparable purity and yield. Quantitative densitometry of SDS polyacrylamide gels showed the light chains of hypertrophy myosin were unchanged in number, molecular weight and stoichiometry. Total protein sulfhydryl content was not different for control and hypertrophy myosins. Calcium-activated ATPase activity of hypertrophy myosin was significantly decreased shen measured at varying ATP concentration. By Lineweaver-Burk analysis, V max of hypertrophy myosin was depressed by 50%, while the apparent K m was 10% lower. When sulfhydryl-dependent ATPase activity was measured as a function of p-chloromercuribenzoate (PCMB) concentration, hypertrophy myosin exhibited a normal activation profile. The ATPase activity of hypertrophy myosin was initially depressed by 50% and was significantly reduced at low PCMB concentrations. However the difference was absent at peak activation, suggesting that the SH 1 sulfhydryls were capable of activation by PCMB to the same level as for control myosin. These data were consistent with a subtle conformational change at or near the active site, rather than with a change in the composition of major subunits of myosin from hypertrophied hearts.