ATPase In Heart Research Articles

In Vivo Action of Ammonia on Ion Transport Function in Liver and Heart Mitochondria of Immersion-Stressed Air-Breathing Fish ( Anabas testudineus Bloch)

Ammonia, as an endogenous respiratory gas, is produced during protein and amino acid metabolism. Accumulation of excess ammonia is toxic, and fishes have developed mechanisms to defend against ammonia toxicity. Here, we tested the in vivo  action of ammonia in an air-breathing fish to find how it modulates mitochondrial ion transport in fish heart and liver.  We thus analysed the activity pattern of mitochondrial ion transporters such as mitochondrial Ca 2+ ATPase, mitochondrial H + ATPase and mitochondrial F 1 F 0  ATPase in heart and liver of air-breathing fish Anabas testudineus  which were kept for immersion-induced hypoxia stress. In addition, plasma metabolites such as glucose and lactate were also quantified. Oral administration of ammonia solution [(NH 4 ) 2 SO 4 ; 50ng g -1 ] for  30 min increased mit.Ca 2+ ATPase activity in heart but lowered its activity in liver mitochondria. A reduced mit.H + ATPase activity was found in heart but in liver its activity showed increase after ammonia treatment. F 1 F 0  ATPase increased significantly in heart but showed reduced activity in liver mitochondria. Administration of ammonia in immersion-stressed fish, however, nullified the excitatory response of heart and liver mitochondria in the immersion-stressed fish. Overall, the data indicate that ammonia can play a significant physiological role in the regulation of mitochondrial ion homeostasis in the liver and heart of air-breathing fish during their acclimation to hypoxia stress.

Electrophysiology of Hypokalemia and Hyperkalemia.

Electrophysiology of Hypokalemia and Hyperkalemia.

Cardiac Nitric Oxide Synthases and Na⁺/K⁺-ATPase in the Rat Model of Polycystic Ovary Syndrome Induced by Dihydrotestosterone.

Nitric oxide synthases (NOSs) and Na(+)/K(+)-ATPase are enzymes essential for regular functioning of the heart. Since both enzymes are under insulin and androgen regulation and since insulin action and androgen level were disturbed in polycystic ovary syndrome (PCOS), we hypothesized that cardiac nitric oxide (NO) production and sodium/potassium transport would be deteriorated in PCOS. To test our hypothesis we introduced animal model of PCOS based on dihydrotestosterone (DHT) treatment of female Wistar rats and analyzed protein expression, phosphorylation or subcellular localization of endothelial NOS (eNOS), inducible NOS (iNOS) and alpha subunits of Na(+)/K(+)-ATPase in the heart. Obtained results indicate that DHT treatment significantly decreased cardiac eNOS protein level and activating phosphorylation at serine 1,177, while inhibitory phosphorylation at threonine 495 was increased. In contrast to expression of eNOS, iNOS protein level in the heart of DHT-treated rats was significantly elevated. Furthermore, cardiac protein level of alpha 1 subunit of the ATPase, as well as its plasma membrane content, were decreased in rats with PCOS. In line with this, alpha 2 subunit protein level in fraction of plasma membranes was also significantly below control level. In conclusion, DHT treatment impaired effectiveness of NOSs and Na(+)/K(+)-ATPase in the female rat heart. Regarding the importance of NO production and sodium/potassium transport in the cardiac contraction and blood flow regulation, it implicates strong consequences of PCOS for heart functioning.

Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats.

Exercise training improves the aging-induced downregulation of myosin heavy chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in the regulation of cardiac contraction and relaxation. Thyroid hormone receptor (TR), a transcriptional activator, affected the regulation of gene expression of MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling contributes to a molecular mechanism of exercise training-induced improvement of MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged heart are affected by exercise training, using the hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo old, swimming training for 8 wk). Trained aged rats showed improvement in cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart were significantly lower in sedentary aged rats than in sedentary young rats and were significantly higher in trained aged rats than in sedentary aged rats. The activity of TR DNA binding to the transcriptional regulatory region in the alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and thyroxine levels were altered in association with changes in the myocardial TR protein levels. These findings suggest that exercise training improves the aging-induced downregulation of myocardial TR signaling-mediated transcription of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of cardiac function in trained aged hearts.

Identification and regulation of the gastric H+/K+-ATPase in the rat heart.

Previous reports indicate that H+/K+-adenosine triphosphatase (ATPase) might be expressed in the heart. The objectives of the present study were to explore the presence of H+/K+-ATPase protein and gene expression in the rat heart and to investigate whether the enzyme could contribute to potassium transport across the sarcolemma. We performed reverse transcription-polymerase chain reaction (RT-PCR) on mRNA from myocardium and isolated cardiomyocytes using primers specific for the gastric H+/K+-ATPase alpha-subunit. The PCR products were sequenced and the predicted gastric H+/K+-ATPase sequence was verified. Western blots from myocardium detected a 34-kDa band and a 94-kDa band, indicating the beta-subunit and alpha-subunit of the gastric H+/K+-ATPase, respectively. Immunocytochemistry detected significant immunoreactivity of the beta-subunit in cardiomyocytes. H+/K+-ATPase-dependent potassium transport was assessed by 86Rb+-uptake in isolated cardiomyocytes. Both ouabain and the selective H+/K+-ATPase inhibitor Schering 28080 reduced 86Rb+-uptake at maximum specific inhibition, by 70 and 25%, respectively; the effects were additive. Competitive RT-PCR analysis indicated a significant upregulation of the myocardial H+/K+-ATPase in heart failure after myocardial infarction. The gastric isoform of H+/K+-ATPase is expressed in rat cardiac myocytes, both at transcript and protein levels. Functional studies indicate that the enzyme could contribute to potassium and pHi regulation in cardiomyocytes.

Mg2+-dependent ATPase activity in cardiac myofibrils from the insulin-resistant JCR:LA-cp rat.

There is a great deal of information presently available documenting a cardiomyopathic condition in insulin-deficient models of diabetes. Less information is available documenting a similar status in non insulin-dependent models of diabetes. We have studied the functional integrity of the myofibrils isolated from hearts of JCR:LA rats. The JCR:LA rat is hyperinsulinemic, hyperlipidemic, glucose intolerant and obese. As such, it carries many of the characteristics found in humans with non insulin-dependent diabetes mellitus. These animals also have many indications of heart disease. However, it is not clear if the hearts suffer from vascular complications or are cardiomyopathic in nature. We examined Mg2+-dependent myofibrillar ATPase in hearts of JCR:LA-cp/cp rats and their corresponding control animals (+/?) and found no significant differences (P> 0.05). This is in striking contrast to the depression in this activity exhibited by cardiac myofibrils isolated from insulin-deficient models of diabetes. Our data demonstrate that myofibrillar functional integrity is normal in JCR:LA-cp rats and suggest that these hearts are not in a cardiomyopathic state. Insulin status may be critical in generating a cardiomyopathic condition in diabetes.

Influences of somatotropin on Na +-K +-ATPase, Mg 2+-ATPase and Ca 2+-ATPases of porcine visceral tissues

Two experiments were conducted to determine the overall influence of porcine somatotropin (pST) administration on the specific activity of visceral tissue ATPases. Pigs were fed a corn-soybean meal-skim milk-based diet approximately 85% of ad libitum, such that for each experiment, control and pST-treated pigs consumed similar amounts of feed. As observed for pigs chronically treated with pST, enhanced growth of visceral tissues was evident in pigs treated for 6 and 14 days (d) with pST. The specific activity of detergent-activated Na −-K +-ATPase (ouabain-sensitive adenosine triphosphatase activity) was determined in fresh tissue homogenates prepared from liver, heart, kidney and duodenum. Treatment with pST was associated with a 19% increase in Na −-K 1-ATPase-specific activity in the liver; specific activity of Mg 2+-ATPase was not influenced by pST. Whole liver Na −-K +-ATPase and Mg 2+-ATPase activities were 35% and 25% greater, respectively, in somatotropin-treated pigs. The specific activities of Na +-K +-ATPase and Mg 2+ -ATPase in heart, kidney and duodenum were similar for controls and pigs treated for 14 d with pST. The specific activities of high- and low-affinity Ca 2+-ATPases in kidney medulla were 20 and 26% lower, respectively, in pigs treated for 14 d with pST compared with controls. In contrast, Ca 2+-ATPases in other tissues, including kidney cortex, were not influenced by pST treatment. These data indicate that some of the observed increase in energy expenditure associated with pST treatment may be attributable to increased organ size as well as to enhanced hepatic Na − and K + flux. While Na +-K +-ATPase activity is specifically enhanced in the liver, pST does not appear to be a general Na −-K −ATPase activator in all tissues and may be associated with depressed activity of Ca 2+-ATPases in the kidney.

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.

Quantitative determination of Na+-K+-ATPase and other sarcolemmal components in muscle cells

A recurring problem in the characterization of plasma membrane enzymes in tissues and cells is whether the samples tested are representative for the entire population of enzyme molecules present in the starting material. Measurements of [3H]-ouabain binding, enzyme activity, and maximum transport capacity all indicate that the concentration of Na+-K+ pumps in mammalian skeletal muscle is high (300-800 pmol/g wet wt). Studies on Na+-K+-ATPase activity in isolated sarcolemma, however, generally give little or no information on total cellular enzyme concentration. Due to the low and variable enzyme recovery (0.2-8.9%), such subcellular preparations may, therefore, give misleading data on factors regulating Na+-K+-ATPase in heart and skeletal muscle cells. As the same isolation and purification procedures are used for the study of other sarcolemmal components (lipids, hormone receptors, enzymes, and other transport systems), this inadequate recovery has general implications for statements on regulatory changes in the sarcolemmal composition of muscle cells. On the other hand, complete quantification of Na+-K+-ATPase in muscle tissue can now be achieved using simple procedures and the entire material (intact muscle fibers, biopsies, and whole homogenates). Recent studies have shown that regulatory changes in the entire population of Na+-K+ pumps in muscle can be quantified in measurements of [3H]-ouabain binding, K+-activated 3-O-methylfluorescein phosphatase activity, as well as maximum ouabain suppressible Na+-K+ transport capacity.

Complete amino-acid sequence of the natural ATPase inhibitor from the mitochondria of the yeast Candida utilis.

The complete alignment of the 63 residues of the mitochondrial ATPase inhibitor from the yeast Candida utilis has been determined. The sequence study was carried out mainly by automatic (liquid and solid-phase) methods. Peptides were obtained by enzymatic digestion with clostripain and purified by reverse-phase high-performance liquid chromatography. The ATPase inhibitor contains three sets of repetitive sequences and eight clusters of charged residues, as also found in the inhibitor of Saccharomyces cerevisiae, with which it shares 58.7% homology of conserved residues. When the two yeast ATPase inhibitor sequences were compared to that of beef heart, 20 residues remained common to the three alignments, although the latter protein contained a long histidine-rich insertion, only found in this inhibitor. Most of the homologous residues were clustered near the center of the protein, which by partial proteolytic digestion of the beef heart ATPase inhibitor [Dianoux, A.C. et al. (1982) FEBS Lett. 140, 223-228] has already been shown to be involved in the biological function.

Some properties of isolated mitochondrial ATPase from salmon heart

1. 1. A soluble Mg-dependent ATPase, similar to the mitochondrial ATPase from beef heart, has been isolated from heart mitochondria of salmon ( Salmo salar). The salmon heart ATPase has 5 subunits with molecular weights similar to the beef heart enzyme, but the Stoke's radius of the intact salmon enzyme is larger. 2. 2. The salmon heart ATPase is less temperature labile than the beef heart enzyme. 3. 3. The salmon heart ATPase is strongly inhibited by ADP, and the inhibition is highly temperature dependent. The ITPase activity is also inhibited by IDP ( K i = 180 μm). 4. 4. 2,4-Dinitrophenol in small concentrations stimulates the ITPase activity as well as the ATPase activity of the “washed” salmon heart enzyme. However, in an enzyme preparation which had been freed of most of the bound nucleotides by dialysis in the presence of glycerol (Roveri et al., 1980) the ITPase activity is not stimulated by 2,4-dinitrophenol.

Decreased myocardial function and myosin ATPase in hearts from diabetic rats.

The effect of diabetes on cardiac function was determined in isolated rat hearts. Diabetes was induced by injection of alloxan (doses ranged from 37.5 to 60 mg/kg body wt), and the heart were removed and perfused in the working heart preparation. Doses of alloxan ranging from 37.5 to 42 mg/kg did not consistently alter cardiac function even though serum glucose was elevated and serum thyroid hormones were reduced. Injection of 45 mg/kg of alloxan caused a large increase in serum glucose and a larger decrease in thyroid hormones. In this case, ventricular function was more consistently depressed after 1-2 wk. Function was not altered 48 h after injection of 60 mg kg of alloxan. However, when animals were given 60 mg/kg of alloxan and then maintained on insulin for 7 days, depressed cardiac function developed within 4 days after the insulin treatment was stopped. The decline in function involved a decrease in heart rate peak systolic pressure, and left ventricular +dP/dt. It was associated with greatly reduced serum thyroid hormones (both T3 and T4) and lower ventricular Ca2+-activated myosin ATPase activity. Fasting of rats for 4 days also resulted in decreased serum T3 and T4, depressed cardiac function (although heart rate was unchanged), and lower Ca2+-activated myosin ATPase activity.

Tissue specificity of dopamine effects on brain ATPases.

Dopamine inhibits Mg2+,Na+,K+- and Na+,K+-ATPase activities but does not modify Mg2+-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na+,K+-, and Mg2+-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. The results indicate tissue specificity of dopamine effect with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.

A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization.

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.

Purification and properties of mitochondrial adenosine triphosphatase of hamster brown adipose tissue

1. 1. Oligomycin-insensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from brown adipose tissue mitochondria. It had a specific activity of 50 units/mg which could be increased up to 85 units/mg by KHCO 3. The isolated enzyme represented less than 0.5% of the initial membrane proteins. 2. 2. The enzyme had a molecular weight equal to beef heart ATPase and was composed of five subunits with molecular weights of 56 200, 54 300, 33 500, 13 400 and 9500 respectively. 3. 3. Isolated ATPase was labile while cold and was activated by the divalent cations Mn 2+, Mg 2+, Co 2+ and Cd 2+. The optimum ATP/MG 2+ ratio found was 1.58 and the enzyme had a maximum activity at pH 8.5; the K m was 220 μM. 4. 4. The ATPase activity was 55% inhibited by aurovertin. The isolated enzyme enhanced the fluorescence of aurovertin, quenched by ATP and Mg 2+ and enhanced by ADP. 5. 5. Oligomycin sensitivity and cold stability of isolated ATPase was restored by its reconstitution with both brown adipose tissue and beef heart particles depleted of ATPase. 6. 6. The results presented demonstrate that the low ATPase activity of brown adipose tissue mitochondria is due to a reduced content of ATPase.

Inhibition of (Na ++K +)-stimulated ATPase of heart by fatty acids

(Na⁺+K⁺)-stimulated ATPase of a plasma membrane fraction from guinea-pig heart is inhibited by fatty acids (octanoate and oleate tested). This inhibition is also observed when ATP is replaced by <i>p</i>-nitrophenylphosphate. The inhibition by fatty acids is competitive with K⁺. These results are in agreement with the earlier experiments conducted by Ahmed and Thomas [<i>1</i>] on brain microsomes. It is suggested that inhibition of (Na⁺+K⁺)-stimulated ATPase may contribute to the arrhythmogenic action of high levels of fatty acids observed under pathological conditions.

Evidence supporting the identity of beef heart mitochondrial chloroform-released adenosine triphosphatase (ATPase) with coupling factor I

Highly purified mitochondrial chloroform-released beef heart ATPase had molecular weight 330 000, five bands (α, β, γ, δ, ε) in sodium dodecyl sulfate gel electrophoresis and could restore the oxidative-phosphorylation function of A particles. Maximal inhibition (90%) of the enzyme by N, N′-dicyclohexylcarbodiimide was achieved at a molar ratio of inhibitor to protein of 30 : 1. Chloroform introduced into an aqueous solution of beef heart coupling factor I protected it from cold inactivation.

Structure of beef heart mitochondrial F 1-ATPase Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands

1. 1. The following bifunctional reagents, dimethylsuberimidate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F 1-ATPase. 2. 2. Treatment of beef heart F 1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis were 115 000, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F 1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by β-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. 3. Cross-linking of beef heart F 1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[ 14 C] ethylmaleimide and of subunit B by chloronitro[ 14C]benzooxodiazole. Bothe products were able to bind N-[ 14 C] ethylmaleimide ; only the 105 000 dalton product was able to bind chloronitro[ 14C]benzooxodiazole. 5. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[ 14 C] ethylmaleimide . Its cleavage, following N-[ 14 C] ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. 6. The above results indicate that the product of molecular weight 115 000 is

The Polypeptides of the Ox Heart Mitochondrial Inner Membrane: Isolation of the Major Component

attempted a reversal of the cross-linking. The stained band was out from the gel and the reversal of the cross-linking by ammonolysis was carried out in the gel slice as described by Hulla & Gratzer (1972). On re-electrophoresis a very faint band which migrated approximately as the a subunit was found. Owing to the small amount of regenerated subunit obtained we could not rule out the presence of other subunits in band A which may not be detectable by Coomassie Blue staining in the quantity likely to be present. To increase the detectability of the regenerated subunits formed on ammonolysis of band A we chemically labelled the band with lZsI in the excised gel slice. Experimental details are given in the legend to Fig. 1 . The products of the reversal were then located by radioautography of the gel. A radioactive band exactly corresponding to the a subunit was obtained (Figs. l e and lf). No other radioactive bands were found, suggesting that band A is derived solely from the a subunit. The estimated molecular weight for band A of 14OOOO indicates that it is probably a dimer of a subunits. This finding demonstrates that a subunits are adjacent to one another in the ATPase molecule. Bragg & Hou (1975) have shown that the predominant species formed after cross-linking the ATPase from Escherichiu coli, which has a similar subunit composition to that of the ox heart enzyme, is a dimer of a and B subunits. Although it is possible that some of the faint bands formed after cross-linking the ox heart ATPase arise from cross-links between the a and subunits, it appears that the arrangements of the subunits in the two molecules is different.

ATPase inhibitor from yeast mitochondria. Purification and properties.

1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.