Mitochondrial alpha-glycerol phosphate dehydrogenase activity in IIA fibres of the rat lateral gastrocnemius muscle; the effect of Ca2+ and ATP

Summary Mitochondrial -glycerol phosphate dehydrogenase is an important enzyme, but it is difficult to extract and purify. We have measured the activity of this enzyme in single type IIA skeletal muscle fibres under initial rate conditions by microdensitometry of the formazan reaction product.The K_m (1.6mm) for the substrate (l--glycerol phosphate) was lower than reported for the extracted enzyme. Further, at low substrate concentrations (3mm), the enzyme was allosterically activated by free Ca²⁺ concentrations of 1 m or greater, and half-maximal stimulation occurred at 0.3 m free Ca²⁺. In the absence of Ca²⁺, there was negative cooperativity of substrate binding with a Hill constant of 0.57, but no cooperativity occurred in the presence of calcium. ATP (10mm) inhibited enzyme activity in the presence of Ca²⁺ but not in its absence.     

Role of magnesium in the (Ca2++Mg2+)-stimulated membrane ATPase of human red blood cells

Summary (Ca²⁺+Mg²⁺)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca²⁺ concentration and at two different but constant Mg²⁺ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value forK _m (for ATP) of 1–2m is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975.J. Membrane Biol. 22:313). Mg²⁺ increases both the maximal rate and the affinity for ATP, whereas Ca²⁺ increases the maximal rate without affectingK _m for ATP.As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1mm substrate concentration is several times faster than maximal rate of (Ca²⁺+Mg²⁺)-ATPase in red cell membranes.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

On the red blood cell Ca2+-pump: An estimate of stoichiometry

Summary Efflux of Ca²⁺ from reversibly hemolyzed human red blood cell ghosts was determined by a Ca²⁺ selective electrode, by atomic absorption spectroscopy, and by the use of⁴⁵Ca. Hydrolysis of ATP was determined by measurement of inorganic phosphate (P_i). At 25°C, ghosts loaded with CaCl₂, MgCl₂, Na₂ATP, and Tris buffer (pH 7.4) extruded Ca²⁺, with mean rates ranging from 58.8±3.5 (sd) to 74.7±8.2 (sd) moles·liter ghosts^–1·min^– depending on the method of Ca²⁺ determination. The ratio of Ca²⁺ transported to P_i released in the presence of ouabain without correction for background ATP splitting was 0.83, 0.83, and 0.80, respectively, for the three methods of Ca²⁺ determination. Correction for the ATPase activity not associated with Ca²⁺ transport resulted in a ratio of 0.91:1. In other experiments, the use of La³⁺ to inhibit the Ca²⁺-pump allowed an estimate of the ATPase activity associated with Ca²⁺ extrusion. In the presence of various concentrations of La³⁺, the ratio of Ca²⁺ pumped to P_i liberated was 0.86 or 1.02, depending on the method of Ca²⁺ determination. It is concluded that the stoichiometry of the Ca²⁺-pump of the RBC plasma membrane is one Ca²⁺ pumped per ATP hydrolyzed.     

Receptor capping in mouse T-lymphoma cells: A Ca2+ and calmodulin-stimulated ATP-dependent process

Summary The roles that Ca²⁺, calmodulin, and ATP play in the redistribution of conconavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable (skinned) to ions and proteins by incubation in a solution containing no added Ca²⁺, 7mm EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca²⁺ concentration from 10^–6–10^–4.9 m. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5×10^–6 to 1.0×10^–4 m) in cell suspensions containing 10^–4 m Ca²⁺ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca²⁺ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9×10^–8–6.3×10^–7 m) to the cell suspension increased the Ca²⁺ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca²⁺ concentration, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca²⁺-calmodulin-activated control system.     

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

Summary NADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. K _m values for Pi in NADH oxidation and phosphorylation are 10^–3 m and 8×10^–4 m, respectively. These K _m values are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 g per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.     

Rundown and reactivation of ATP-sensitive potassium channels (KATP) in mouse skeletal muscle

Dissociated single fibers from the mouse flexor digitorum brevis (FDB) muscle were used in patch clamp experiments to investigate the mechanisms of activation and inactivation of K_ATP in mammalian skeletal muscle. Spontaneous rundown of channel activity, in many excised patches, occurred gradually over a period of 10–20 min. Application of 1.0 mm free-Ca²⁺ to the cytoplasmic side of the patch caused irreversible inactivation of K_ATP within 15 sec. Ca²⁺-induced rundown was not prevented by the presence of 1.0 m okadaic acid or 2.0 mg ml^– of an inhibitor of calcium-activated neutral proteases, a result consistent with the conclusion that phosphatases or calcium-activated neutral proteases were not involved in the rundown process. Application of 1.0 mm Mg.ATP to Ca²⁺inactivated K_ATP caused inhibition of residual activity but little or no reactivation of the channels upon washout of ATP, even in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase (10 U ml^–1). Mg.ATP also failed to reactivate K_ATP, even after only partial spontaneous rundown, despite the presence of channels that could be activated by the potassium channel opener BRL 38227. Nucleotide diphosphates (500 m; CDP, UDP, GDP and IDP) caused immediate and reversible opening of Ca²⁺-inactivated K_ATP. Reactivation of K_ATP by ADP (100 m) increased further upon removal of the nucleotide. In contrast to K_ATP from cardiac and pancreatic cells, there was no evidence for phosphorylation of K_ATP from the surface sarcolemma of dissociated single fibers from mouse skeletal muscle. The small degree of activation occasionally observed following application of 10 m or 1.0 mm Mg.ATP could have been due to the generation of ADP from ATP hydrolysis and not through phosphorylation. Data are consistent with the suggestion that Ca²⁺ inactivation of K_ATP involves a gating mechanism that can be reopened by nucleotide diphosphates.M.H. is supported by the Medical Research Council.     

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro

Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 m monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 m, monensin also reduced net, active movements of K⁺, Na⁺, Cl^– and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 m) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.     

Effects of pyridine nucleotides on the gating of ATP-sensitive potassium channels in insulin-secreting cells

Summary The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K⁺ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 m and less, each promoted channel opening whereas high concentrations, 500 m and above, evoked channel closure. The degree of K⁺ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K⁺ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K⁺ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1mm ADP and 4mm ATP, 10 to 100 m concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 m and above were found to evoke channel closure. In the presence of 2mm ATP and 0.5mm ADP, however, 10 to 100 m concentrations of the pyridine nucleotides were able to activate K⁺ channels.     

The mode of inhibition of oxidative phosphorylation by efrapeptin (A23871): measurement of substrate effects on rates of inactivation by a tight-binding inhibitor.

Equations are derived for predicting the effects of substrate concentration on the inactivation rate constants of tight-binding competitive and uncompetitive inhibitors. These relationships are used to study the inhibition of mitochondrial oxidative phosphorylation by efrapeptin. The results show that the apparent rate constant for efrapeptin inactivation of ATP synthesis decreases with increase in phosphate concentration. The reciprocal of the observed rate constant varies linearly with changes in the level of phosphate as predicted for a competitive inhibitor. The concentrations of ADP during ATP synthesis and of ATP during ATP hydrolysis, on the other hand, have no effect on the rate of inactivation by efrapeptin. This is in contrast to previous observations that adenine nucleotide substrates influence the level of efrapeptin bound at equilibrium (R. L. Cross, and W. E. Kohlbrenner, 1978, J. Biol. Chem.253, 4865–4873). The results suggest that efrapeptin interacts primarily at the phosphate binding site and that adenine nucleotides may influence equilibrium binding of efrapeptin by affecting the rate of dissociation of the inhibitor. Studies of efrapeptin inhibition of ATP synthesis under pseudo-first-order conditions show that the onset of inhibition is first order with respect to efrapeptin. The maximum apparent rate constant for efrapeptin binding, obtained by extrapolation to zero phosphate concentration, is 1.5 × 10⁵m^?1 s^?1. Also described is a computer program for calculating the concentrations of complexes formed in a mixture of interacting species. The program may be used for most multiple-equilibrium calculations and permits the estimation of the levels of protonated complexes at any pH. The program was used to select Mg²⁺ concentrations which ensure that a large and relatively constant fraction of added ADP is present as MgADP. In the range of phosphate and ADP concentrations commonly used in studies of oxidative phosphorylation a 3 mm excess of Mg²⁺ relative to ADP was found sufficient to maintain high levels of MgADP at pH 8.0.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide

Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na⁺+K⁺)-ATPase and K⁺-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na⁺+K⁺)-ATPase is impeded by either Na⁺ or K⁺ whereas only K⁺ impedes protection by ATP of K⁺-activated phosphatase. On the other hand, Na⁺ or K⁺ slightly protects Ca²⁺-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca²⁺. This effect is half-maximal at less than 1 m Ca²⁺ and does not require ATP, which suggests that sites with high affinity for Ca²⁺ exist in the Ca²⁺-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K⁺ or Na⁺ in the presence of Ca²⁺ parallels that of the Ca²⁺-dependent activities, suggesting that the Ca²⁺-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

Über eine Wirkung von Natrium-Ionen auf die Phosphataufnahme und die lichtabhängige Phosphorylierung von Ankistrodesmus braunii

Summary The uptake of labelled phosphate, especially the incorporation in the organic, in TCA soluble phosphate compounds of the unicellular green alga Ankistrodesmus braunii is markedly stimulated by Na⁺ more in the light but is stimulated in the dark as well (Na⁺-effect). This stimulation depends on the phosphate concentration and on the sodium concentration of the medium (optimum 10^-3M NaCl) and appears in short-time incorporations (1 min) only at low phosphate concentrations (10^-7 to 10^-5 m PO₄). In addition the Na⁺-effect depends on temperature and almost disappears at 1°C. The incorporation of ³²P in the dark is strongly inhibited by 2,4-dinitrophenol (DNP) and under this condition only a very samll increase of the ³²P incorporation by Na⁺ can be measured. In the light however the same concentration of DNP has only a low effect on ³²P incorporation in case no Na⁺ is present in the medium. If Na⁺ is present in the medium, the effect of DNP on ³²P incorporation is increased in the light. The Na⁺-effect in the light is also inhibited by di-chlorophenyl-1,1-dimethylurea (DCMU) in N₂-atmosphere. High concentrations of g-Strophantin (10^-3 m) inhibit the uptake of phosphate by Ankistrodesmus; the inhibition is more increased in the presence of KCl than in the presence of NaCl. The results clearly indicate, that Na⁺ will not effect the incorporation of labelled phosphate by means of influencing passive processes of phosphate diffusion or phosphate exchange, but acts on different energy-requiring processes of phosphorylation in dark and light. At present one could conclude, that Na⁺ acts less through a mechanism of a sodium pump, but rather affects the formation of energy-rich compounds (in the dark by way of the oxydative phosphorylation, in the light perhaps by means of the non-cyclic photosynthetic phosphorylation).     

Structural characteristics of the saxitoxin receptor on nerve

Summary The effects of uranyl ion (UO ₂ ²⁺ ; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB⁺; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00±0.05 and 13.5mm Ca²⁺, addition of 10.0 m UO ₂ ²⁺ +5.0nm STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79±0.04 (viz., mean±sem) of control value] than did addition of 5.0nm STX alone (0.72±0.05). Under the same conditions of pH and Ca²⁺ concentration, 1.0mm MB⁺ had approximately the same effect: 1.0mm MB⁺+5.0nm STX, 0.76±0.03; 5.0nm STX alone, 0.70±0.04. However, at pH 7.00±0.05 and lower Ca²⁺ concentrations, 1.0mm MB⁺ significantly reduced STX potency. Using 6.0mm Ca²⁺: 1.0mm MB⁺+5.0nm STX, 0.92±0.01; 5.0nm STX alone, 0.68±0.08. Using 3.0mm Ca²⁺, the corresponding values were 0.94±0.03 and 0.67±0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

Summary The ultrastructural localization of Ca²⁺, Mg²⁺-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl₂ and 2.4mm Pb(NO₃)₂ in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca²⁺ or Mg²⁺ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate.     

Control of mitochondrial respiration in muscle

Summary Control of mitochondrial respiration depends on ADP availability to the F₁ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · P_i which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca²⁺ concentrations. The Ca^2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca²⁺ by activation of Ca²⁺-sensitive dehydrogenases. The Ca²⁺-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca^2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.     

Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers

Summary Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] _o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] _o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is 20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca²⁺ artificial seawater (ASW) or in Ca²⁺-free ASW decreases approximately linearly with increasing logarithm of [K] _o . The slope of the plot is slightly steeper for fibers bathed in Ca²⁺-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK _o depends on the external Na⁺ and Ca²⁺ concentrations, as well as on external pH, but is independent of external Mg²⁺ concentration. External application of 10^–4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca²⁺ by injection of 250mm EGTA before exposure of the fiber to 100mm K _o leads to 60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K _o by 50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na₂ before elevation of [K] _o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K _o for time periods ranging from 30 sec to 10 min causes a small (60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K _o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca²⁺ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.     

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate

Summary In a study of the Ca²⁺ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca²⁺ uptake was linear up to a medium Ca²⁺ concentration of 30 m. The half maximal transport rate occurred at 34 m Ca²⁺. The Ca²⁺ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg²⁺ had little effect on Ca²⁺ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca²⁺/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca²⁺ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca²⁺. The Ca/Pi ratio of the extrudate was very similar to the ratio of these ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca²⁺ binding was elevated and a high percentage of the total Ca²⁺ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca²⁺ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca²⁺ may form calcium phosphate granules described by other investigators.