New insights into the regulation of plant succinate dehydrogenase. On the role of the protonmotive force.

Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K(1/2) approximately 3 microm) and ADP. The carboxyatractyloside insensitivity of these stimulatory effects indicated that they occur at the cytoplasmic side of the mitochondrial inner membrane. Importantly, our novel approach revealed that the enzyme is also activated by oligomycin (K(1/2) approximately 16 nm). Time-resolved kinetic measurements of succinate dehydrogenase activation by succinate furthermore revealed that the activity of the enzyme is negatively affected by potassium. The succinate-induced activation (+/-K(+)) is prevented by the presence of an uncoupler. Together these results demonstrate that in vitro activity of succinate dehydrogenase is modulated by the protonmotive force. We speculate that the widely recognized activation of the enzyme by adenine nucleotides in plants is mediated in this manner. A mechanism that could account for such regulation is suggested and ramifications for its in vivo relevance are discussed.

The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria. Interplay between quinol-oxidizing and quinone-reducing pathways.

The dependence of electron flux through quinone-reducing and quinol-oxidizing pathways on the redox state of the ubiquinone (Q) pool was investigated in plant mitochondria isolated from potato (Solanum tuberosum cv. Bintje, fresh tissue and callus), sweet potato (Ipomoea batatas) and Arum italicum. We have determined the redox state of the Q pool with two different methods, the Q-electrode and Q-extraction techniques. Although results from the two techniques agree well, in all tissues tested (with the exception of fresh potato) an inactive pool of QH2 was detected by the extraction technique that was not observed with the electrode. In potato callus mitochondria, an inactive Q pool was also found. An advantage of the extraction method is that it permits determination of the Q redox state in the presence of substances that interfere with the Q-electrode, such as benzohydroxamate and NADH. We have studied the relation between rate and Q redox state for both quinol-oxidizing and quinone-reducing pathways under a variety of metabolic conditions including state 3, state 4, in the presence of myxothiazol, and benzohydroxamate. Under state 4 conditions or in the presence of myxothiazol, a non-linear dependence of the rate of respiration on the Q-redox state was observed in potato callus mitochondria and in sweet potato mitochondria. The addition of benzohydroxamate, under state 4 conditions, removed this non-linearity confirming that it is due to activity of the cyanide-resistant pathway. The relation between rate and Q redox state for the external NADH dehydrogenase in potato callus mitochondria was found to differ from that of succinate dehydrogenase. It is suggested that the oxidation of cytoplasmic NADH in vivo uses the cyanide-resistant pathway more than the pathway involving the oxidation of succinate. A model is used to predict the kinetic behaviour of the respiratory network. It is shown that titrations with inhibitors of the alternative oxidase cannot be used to demonstrate a pure overflow function of the alternative oxidase.

Characterisation of PHSP1, a cDNA encoding a mitochondrial HSP70 from Pisum sativum.

A pea cDNA clone, PHSP1, encoding a member of the HSP70 gene family has been isolated. DNA sequence analysis indicates that the protein encoded by PHSP1 is a homologue of the mitochondrial HSP70 proteins, SSP1 from Schizosaccharomyces pombe and SSC1 from S. cerevisiae. It contains an amino-terminal extension of 50 amino acids, rich in basic and hydroxyl amino acids, similar to other plant mitochondrial leader sequences. Western blot analysis indicates that the PHSP1 protein is associated only with mitochondria and not with any other sub-cellular organelle or cytoplasm. Further confirmation of its location within mitochondria was obtained from in vitro protein translocation experiments into purified Pisum sativum mitochondria. It was observed that the precursor protein was efficiently imported and that it is processed to produce a protein with an Mr of the anticipated size of the mature protein. Results are discussed with respect to the structure and function of the mitochondrial HSP70 protein.

Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision.

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.

Immunological analysis of plant mitochondrial NADH dehydrogenases.

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.

Trifluoperazine inhibition of electron transport and adenosine triphosphatase in plant mitochondria.

Trifluoperazine inhibits ADP-stimulated respiration in mung bean (Phaseolus aureus) mitochondria when either NADH, malate, or succinate serve as substrates (IC50 values of 56, 59, and 55 microM, respectively). Succinate:ferricyanide oxidoreductase activity of these mitochondria was inhibited to a similar extent. The oxidation of ascorbate/TMPD was also sensitive to the phenothiazine (IC50 = 65 microM). Oxidation of exogenous NADH was inhibited by trifluoperazine even in the presence of excess EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] (IC50 = 60 microM), indicating an interaction with the electron transport chain rather than with the dehydrogenase itself. In contrast, substrate oxidation in Voodoo lily (Sauromatum guttatum) mitochondria was relatively insensitive to the phenothiazine. The results suggest the bc1 complex to be a major site of inhibition. The membrane potential of energized mung bean mitochondria was depressed by micromolar concentrations of trifluoperazine, suggesting an effect on the proton-pumping capability of these mitochondria. Membrane-bound and soluble ATPases were equally sensitive to trifluoperazine (IC50 of 28 microM for both), implying the site of inhibition to be on the F1. Inhibition of the soluble ATPase was not affected by EGTA, CaCl2, or exogenous calmodulin. Trifluoperazine inhibition of electron transport and phosphorylation in plant mitochondria appears to be due to an interaction with a protein of the organelle that is not calmodulin.

Phosphate dependent, ruthenium red insensitive CA2+ uptake in mung bean mitochondria.

Energy linked Ca2+ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca2+, we observe a respiration-linked uptake of Ca2+ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca2+ uptake. The results suggest that although plant mitochondria accumulate Ca2+ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.

The effect of calcium on the respiratory responses of mung bean mitochondria.

Purified mung bean hypocotyl mitochondria were examined for their capacity to carry out respiration-dependent accumulation of calcium. The addition of 0.1-1.0 mM calcium to mung bean mitochondria supplemented with succinate gave no stimulation of state 4 respiration even in the presence of inorganic phosphate and the ionophoretic antibiotic A-23187. Even at high calcium concentrations, no transient changes in the respiratory activity occurred and subsequent addition of ADP initiated a further state 3 response. Although the additions of calcium resulted in a rapid H+ ejection, it was insensitive to lanthanum and uncoupling agents. Similarly, additions of calcium failed to initiate any transient changes in the oxidation-reduction states of either pyridine nucleotides or cytochrome b. Direct spectrophotometric recordings of absorbance changes of murexide revealed no respiration-linked calcium transport. It is proposed that although mung bean mitochondria possess a respiration-linked electrochemical potential gradient it would appear that this potential cannot be expressed as calcium transport even at high ion concentrations, probably due to a low calcium membrane permeability.