Hydrolysis of inositol phosphates by plant cell extracts.

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.

Luminescence from the carbon monoxide derivative of Agaricus bispora tyrosinase.

The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an edible white mushroom, has been studied. At room temperature the emission appears as a single smooth peak centered at 530 nm with FWHM of 2700 cm-1 and a lifetime of 36 microseconds. The lifetime and wavelength of the emission are virtually unchanged on lowering the temperature from 298 to 77 degrees K. Solvent composition affects the wavelength of emission minimally. The emission is quenched by oxygen but not by a series of substrate analogs, inhibitors, or Lewis bases. The emission further appeared independent of aggregation state of the enzyme or isozyme type. A comparison of these data is made with those obtained by other researchers for the tyrosinase from Neurospora crassa and for several hemocyanins. The comparison supports the hypothesis that regulation of enzymatic activity does not take place within the coordination sphere of the copper atom observed. In addition, it suggests that the 550- to 560-nm emissions previously observed may not be considered characteristic of all CO derivatives of coupled binuclear copper proteins.

Evaluation of Reductive Release as a Mechanism for Iron Uptake from Ferrioxamine B by Chlorella vulgaris.

The involvement of ferric reduction in the iron uptake mechanism of iron-stressed Chlorella vulgaris from ferrioxamine B was investigated. Some comparative data for ferric-citrate was also obtained. EPR and a spectrophotometric assay were used to measure Fe(3+) reduction. These two methods differed in the absolute quantity but not in effectors of ferric reduction. The mechanism governing ferric reduction was investigated by use of respiratory inhibitors, uncouplers, alternative electron acceptors, and ATPase inhibitors. Reduction appears to play a role in iron uptake from both Fe(3+)-deferrioxamine B and Fe(3+)-citrate; however, the involvement of photoreduction in Fe(3+)-citrate uptake implies multiple reductive mechanisms could be involved.

Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris.

Iron uptake from two Fe(3+)-hydroxamate siderophores, ferrioxamine B and Fe(3+)-rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe(3+)-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates.

Correction of Freezing Artifacts in the Application of Electron Paramagnetic Resonance for Quantification of the Reduction of Ferric Hydroxamate by Chlorella vulgaris.

Electron paramagnetic resonance has been used to quantitate reduction of ferric ion bound to hydroxamate siderophores by iron-stressed fungi (DJ Ecker et al. 1982 J Biol Chem 257:8623-8626) and by iron-stressed Chlorella vulgaris (ATCC 11468) (FCT Allnutt, WD Bonner 1984 J Plant Nutr 7: 427-435). Application of this technique to quantitation of bound ferric ion has been questioned due to the possibility of freezing artifacts. Dipole-dipole interaction which resulted in broadening of the g = 4.3 signal was observed. Quantitation was affected when peak to trough amplitude was applied to quantitate the ferric ion but could be corrected by the application of internal standards and maintenance of constant cell content in the samples.

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria.

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.

Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH.

Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30 degrees C) or a cold (13 degrees C) temperature regime, and then their respiration rates were measured as O(2) uptake at 25 degrees C. The capacity of the alternative pathway (KCN-insensitive O(2) uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH.

Energetics of proline transport in corn mitochondria.

The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea mays L. Mo17 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in A(520)) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog l-thiazolidine-4-carboxylic acid.The energetics of proline transport was investigated by measuring the effects of FCCP (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and valinomycin on mitochondrial swelling and substrate oxidation. Proline transport and resulting oxidation were found to be partially dependent upon the energy of the electrochemical gradient. At low proline concentrations, entry was found to be primarily independent of the gradient (based on insensitivity to FCCP), whereas at higher proline concentrations a gradient-dependent mechanism became involved. Results with valinomycin indicated that proline transport and oxidation are dependent upon the pH potential across the membrane rather than the electrical (membrane) potential.

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores.

The dependence on redox potential (Eh) of the steady-state photophosphorylation rate in chromatophores of Rhodopseudomonas sphaeroides Ga was measured using slowly equilibrating (and hence less interfering) redox mediators. The remaining interference of the mediators was taken into account by extrapolating to zero mediator concentration. The extents of cytochrome redox reactions (in the presence of antimycin) and of the carotenoid shift were similarly measured. The redox titration of cytochrome c oxidation is consistent with a requirement for prior cytochrome c2 reduction and prior Q1 (primary electron acceptor) oxidation, while the titration of cytochrome b reduction is consistent with a requirement for prior (BChl)2 reduction and prior cytochrome b560 oxidation. The steady-state carotenoid shift extent is a much more broadly peaked function of Eh than is the extent following a single-turnover flash, indicating that the transmembrane electrical potential difference can be built up to significant levels by minimal rates of electron flow. The photophosphorylation rate, in contrast, is much more strongly Eh dependent, supporting the concept of a threshold membrane energization below which phosphorylation cannot occur. Earlier work by others showing a requirement for equilibrium cytochrome c2 reduction and Q1 oxidation is clearly confirmed. A much greater enhancement in phosphorylation rate was found at low Eh than has heretofore been reported. This threefold enhancement is discussed in relation to the equilibrium redox states of the ubiquinone-10 complement of the chromatophore membrane.

Measurements of membrane potentials in plant mitochondria with the safranine method.

The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaseolus aureus) and Voodoo lily (Sauromatum guttatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K(+) diffusion potential and was found to be linearly related to the developed potential within the range of 50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potential, whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential.

A comparison of the phosphorylation potential and electrochemical proton gradient in mung bean mitochondria and phosphorylating sub-mitochondrial particles.

The phosphorylation potential (delta Gp) and the electrochemical proton gradient (delta muH+) normally maintained during respiration or ATP hydrolysis by mung bean hypocotyl mitochondria and phosphorylating sub-mitochondrial particles have been investigated. Phosphorylation potential experiments using safranine and oxonol-VI, as membrane potential markers for mitochondria and sub-mitochondrial particles, respectively, suggest that the 'null point' delta Gp (i.e., the phosphorylation potential at which no change in optical signal occurred) corresponds to a value of 15.2 +/- 0.7 kcal/mol in mitochondria and 11.2 +/- 0.3 kcal/mol in sub-mitochondrial particles. The value of delta muH+ generated by the hydrolysis of ATP was estimated using ion distribution techniques. In each case a rapid centrifugation technique was used to separate the organelle from the suspending medium. The total delta muH+ generated in each case was approx. 200 mV being composed of both membrane potential and pH components. A comparison of delta muH+ with delta Gp indicates that the apparent H+/ATP ratio in mung bean mitochondria is 3.4 +/- 0.2 while in phosphorylating sub-mitochondrial particles it is 2.2 +/- 0.1.

Electron and proton transport in the ubiquinone cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. Patterns of binding and inhibition by antimycin.

The effect of antimycin on the ubiquinone cytochrome b-c2 (Q b-c2) oxidoreductase of the photosynthetic bacterium Rhodopseudomonas sphaeroides has been studied under controlled oxidation-reduction potential (Eh) conditions by equilibrium measurements and by rapid kinetic analysis of single turnover flash.induced electron and proton translocations. 1. Antimycin shifts the alpha-band of ferro b50 (lambda max 560 nm) by 1 to 2 nm toward the red but has no apparent effect on the equilibrium oxidation-reduction midpoint potential of the cytochrome. 2. This red shift is proportional to the antimycin added until a "titer" of 0.7 +/- 0.1 antimycin per reaction center (RC) is approached. With a similar titer antimycin essentially abolishes the following millisecond reactions activated by saturating single turnover flashes: reduction of ferri c2, oxidation of ferro b, Phase III of the membrane-potential-indicating band shift of endogenous carotenoid pigments, and the uptake of 1 of the 2 protons taken up per electron transferred. Such titrations indicate that the binding (KD approximately 10(-9) m) and mode of inhibition of antimycin are noncooperative and are independent of the membrane's coupling status and of the pH and Eb over the range in which electron transport is operative. 3. In the presence of excess antimycin a partial recovery of ferri c2 reduction is seen when the intensity of the flash is diminished, but only at Eh values such that Z (a special quinone serving as reductant for ferri c2) is reduced but b50 is oxidized before activation. These results are consistent with the following model. Each Q b-c2 oxidoreductase complex includes one antimycin binding site, one b50, and one Z. These complexes and the c2 . RC complexes, present in an 0.7:1 ratio, are to some degree mobile with respect to each other. Ferri b50 can be reduced either via the quinones of the RC or via Z in a reaction also involving c2. The former route is kinetically dominant in the presence of antimycin, but the latter route is the means for "oxidant-induced reduction" and depends on the collisional interaction of the oxidoreductase and c2 . RC complexes. Antimycin interferes with neither of these two routes but does inhibit the oxidation of ferro b50; all the other inhibitory effects are consequent on this.

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa.

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations. Of the ferredoxin-type iron-sulfure centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two 'high potential iron sulfur' centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished. The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.

EPR studies of higher plant mitochondria. II. Center S-3 of succinate dehydrogenase and its relation to alternative respiratory oxidations.

1. An electron paramagnetic resonance study of the high potential iron sulfur (HiPIP-type) Center S-3 of higher plant mitochondria is described. This center is the major HiPIP-type center associated with plant mitochondria and it displays physical properties which are similar to its mammalian counterpart. It has a pH-independent midpoint potential of +65 +/- 10 mV between pH 6.0 and 8.5. 2. The behavior of Center S-3 in a variety of steady-state conditions suggests that it is of physiological significance in electron transport. Furthermore, it can be shown that the alternative oxidase, which is present in many higher plant mitochondria, tends to keep this center oxidized in the presence of succinate and cyanide. This indicates that the alternative oxidation site is on the electron-donating side of the Center S-3. 3. Salicylhydroxamic acid, an inhibitor of the alternative pathway, does not affect the midpoint potential, signal size or shape, or temperature and power saturation profiles of Center S-3, suggesting that direct autoxidation of this center cannot account for alternative oxidase activity. This is further confirmed by the finding that the presence of succinate dehydrogenase is not necessary for alternative oxidase activity with NADH as respiratory substrate in submitochondrial particles.

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.