Physical and biochemical energy balance during an isometric tetanus and steady state recovery in frog sartorius at 0 degree C

Frog sartorius muscle stimulated isometrically for 3 s every 256 s to attain a steady state in which initial heat (QI), recovery heat (QR), rate of O2 consumption (JO2), and isometric force (PO) generated are constant for each cycle. For a 3-s tetanus given every 256 s, JO2 was 0.106 mumol/(min . g blotted weight), approximately 71% of the maximum rate observed, whereas lactate production was negligible under these conditions. QI, QT(= QI + QR), and QT/QI were 88.2, 181.5, 2.06 mJ/g blotted weight, respectively. The high-energy phosphate breakdown (delta approximately P) breakdown during the first 3-s tetanus was not different from that during a contraction in the steady state and averaged 1.1 mumol/g blotted weight. Less than half of the initial heat could be accounted for in terms of the extent of the known chemical reactions occurring during contraction. From the stoichiometry of the theoretical biochemical pathways, the amount of ATP synthesized in the steady state exceeds delta approximately P during contraction by more than twofold, corresponding to an apparent ADP:O ratio of 1.5. If it is assumed that carbohydrate oxidation is the only net chemical reaction in the steady state, the total heat production can be explained on the basis of the measured JO2. Under this assumption, heat production during recovery was less than that expected on the basis of the oxygen consumption and delta approximately P during contraction. These observations support the hypothesis that the unexplained enthalpy production and low apparent ADP:O ratio are causally related, i.e., that the reaction(s) producing the unexplained heat during contraction is reversed during the recovery period.     

The relationship between initial creatine phosphate breakdown and recovery oxygen consumption for a single isometric tetanus of the frog sartorius muscle at 20 degrees C

A previous paper (Mahler, M. 1978 J. Gen. Physiol. 71:559--580) describes the time-course of the suprabasal rate of oxygen consumption (delta QO2) in the sartorius muscle of R. pipiens after isometric tetani of 0.1--1.0 s at 20 degrees C. To test whether these were the responses to impulse changes in the rate of ATP hydrolysis, we compared the total suprabasal oxygen consumption during recovery (delta[O2]) with the amount of ATP hydrolyzed during a contraction, measured indirectly as the decrease in creatine phosphate (delta[CP]O). If suprabasal ATP hydrolysis during recovery is negligible in comparison with that during contraction, delta[CP]0/delta[O2] should approximate the P:O2 ratio for oxidative metabolism, which has an expected value of 6.1--6.5. We found: formula; see text. We conclude that in this muscle at 20 degrees C: (a) after a tetanus of 0.2--1.0 s, delta QO2(t) can be considered the response to an impulse increase in the rate of ATP hydrolysis; (b) the reversal during recovery of unidentified exothermic reactions occurring during the contraction (Woledge, R. C. 1971. Prog. Biophys. Mol. Biol. 22:39--74) can be coupled to an ATP hydrolysis that is at most a small fraction of delta[CP]0; (c) the pooled mean for delta[CP]0/delta[O2], 6.58 +/- 0.55, sets an experimental lower bound for the P:O2 ratio in vivo.     

Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder

Caveolin-1 (Cav1), a structural protein of caveolae, plays cell- and context-dependent roles in signal transduction pathway regulation. We have generated a knockout mouse homozygous for a null mutation of the Cav1 gene. Cav1 knockout mice exhibited impaired urinary bladder contractions in vivo during cystometry. Contractions of male bladder strips were evoked with electric and pharmacologic stimulation (5–40 Hz, 1–10 μM carbachol, 10 mM ,β-methylene ATP, 100 mM KCl). Acetylcholine (ACh) and norepinephrine (NE) release from bladder strips were measured with a radiochemical method by incubating the strips with ¹⁴C-choline and ³H-NE prior to electric stimulation, whereas ATP release was measured using the luciferin-luciferase assay with a luminometer. A 60–75% decline in contractility was observed when Cav1 knockout muscle strips were stimulated with electric current or carbachol, compared to wildtype muscle strips. No difference in contractility was noted when contractions were evoked either by the purinergic agonist ,β-methylene ATP, or by extracellular potassium. To investigate the relative contribution of non-cholinergic activity to bladder contractility, the amplitude of the electric stimulation-evoked contractions was compared in the presence of the muscarinic antagonist atropine (1 μM). While the non-muscarinic (purinergic) response was unaltered, muscarinic cholinergic response was principally disrupted in Cav1 knockout mice. The loss of Cav1 gene expression was also associated with a 70% reduction in ACh release. NE and ATP release was not altered. It is concluded that the loss of caveolin-1 is associated with disruption of M₃ muscarinic cholinergic activity in the bladder. Both pre-junctional (acetylcholine neurotransmitter release from neuromuscular junctions) and post-junctional (M₃ receptor-mediated signal transduction in bladder smooth muscles) mechanisms are disrupted, resulting in impaired bladder contraction.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P_i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P_i concentration was not further diminished (even though CO₂-dependent O₂ evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P_i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO₂-dependent O₂ evolution ceased with 5 min of the onset of illumination. The decline in O₂ evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q_E). Measurements of stromal metabolite concentrations showed that the ATP/ADP ratio was decreased in the P_i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P_i. The stromal concentration of glycerate 3-phosphate was comparable in the P_i-deficient chloroplasts and those to which P_i had been supplied. Chloroplasts which were illuminated in P_i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P_i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P_i was added to chloroplasts illuminated in the absence of P_i, both non-photochemical quenching (q_E), photochemical quenching (q_Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P_i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P_i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P_i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P_i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O₂ evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P_i.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity

Isolated hepatocytes incubated with selenite (30–100 μM) exhibited changes in the glutathione redox system as shown by an increase in O₂ consumption, oxidation of glutathione and loss of NADPH. Selenite (50 μM) raised O₂ consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 μM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl₂ and KCN prevented the increase in O₂ uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O₂ consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O₂ consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O₂ in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 μM) there were indications that O₂ reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 μM) it appeared that O₂ reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite.     

Synthesis of free ATP from membrane-bound ATP in chromatophores of Rhodospirillum rubrum

Chromatophores of Rhodospirillum rubrum were preincubated with ³²P_i in the absence of added nucleotides. Particles and reaction mixture were then separated by sucrose density gradient centrifugation. The labeled chromatophores thus obtained esterify ³²P_i into acid-soluble ATP (ATP_as) on the addition of ADP in the dark. Additional firmly bound ATP (ATP_fb) can be liberated on sodium dodecylsulfate treatment. Coinciding with the formation of acid-soluble ATP there is a decrease in the amount of firmly bound ATP. The isotopic concentration experiments in which labeled chromatophores were incubated with carrier-free ³²P_i and ADP in the dark, show that ATP_as might arise from ATP_fb not by a direct γ-phosphate transfer but by an esterification of the added ADP and free phosphate with a concomitant hydrolysis of the ATP_fb. On this basis we have proposed a new working hypothesis for the last step of electron transport-linked phosphorylations. It includes the following reactions: + P*_i → P* (i.e., ATP_fb) P* + ADP + P**_i → ATP**_as + P*_i

The hypothesis is compatible with the concept of conformational energy conservation.     

Wavelength-dependent quantum yield of ATP synthesis and NADP reduction in normal and dichlorodimethylphenylurea-poisoned chloroplast

At short wavelengths (525–690 mμ) the direct measurement of the quantum yield of the photoreduction of NADP⁺ in normal O₂-evolving spinach chloroplasts is constant ( approx. 0.3 equiv/hv). At short wavelengths (<690 mμ) the quantum yield for NADP⁺ reduction in 3(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts supplied with the ascorbate-2,6-dichlorophenolindophenol couple (donor system) is approx. half as efficient as the normal system. At long wavelengths the quantum yield of NADP⁺ reduction in the donor system increases by a factor of 2 ( approx. 0.3 equiv/hv) when compared with the corresponding yield for the donor system at short wavelengths ( approx. 0.15 equiv/hv).

Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.

Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.

These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O₂-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP.     

Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans

A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme.

1. 1. The preparations catalysed ATP synthesis coupled to O₂ uptake or NO₃⁻ reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO₃⁻ ratios were about 0.9 and 0.06, respectively.

2. 2. Addition of ADP or P_i to the reaction mixture increased the rates of NADH-dependent O₂ uptake and NO₃⁻ reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport.

3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O₂ and NO₃⁻ reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO₃⁻ to NO₂⁻.

First-order kinetics of muscle oxygen consumption, and an equivalent proportionality between QO2 and phosphorylcreatine level. Implications for the control of respiration

In frog sartorius muscle, after a tetanus at 20 degrees C, during which an impulse-like increase occurs in the rate of ATP hydrolysis, the rate of O2 consumption (QO2) reaches a peak relatively quickly and then declines monoexponentially, with a time constant not dependent on the tetanus duration (tau = 2.6 min in Rana pipiens and 2.1 min in Rana temporaria). To a good approximation, these kinetics are those of a first-order impulse response, and the scheme of reactions that couple O2 consumption to extramitochondrial ATP hydrolysis thus behaves as a first-order system. It is first deduced and then demonstrated directly that while QO2(t) is monoexponential, it changes in parallel with the levels of creatine and phosphorylcreatine, with proportionality constants +/- 1/tau p, where p is the P/O2 ratio in vivo. From this, it is further deduced that the mitochondrial creatine kinase (CK) reaction is pseudo-first order in vivo. The relationship between [creatine] and QO2 predicted by published models of the control of respiration is markedly different from that actually observed. As shown here, the first-order kinetics of QO2 are consistent with the hypothesis that respiration is rate-limited by the mitochondrial CK reaction; this has as a corollary the "creatine shuttle" hypothesis.     

Fluorescence characteristics of lyophilized maize chloroplasts suspended in buffer

Fluorescence transients were measured in lyophilized maize chloroplasts (suspended in Tris-maleate buffer (pH 6.6)) after extraction with heptane. (The fluorescence characteristics before extraction were qualitatively similar to those in the fresh chloroplasts.) The initial fluorescence level (m) in the (dry) heptane-extracted sample remained the same as in the unextracted material, but the variable fluorescence (Δm) was drastically diminished. A portion of variable fluorescence, however, could be restored by adding Na₂S₂O₄. If the heptane extraction was made in the presence of water (wet), the m level was almost as high as (or higher than) the final level (M) of the unextracted sample, and Δm was reduced. The “jet” of O₂ (that measures the pool size of the intersystem intermediate A) and the “microjet” (that measures the pool size of the reaction center complex E), present in the unextracted samples, were absent in both types of extracted samples. Some of the above data may be interpreted in a hypothesis in which two quenchers (Q₁ and Q₂) control the fluorescence (O → P) of chloroplasts — the reduction of Q₁ being responsible for the rapid and that of Q₂ for the slow fluorescence rise.     

Oxygen photoreduction and variable fluorescence during a dark-to-light transition in Chlorella pyrenoidosa

When dark-adapted (5 min in the dark) Chlorella cells were deposited on a bare platinum electrode, treated with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and illuminated, O₂ was consumed after a lag time of about 250 ms. The comparison of the O₂ consumption kinetics with the fluorescence O-I-D-P-S transition (the fast change in chlorophyll fluorescence which occurs after the onset of illumination of dark-adapted algae and is over within 2 s) observed in untreated algae indicates that no O₂ is consumed during the fluorescence rise and that O₂ uptake is initiated approximately when the maximum level of fluorescence P is reached. Mass spectrometry measurements of O₂ exchange (using ¹⁸O₂) were performed during dark to light transition with DCMU-untreated Chlorella cells. Under these conditions, O₂ reduction began after a lag time (about 200–400 ms) and stopped after about 5 s of illumination. The above experiments clearly show that the reduction of O₂ starts nearly at the same time that the fluorescence P-S decline. On the other hand, we show that the reduction of CO₂ does not interfere in the fluorescence O-I-D-P-S transient. We found the same apparent affinity for O₂ (about 57 μM) for both the fluorescence P-S decline and the reduction of O₂. At least three consecutive short (2 μs) saturating flashes were required to affect the fluorescence transient significantly and also to induce a significant uptake of O₂. Moreover, parabenzoquinone, an artificial Photosystem I electron acceptor, inhibited both the fluorescence D-P rise and the 250 ms lag time observed in the reduction of O₂. We conclude from the above results that in the early stages of the illumination of dark-adapted algae, some Photosystem I electron acceptors are in an inactive form. In this form, the electron transport chain is unable to reduce either O₂ or CO₂. This would lead to the accumulation of electrons on the Photosystem II acceptors (principally Q⁻_A and the plastoquinone pool) and therefore explains the fluorescence D-P rise. The light activation, probably achieved through the reduction of at least two electron acceptors, first allows the reduction of O₂, and therefore explains the P-S fluorescence decline. By accepting electrons before the site of regulation and mediating rapid O₂ reduction, parabenzoquinone avoids the accumulation of electrons and therefore inhibits the D-P fluorescence rise.     

A charge-transfer interpretation of the interactions of hemoglobin with oxygen and carbon monoxide

It is shown that the use of the charge-transfer theory to describe the interaction between hemoglobin and O₂ or CO leads to the prediction of a linear relationship between log K and the quantity (λ_HbO2 − λ_HbC), where K is the equilibrium constant of the reaction HbO₂ + CO af HbCO + O₂ and λ_HbO2 and λ_HbCO are the wavelengths of the so-called -bands in the absorption spectra of oxyhemoglobin and carboxyhemoglobin. Such a linear relation was long ago observed experimentally for a number of vertebrate hemoglobins, but was never satisfactorily explained. The fact that the charge-transfer theory does explain this seemingly unlikely experimental relationship provides support for the view that the interaction between hemoglobin and O₂ or CO may be regarded and treated as a charge-transfer process.     

Reappraisal of diffusion, solubility, and consumption of oxygen in frog skeletal muscle, with applications to muscle energy balance

Previously we tested the validity of the one-dimensional diffusion equation for O2 in the excised frog sartorius muscle and used it to measure the diffusion coefficient (D) for O2 in this muscle and the time course of its rate of O2 consumption (Qo2) after a tetanus (Mahler, 1978, 1979, J. Gen. Physiol., 71:533-557, 559-580, 73:159-174). A transverse section of the frog sartorius is in fact well fit by a hemi-ellipse with width divided by maximum thickness averaging 5.1 +/- 0.2. Using the previous techniques with the two-dimensional diffusion equation and this hemi-elliptical boundary yields a value for D that is 30% smaller than reported previously; the revised values at 0, 10, and 22.8 degrees C are 6.2, 7.9, and 10.8 X 10(-6) cm2/s, respectively. After a tetanus at 20 degrees C, Qo2 rose quickly to a peak and then declined exponentially, with a time constant (tau) approximately 15% faster than that reported previously; tau averaged 2.1 min in Rana temporaria and 2.6 min in Rana pipiens. A technique was devised to measure the solubility (alpha) of O2 in intact, respiring muscles, and yielded alpha (muscle)/alpha (H2O) = 1.26 +/- 0.04. With these modifications, the values for O2 consumption obtained with the diffusion method were in agreement with those measured by the direct method of Kushmerick and Paul (1976, J. Physiol. [Lond.]., 254:693-709). Using results from both methods, at 20 degrees C the ratio of phosphorylcreatine split during a tetanus to O2 consumption during recovery ranged from 5.2 to 6.2 mumol/mumol, and postcontractile ATP hydrolysis was estimated to be 13.6 +/- 4.1 (n = 3) nmol/mumol total creatine.     

The rate of ATP synthesis catalyzed by reconstituted CF0F1-liposomes: Dependence on ΔpH and Δψ

The kinetics of proton-transport coupled ATP synthesis in CF₀F₁ reconstituted into asolectin liposomes was investigated upon energization of the membrane by an artificially generated ΔpH and Δψ. With a rapid mixing system the rate of ATP synthesis was measured at short reaction times (under 200 ms) where all parameters (ΔpH, Δψ, substrate and product concentrations) remain practically constant at their initial values. The rate of ATP synthesis depends, in a sigmoidal way, on ΔpH, the maximal rate being 200 ATP per CF₀F₁ per s. At constant ΔpH, an additional diffusion potential increases the rate until the maximal rate is reached.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Studies on the “facilitated flux” of oxygen through hemoglobin solutions using a polarographic method

The phenomenon of hemoglobin-facilitated O₂ diffusion was studied by a polarographic method.

Polarograms relative to the reduction process of O₂ have been obtained at pH 7.2 (phosphate buffer, 30°) in the presence of various hemoglobin concentrations (Hb_tot*) and at various O₂ partial pressures (from 8 to 360 mm Hg).

Analogous experiments were performed at pH 6.4 and 8.1 (at constant ionic strength). Graphs of the limiting current values (at E = −1.5 V versus the saturated calomel electrode), relative to the overall reduction process of oxygen, plotted versus P_O2 (at Hb_tot* = constant), show some characteristic trends. The influence of pH on the features of the experimental curves is discussed.

Experimental results suggest that the diffusions of O₂, oxyhemoglobin and hemoglobin, as well as the kinetics of dissociation and association of O₂ with hemoglobin, are effective in determining the “facilitated flux”.

The corresponding nonlinear differential system is solved under some simplifying assumptions, and an expression for the flux, and consequently for the current, is obtained which is consistent with the experimental findings.

Furthermore, it is shown that the dissociation curve of oxyhemoglobin can be obtained from these polarographic experiments on the basis of this theory. Agreement with tensiometric data was satisfactory.