Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E1-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since from a series of trialkyltin chlorides, tri-n-propyltin, tri-n-butyltin and tri-n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri-n-octyltin affect neither of them; other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri-n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.     

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 μM. From a concentration of 0.1 μM, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E₁ is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.     

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 microM. From a concentration of 0.1 microM, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E1 is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

The inhibitory effect of cyclic AMP on phosphatidylinositol kinase is not mediated by the cAMP dependent protein kinase

Addition of cAMP to cells has been shown to inhibit phosphatidylinositol (PI) metabolism. cAMP has been reported to inhibit an enzyme in this pathway, PI kinase and it has been suggested that this inhibition is due to phosphorylation of PI kinase by the cAMP dependent protein kinase (PKA). In the present study we directly investigated if the inhibitory effect of cAMP was mediated by PKA. In membranes derived from murine hepatocytes we found that cAMP inhibited PI kinase but other adenine derivatives were more potent inhibitors. Moreover, it was found that the effects of the derivatives were unlikely to be due secondarily to the production of cAMP via their interaction with adenosine receptors. Through studies employing an inhibitor of PKA, mutant cells lacking PKA, and addition of purified catalytic subunit of PKA, we found that the inhibitory effect of cAMP was not mediated by PKA. In addition, the inhibitory effect of cAMP and adenosine was retained upon partial purification of PI kinase. Pulse chase experiments affirmed that the inhibitory effect was not due to breakdown of PI but rather to inhibition of its synthesis. We conclude that the inhibitory effect of cAMP and related compounds on PI kinase is not mediated by PKA dependent phosphorylation but rather appears to be a direct effect of these agents.     

Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid-soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of ¹⁴C-leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.     

Cellular energy dependent agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin.

Effect of various metabolic inhibitors on the agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin was studied using a quantitative assay method for agglutination in which turbidity of cell suspension is measured. Cell agglutination was inhibited by low temperature, cytochalasin B and inhibitors of energy generating systems without affecting lectin binding, and agglutination was not affected by hydroxyurea, actinomycin D or cycloheximide. The inhibitors of energy generating systems decreased the cellular ATP level and inhibited macromolecular synthesis under the conditions where they inhibited the agglutinations. In contrast, cytochalasin B did not depress the cellular ATP level nor inhibit RNA and protein syntheses. These results suggest that the agglutination is associated with cellular energy dependent processes other than macromolecular synthesis; probably with some cellular surface movements participated by microfilament activity.     

Cellular energy dependent agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin

Effect of various metabolic inhibitors on the agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin was studied using a quantitative assay method for agglutination in which turbidity of cell suspension is measured. Cell agglutination was inhibited by low temperature, cytochalasin B and inhibitors of energy generating systems without affecting lectin binding, and agglutination was not affected by hydroxyurea, actinomycin D or cycloheximide. The inhibitors of energy generating systems decreased the cellular ATP level and inhibited macromolecular synthesis under the conditions where they inhibited the agglutinations. In contrast, cytochalasin B did not depress the cellular ATP level nor inhibit RNA and protein syntheses. These results suggest that the agglutination is associated with cellular energy dependent processes other than macromolecular synthesis; probably with some cellular surface movements participated by microfilament activity.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

The influence of cyclic nucleotides on macromolecular synthesis in cultured neural retina cells

1. The effect of cyclic nucleotides on aggregates of dispersed embryonic neural retina cells was examined in order to study their influence upon macromolecular synthesis, i.e. protein and DNA. 2. Cyclic AMP, dibutyryl cAMP, cyclic GMP and dibutyryl cGMP were used at various concentrations (5 x 10(-4) -5 mM). 3. The incorporation of labeled precursors into DNA and protein were used to monitor the effect of cyclic nucleotides on cultured aggregates. 4. All nucleotides exhibited a stimulatory effect at 5 x 10(-4) and 5 x 10(-3) mM on macromolecular synthesis, with resulting growth and proliferation of chick neural retina cells. 5. High concentrations (5 x 10(-1) and 5 mM) of cyclic nucleotides exhibited an inhibitory effect upon macromolecular synthesis and a marked cytotoxic effect.     

Effect of inhibitors of energy metabolism and protein synthesis on the process of neutral red segregation in frog erythrocytes

Effects of inhibitors of energy metabolism and protein synthesis on Neutral red segregation in frog erythrocytes were studied. Inhibitors of both glycolysis and respiration significantly reduced formation of segregation zones. This influence was most striking with antimycin A, rotenone and cyanide. This indicates that intact respiratory pathways may play an important part in the process of Neutral red segregation. Such uncouplers as FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and 2,4-dinitrophenol (DNP) as well as inhibitors of oxidative phosphorylation (arsenate and azide) are also very effective in inhibiting the Neutral red segregation at low concentrations. The effects of these uncouplers and of olygomycin suggest an important role of ATP as an energy source for the segregation process. An inhibitor of protein synthesis, such as cycloheximide, produces some reduction in segregation zones formation. Trapping of Neutral red by protonation could readily explain the high level of this dye accumulation in nucleated erythrocytes. The fact that low concentrations of FCCP and DNP inhibit the process of segregation brings a supporting evidence for the possibility of the ATP-driven proton pump involved in Neutral red segregation.     

Effects of vasopressin and prostaglandin synthesis inhibition on cyclic AMP accumulation in the dog renal inner medulla

The interaction of arginine vasopressin (AVP) and endogenous prostaglandins on cAMP production was investigated in the dog. Cyclic AMP content of dog inner medullary tissue slices exposed to different concentrations of AVP in the presence and absence of various prostaglandin synthesis inhibitors was determined. If the slices were incubated in isotonic media with 95% O2 and 5% CO2 gas phase, inhibition of prostaglandin synthesis decreased cAMP accumulation. A significant correlation was found between the decrements in cAMP content and basal cAMP levels. AVP-induced increments in cAMP accumulation was, however, unaffected by prostaglandin synthesis inhibitors. If incubation was performed in a hypertonic medium and at low O2 concentration, basal cAMP content was significantly reduced and it was not altered by inhibition of prostaglandin synthesis. The cAMP response to AVP was practically identical in the presence and absence of prostaglandin synthesis inhibitors. In conscious dogs AVP and indomethacin in itself had no effect on urinary cAMP excretion, but there was a significant decrease if the two compounds were combined. These results fail to lend support the hypothesis that endogenous prostaglandins modulate AVP-induced cAMP accumulation in the inner medulla.     

Metabolic and electrochemical mechanisms of dimeric naphthoquinones cytotoxicity in breast cancer cells

Cancer cells reprogram their metabolism due to genetic alteration to compensate for increased energy demand and enhanced anabolism, cell proliferation, and protection from oxidative damage. Here, we assessed the cytotoxicity of three dimeric naphthoquinones against the glycolytic MCF-7 versus the oxidative MDA-453 breast carcinoma cell lines. Dimeric naphthoquinones 1 and 2 impaired MDA-453, but not MCF-7, cell growth at IC(50)=15 μM. Significant increase in reactive oxygen species, decrease in oxygen consumption and ATP production were observed in MDA-453 cells but not in MCF-7 cell. These findings suggest that oxidative stress and mitochondrial dysfunction are mechanisms by which these agents exert their cytotoxic effects. Cyclic voltammetry and semi-empirical molecular orbital calculations further characterized the electrochemical behavior of these compounds. These results also suggest that dimeric naphthoquinones may be used to selectively target cancer cells that depend on oxidative phosphorylation for energy production and macromolecular synthesis.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Rapid turnover of endogenous endonuclease activity in thymocytes: effects of inhibitors of macromolecular synthesis

Previous work has shown that inhibitors of protein or mRNA synthesis block endonuclease activation in thymocytes undergoing programmed cell death. In the present study we used isolated nuclei to investigate the effects of cycloheximide and actinomycin D, inhibitors of protein and mRNA synthesis, respectively, on endogenous endonuclease activity in thymocytes. We observed a rapid loss of Ca2(+)-dependent endonuclease activity in nuclei isolated from thymocytes treated with these inhibitors. In contrast, pretreatment of cells with antipain and leupeptin, inhibitors of proteases, prevented the depletion of endonuclease activity in the nuclei, suggesting that proteolysis was involved. The effects of cycloheximide and actinomycin D were mimicked by incubating thymocytes with treatments known to exert their effects via activation of protein kinase C. Our results suggest that endonuclease activity in thymocyte nuclei undergoes rapid, spontaneous turnover. Agents interfering with macromolecular synthesis may therefore block DNA fragmentation in thymocytes by depleting nuclei of endogenous endonuclease activity.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Substrate level versus oxidative phosphorylation in the generation of ATP in Thiobacillus denitrificans

Particulate fractions of Thiobacillus denitrificans catalyse the phosphorylation of ADP to ATP during the oxidation of various inorganic sulphur compounds or NADH via an electron transport chain. On the other hand, a soluble cell-free fraction synthesized ATP from APS and inorganic phosphate.The production of ATP was verified either by the firefly luciferin-luciferase enzyme system or by the incorporation of ³²Pi into ATP. During the oxidation of sulphide, sulphite and NADH the production of ATP from ADP by particulate fractions is inhibited by compounds that inhibit electron transfer and by uncouplers of oxidative phosphorylation. However, these compounds had little effect on the production of ATP from AMP during the oxidation of sulphite by the soluble fraction. NADH was the most effective electron donor for oxidative phosphorylation. The soluble fraction contained high activities of ATP sulphurylase, inorganic pyrophosphatase and adenylate kinase but ADP sulphurylase activity was relatively low. The effects of inhibitors on ATP production from APS and Pi are compared with those on adenylate kinase and ATP sulphurylase.Abbreviations APS adenosine-5-phosphosulphate - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide