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Taurine, which is abundant in mammalian tissues, especially in the heart, is essential for cellular osmoregulation. We previously reported that taurine deficiency leads to changes in the levels of several metabolites, suggesting that alterations in those metabolites might compensate in part for tissue taurine loss, a process that would be important in maintaining cardiac homeostasis. In this study, we investigated the molecular basis for changes in the metabolite profile of a taurine-deficient heart using pathway analysis based on the transcriptome and metabolome profile in the hearts of taurine transporter knockout mice (TauTKO mice), which have been reported by us. First, the genes associated with transport activity, such as the solute carrier (SLC) family, are increased in TauTKO mice, while the established transporters for metabolites that are elevated in the TauTKO heart, such as betaine and carnitine, are not altered by taurine deficiency. Second, the integrated analysis using transcriptome and metabolome data revealed significant increases and/or decreases in the genes involved in Arginine metabolism, Ketone body degradation, Glycerophospholipid metabolism, and Fatty acid metabolism in the KEGG pathway database. In conclusion, these pathway analyses revealed genetic compensatory mechanisms involved in the control of the metabolome profile of the taurine-deficient heart.

Removal of Ca2+ from the incubation medium of cultured rat cardiac fibroblasts causes cellular morphological changes, such as the formation of blebs, the ballooning of the cell membrane and the detachment from the culture dish. A 24 hr preincubation with 20 mM taurine blocked the Ca2+ depletion-induced detachment of the cardiac fibroblasts. However, taurine treatment did not prevent other morphological changes induced by Ca2+ depletion. The data suggest that taurine plays an important role in cell adhesion in the heart.

Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na+]i. The data suggest that taurine efflux is regulated by [Na+]i and cellular osmolality, but not by cellular pH.

Taurine regulates an unusual number of biological phenomena, including heart rhythm, contractile function, blood pressure, platelet aggregation, neuronal excitability, body temperature, learning, motor behavior, food consumption, eye sight, sperm motility, cell proliferation and viability, energy metabolism and bile acid synthesis. Many of these actions are associated with alterations in either ion transport or protein phosphorylation. Although the effects on ion transport have been attributed to changes in membrane structure, they could be equally affected by a change in the activity of the affected transporters. Three common ways of altering transporter activity is enhanced expression, changes in the phosphorylation status of the protein and cytoskeletal changes. Interestingly, all three events are altered by osmotic stress. Since taurine is a key organic osmolyte in most cells, the possibility that the effects of taurine on ion transport could be related to its osmoregulatory activity was considered. This was accomplished by comparing the effects of taurine, cell swelling and cell shrinkage on the activities of key ion channels and ion transporters. The review also compares the phosphorylation cascades initiated by osmotic stress with some of the phosphorylation events triggered by taurine depletion or treatment. The data reveal that certain actions of taurine are probably caused by the activation of osmotic-linked signaling pathways. Nonetheless, some of the actions of taurine are unique and appear to be correlated with its membrane modulating and phosphorylation regulating activities.

The amino acid, taurine, is an important nutrient found in very high concentration in excitable tissue. Cellular depletion of taurine has been linked to developmental defects, retinal damage, immunodeficiency, impaired cellular growth and the development of a cardiomyopathy. These findings have encouraged the use of taurine in infant formula, nutritional supplements and energy promoting drinks. Nonetheless, the use of taurine as a drug to treat specific diseases has been limited. One disease that responds favorably to taurine therapy is congestive heart failure. In this review, we discuss three mechanisms that might underlie the beneficial effect of taurine in heart failure. First, taurine promotes natriuresis and diuresis, presumably through its osmoregulatory activity in the kidney, its modulation of atrial natriuretic factor secretion and its putative regulation of vasopressin release. However, it remains to be determined whether taurine treatment promotes salt and water excretion in humans with heart failure. Second, taurine mediates a modest positive inotropic effect by regulating [Na+]i and Na+/Ca2+ exchanger flux. Although this effect of taurine has not been examined in human tissue, it is significant that it bypasses the major calcium transport defects found in the failing human heart. Third, taurine attenuates the actions of angiotensin II on Ca2+ transport, protein synthesis and angiotensin II signaling. Through this mechanism taurine would be expected to minimize many of the adverse actions of angiotensin II, including the induction of cardiac hypertrophy, volume overload and myocardial remodeling. Since the ACE inhibitors are the mainstay in the treatment of congestive heart failure, this action of taurine is probably very important.

Male Wistar-Kyoto rats were given either tap water (control) or 3% beta-alanine (taurine-depleted) for three weeks. To prepare for the kidney function studies, the animals were then implanted with femoral vessels and bladder catheters. Two days after surgery, each rat was given an intravenous infusion of saline at the rate of 50 microliter/min and urine samples were collected at specific time intervals. An isotonic saline solution (0.9% NaCl) was infused for determination of baseline parameters and was followed by the infusion of a hypotonic saline solution (0.45% NaCl). Two days later, the infusion protocol was repeated in the same animals; however, a hypertonic saline solution (1.8% NaCl) was substituted for the hypotonic saline solution. Renal excretion of fluid and sodium increased in the control, but not taurine-depleted, rats during the hypotonic saline infusion. Interestingly, diuretic and natriuretic responses were similar between the groups during hypertonic saline infusion. The results suggest that taurine-depletion in rats affects renal excretory responses to a hypotonic, but not a hypertonic, saline solution.

Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5 mM beta-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after beta-alanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30 mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.

Angiotensin II modulates several aspects of cardiac function, including myocardial contractility, heart rate and myocyte growth. Most of these actions are intimately associated with alterations in calcium transport. Since taurine also modulates calcium transport, we examined possible interactions between taurine and angiotensin II at the level of the major cellular extruder of calcium, the Na−-Ca2+ exchanger. Over a concentration range of 0.5–25 mM, Turne served as an effective inhibitor of angiotensin II-mediated stimulation of the exchanger. An Arrhenius plot of Na+-Ca2+ exchange activity revealed that angiotensin II (2 nM) increased transporter activity by reducing the activation energy of the transport process. Taurine (25 mM) inhibited the angiotensin II effect by partially preventing the reduction in activation energy. However, neither agent significantly altered the transition temperature, ruling out a change in membrane fluidity or an alteration in the rate limiting step of the transporter as a cause of the observed effects. Since the Na+-Ca2+ exchanger plays an important role in the handling of [Ca2+]i by the myocardium, the effect of taurine on angiotensin II's modulation of contractile function was also examined. Hearts perfused with buffer containing angiotensin 11 experienced a slight positive isotropic effect in the absence of taurine but this was converted to a negative inotropic effect in the presence of taurine. The data suggest that Turine inhibits some, but not all of the actions of angiotensin II. The possibility that a phosphorylation event is the site of the angiotensin II-taurine interaction is discussed.

The effect of taurine on the cellular distribution of [Ca2+]i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca2+ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca2+-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca2+]; rapidly fell to a level of 3–6 × 10−8M. Subsequent restoration of medium calcium increased [Ca2+]i to level 4–7 times normal. Large increases in [Ca2+]i were observed in most cells and were associated with the development of contracture and bleb formation.

Taurine mediates a plethora of membrane-linked effects in excitable tissues. To account for these multiple actions, four hypotheses have been proposed. One theory is based on the observation that taurine diminishes the inflammatory response of several cytotoxic oxidants. It is proposed that a reduction in the extent of membrane oxidative injury contributes to these cytoprotective actions. The second theory maintains that alterations in protein phosphorylation may underlie certain effects of taurine, particularly its effect on calcium transport. The third hypothesis assumes that the interaction of taurine with the neutral phospholipids leads to altered membrane calcium binding and function. The final theory ties the actions of taurine to inhibition of phospholipid N-methylation and the resulting changes in membrane composition and structure. While each of these hypotheses has merit, none of them can fully explain the membrane actions of taurine. Further studies are required to ascertain the importance of each theory.

The present study correlates the insulin resistance seen in the myocardium of rats with streptozotocin-induced non-insulin-dependent diabetes mellitus with insulin secretory defects, hyperglycaemia and disease duration. Two-day-old male Wistar rats were given a bolus injection of streptozotocin (0.09 mg/g body weight) which caused glucose intolerance when these animals reached adulthood. Although these rats developed a progressive resistance to the actions of insulin in the heart this did not correlate with the development of glucose intolerance. However, a correlation was seen with a shift in insulin secretory response from hyper- to hypo-secretion which developed between 6 and 14 months of age. Moreover, this shift in secretory pattern can be associated with the onset of a cardiac mechanical malfunction.

Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na(+)](i). The data suggest that taurine efflux is regulated by [Na(+)](i) and cellular osmolality, but not by cellular pH.

Taurine depletion was induced by either incubation of isolated myocytes with 5 mM beta-alanine or feeding rats with water containing 3% beta-alanine. Hearts of taurine depleted rats exhibited an impairment in myocardial relaxation, associated with a decrease in Na(+)-Ca2+ exchanger activity. Exposure of the heart to angiotensin II, an activator of the Na(+)-Ca2+ exchanger, eliminated the relaxation defect. In agreement with the contractile results, taurine depletion prolonged the calcium transient, an effect which was partially eliminated by exposure to angiotensin II. Although peak systolic [Ca2+]i was modestly depressed in the taurine depleted myocyte, peak ventricular pressure was normal. This may be related to an elevation in pHi induced by taurine depletion. Angiotensin II had little effect on contractility of the taurine depleted heart, presumably because of two opposing effects, a reduction in pHi and an increase in [Ca2+]i. Thus, taurine depletion alters contractile function and ion transport and both of these effects are modulated by exposure of the heart to angiotensin II.

The activity of the beta isoform of protein kinase C (PKC beta) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC beta contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC beta activity in the membrane. Associated with the improvement in PKC beta activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC beta in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.

The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37,000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of 32P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins. Through this mechanism they serve as important modulators of the sarcolemmal Ca2+ pump.

The rate of regeneration of reduced RNase by glutathione was examined in the presence of several added substances: substrate, phospholipid, other proteins, bacterial ribosomes, and neutral salts. Of these, only neutral salts showed substantial effects. K2HOP4 and (NH4)2SO4 strongly accelerated regeneration, the alkali chlorides showed moderate acceleration or inhibition, while LiBr and KSCN strongly inhibited. The t1/2 for regeneration in 1 M Pi is 4 min compared to 75 min in the absence of Pi; in 0.5 M KSCN t1/2 greater than 100 min. The pattern of specific salt effects is similar to a Hofmeister series. There is a strong parallel between the pattern of specific salt effects on the kinetics of RNase regeneration and the pattern of effects of the same salts on the equilibrium stability of biopolymers. This suggests that the role of salts in the regeneration is to stabilize or destabilize rate-limiting folding intermediates. Pi-accelerated glutathione regenerations showed a broad temperature optimum from 30-37 degrees. In strong contrast with the virtual concentration independence of the Pi-free controls, with Pi = 1 M, both rates and yields of RNase activity were decreased markedly at [RNase] greater than 2 x 10(-6) M. Phosphate and pyrophosphate showed additive, and in some cases, synergistic accelerations. These results suggest that specific ion binding occurs in addition to general solvent effects.

The effect of several phenothiazines on the extent of cellular damage resulting from the calcium paradox was examined. Hearts treated with trifluoperazine, a potent calmodulin inhibitor, exhibited less cellular damage than untreated myocardium as reflected by light microscopy, high-energy phosphate content and the loss of protein and creatine phosphokinase into the perfusate. A dose response of this effect revealed a maximal response at about 1 microM trifluoperazine, a concentration which lies well within the range generally attributed to calmodulin inhibition. Several other lines of evidence were also obtained suggesting a possible role for calmodulin in calcium-overload induced necrosis. First, the phenothiazines had little influence on membrane changes believed responsible for altered calcium permeability. Second, trifluoperazine was without major effect unless included in the reperfusion buffer, indicating that the drug is only effective during the phase associated with calcium overload. Finally, less protection was afforded hearts exposed to phenothiazines such as chlorpromazine and promethazine, which are weaker inhibitors of calmodulin, than those treated with the potent inhibitor trifluoperazine. While other interpretations are possible, these studies are consistent with a role for calmodulin in calcium overload-induced heart failure.

With the glutathione system that leads to rapid regeneration of reduced lysozyme (Saxena, V. P., and Wetlaufer, D. B. (1971) Biochemistry 9, 5015), reduced pancreatic ribonuclease (RNase) regenerated activity in high yield (greater than 90%) but at a considerably lower rate (t1/2 approximately 75 min). Systematic examination of the effects upon regeneration of the concentrations and ratios of reduced and oxidized glutathione (GSH and GSSG) showed the same broad optima for RNase as were earlier found for lysozyme: [GSSG] = 5 X 10(-4) M, [GSH] = 5 X 10(-3) M. Regeneration of reduced RNase by air oxidation was shown to be inhibitable by 10(-4) M EDTA, whereas the glutathione regeneration was unaffected by EDTA. In addition the air-oxidative regeneration showed a strong temperature dependence, in contrast with the glutathione system. The mechanisms of these two kinds of regenerations are therefore different. Six potentially catalytic metal ions were tested in the air-oxidative regeneration of RNase: Cu2+, Co2+, Mn2+, Fe3+, Zn2+, and Ni2+. Of these, only Cu2+ enhanced the rate of regeneration of RNase activity, although both Cu2+ and Co2+ catalyzed thioloxidation of reduced RNase. The rates and yields of RNase regenerations were independent of protein concentration from 3 X 10(-7) M to 1.2 X 10(-5) M in the glutathione system. Preincubation of freshly dissolved reduced RNase under nonoxidizing conditions before adding glutathione did not change the rate or extent of regeneration. Studies of its pH dependence showed that the glutathione regeneration depends on the deprotonation of prototropic groups with 7.5 less than pK less than 8.0. The major ion exchange chromatographic peaks from glutathione and air-oxidative regenerations appeared to be identical with native RNase, by the criteria of specific activity, chromatographic mobility, and circular dichroic spectra. The glutathione system permits regeneration at much higher RNase concentration than the air regeneration, with rates and yields comparable to the greatest reported for air regeneration.

Taurine is a β-amino acid found in very high concentration in excitable tissues [1]. Although the amino acid was discovered over 150 years ago, the only established role of taurine is conjugation with bile acids [1]. Yet, recent evidence has led to the view that taurine must have other physiological functions. One idea that has received considerable attention is that taurine serves as a stabilizer of biological membranes. This concept was originally proposed by Huxtable and Bressler [2], who showed that taurine protected sarcoplasmic reticulum from damage caused by either isolation of the membrane fraction or incubation of the membrane preparation with phospholipase C. Kramer et al. [3] provided further support for this hypothesis when they discovered that reduction in cellular damage resulting from the treatment of failing hearts with taurine was associated with an improvement in sarcolemmal function. Membrane stabilization has also been implicated in the ability of taurine to protect rod outer segments against light-induced lipid peroxidation [4], lymphoblastoid cells against iron-ascorbate-induced damage [5], and spermatozoa against loss of motility [6].

Single doses of ethanol (5 g/kg, intragastric) produce oxidative stress in the liver as well as in the heart. The metabolism of acetaldehyde through xanthine oxidase appears to play an important role in the production of oxidative stress in the heart, but it has only a contributory role in the liver. It is suggested that, as oxidative stress through lipid peroxidation may produce organ pathology, the metabolic pathway of acetaldehyde through xanthine oxidase may be one of the mechanisms which mediate cardiac pathology in alcoholism.

Recent studies have established a major role for oxygen-derived free radicals in post ischemic tissue injury to the intestine. During ischemia, there appears to be a calcium-triggered, protease-dependent conversion of the native xanthine dehydrogenase to a superoxide-producing xanthine oxidase. The catabolic degradation of ATP during ischemia provides an oxidizable substrate, hypoxanthine. On reperfusion, molecular oxygen is resupplied and a burst of superoxide production ensues, resulting in extensive tissue damage. The same mechanism appears to occur in myocardial ischemia. Xanthine dehydrogenase rapidly converts to the oxidase during nonperfusion in the rat heart. In the isolated perfused working rat heart model, 40 min of anoxia followed by reoxygenation results in substantial release of creatine kinase. The release of creatine kinase is blocked almost completely by pretreatment of the rats with allopurinol, a specific inhibitor of xanthine oxidase.

Disulfide-containing proteins offer unique advantages for mechanistic studies of the formation of native three-dimensional structure from unordered, reduced precursors. The main advantage is that covalent intermediates are formed; by characterizing these intermediates, one obtains substantial information about the reaction pathway. Thiol-disulfide interchange is a major component of most oxidative mechanisms carrying thiol to disulfide; thus, it required some attention in its own right. Afinsen's descriptions of a "shuffle-ase" enzyme led us to examine the rates of the uncatalyzed exchange under physiologically plausible conditions. Somewhat surprisingly, we found that the rates for formation of several native proteins in uncatalyzed systems containing GSSG and GSH are as great as with the "shuffle-ase" enzyme, suggesting that a substantial portion of biological thiol oxidations proceed by uncatalyzed exchange. While thiol-disulfide exchange of course results in no net change in the oxidation level of a system, catalytic linkage of thiol or disulfide to other redox systems provides a mechanism for achieving net changes.

The role of taurine in maintaining calcium and potassium homeostasis in excitable tissues is discussed. These effects of taurine appear to be related to its interaction with a low affinity binding protein of the cell membrane. This conclusion is based on the observation that the sulfinic acid analog of taurine, hypotaurine, also interacts with this protein and mimics these actions of taurine, whereas two other analogs, beta-alanine and isethionic acid, have little affinity for the binding protein and fail to exhibit taurine-like activity. The possibility that a relationship exists between the low affinity protein and sarcolemmal calcium pools is considered.

Examination of hearts and livers of rats fed ethanol for 25-30 weeks showed significant increases in catalase and glutathione peroxidase activity. Further examination revealed that the xanthine dehydrogenase/oxidase activity ratio in both tissues were decreased, suggesting that an interconversion of the dehydrogenase into oxidase might have occurred. Such an interconversion would be expected to enhance the formation of superoxide anions during acetaldehyde metabolism by xanthine oxidase. Since a role of oxidative or free radical damage in the etiology of ethanol-induced liver pathology is becoming increasingly apparent, the observation that the biochemical changes in the heart and liver are comparable suggests that oxidative damage is involved in alcoholic pathology of the heart as well as liver.

The relationship between energy metabolism and the extent of irreversible ischemic damage was examined in an isolated perfused working rat heart. The amount of cardiac work recovered after reperfusion of hearts exposed to severe global ischemia was dependent upon both the duration of ischemia and the type of substrate provided (either 5 mM glucose or 5 mM glucose + acetate). There appear to be two distinct phases in the ability to recover mechanical function in the reperfused ischemic heart. The second phase corresponds to the onset of severe irreversible tissue damage. Irreversible mitochondrial damage was not found to correspond with the onset of heart failure since the ATP/ADP ratio remained constant in the reperfused myocardium. Furthermore, there does not appear to be a direct correlation between the total ATP content and the extent of irreversible damage, either during ischemia or following reperfusion. However, the total adenine nucleotide content during ischemia showed dramatic changes which correspond temporally with the initiation of the second phase of damage. The observation that the adenine nucleotide pool becomes further depleted during reperfusion suggests that alterations in the salvage pathway for adenine nucleotide synthesis have occurred. Loss of adenine nucleotides appears to be an excellent marker for irreversible heart failure. Acetate provides some protection the the ischemic myocardium. The mechanism by which acetate mediates this protective effect is discussed.

With the glutathione system that leads to rapid regeneration of reduced lysozyme (Saxena, V. P., and Wetlaufer, D. B. (1971) Biochemistry 9, 5015), reduced pancreatic ribonuclease (RNase) regenerated activity in high yield (greater than 90%) but at a considerably lower rate (t1/2 approximately 75 min). Systematic examination of the effects upon regeneration of the concentrations and ratios of reduced and oxidized glutathione (GSH and GSSG) showed the same broad optima for RNase as were earlier found for lysozyme: [GSSG] = 5 X 10(-4) M, [GSH] = 5 X 10(-3) M. Regeneration of reduced RNase by air oxidation was shown to be inhibitable by 10(-4) M EDTA, whereas the glutathione regeneration was unaffected by EDTA. In addition the air-oxidative regeneration showed a strong temperature dependence, in contrast with the glutathione system. The mechanisms of these two kinds of regenerations are therefore different. Six potentially catalytic metal ions were tested in the air-oxidative regeneration of RNase: Cu2+, Co2+, Mn2+, Fe3+, Zn2+, and Ni2+. Of these, only Cu2+ enhanced the rate of regeneration of RNase activity, although both Cu2+ and Co2+ catalyzed thioloxidation of reduced RNase. The rates and yields of RNase regenerations were independent of protein concentration from 3 X 10(-7) M to 1.2 X 10(-5) M in the glutathione system. Preincubation of freshly dissolved reduced RNase under nonoxidizing conditions before adding glutathione did not change the rate or extent of regeneration. Studies of its pH dependence showed that the glutathione regeneration depends on the deprotonation of prototropic groups with 7.5 less than pK less than 8.0. The major ion exchange chromatographic peaks from glutathione and air-oxidative regenerations appeared to be identical with native RNase, by the criteria of specific activity, chromatographic mobility, and circular dichroic spectra. The glutathione system permits regeneration at much higher RNase concentration than the air regeneration, with rates and yields comparable to the greatest reported for air regeneration.

Disulfide-containing proteins offer unique advantages for mechanistic studies of the formation of native three-dimensional structure from unordered, reduced precursors. The main advantage is that covalent intermediates are formed; by characterizing these intermediates, one obtains substantial information about the reaction pathway. Thiol- disulfide interchange is a major component of most oxidative mechanisms carrying thiol to disulfide; thus, it required some attention in its own right. Anfinsen’s descriptions of a “shuffle-ase” enzyme led us to examine the rates of the uncatalyzed exchange under physiologically plausible conditions. Somewhat surprisingly, we found that the rates for formation of several native proteins in uncatalyzed systems containing GSSG and GSH are as great as with the “shuffle-ase” enzyme, suggesting that a substantial portion of biological thiol oxidations proceed by uncatalyzed exchange. While thiol-disulfide exchange of course results in no net change in the oxidation level of a system, catalytic linkage of thiol or disulfide to other redox systems provides a mechanism for achieving net changes.

It is well established that calcium serves as the coupler of excitation and contraction in muscle (Dhalla et al., 1977; Katz, 1977; Langer, 1976; and Williamson and Schaffer, 1976). The calcium necessary for maximal contraction appears to be derived from two calcium pools. Pool I calcium is supplied from the extracellular fluid and enters the cell during the plateau phase of the action potential. This pool is thought to be located on the sarcolemmal membrane (Langer, 1978; Limas, 1977; and Williamson et al., 1975). Pool II calcium appears to be separate from the sarcolemma since the calcium in this pool does not rapidly exchange with calcium from the extracellular space unless the muscle is stimulated. Most investigators feel that this latter pool is associated with the terminal cisternae of the sarcoplasmic reticulum.