Swelling and contraction of corn mitochondria

A survey has been made of the properties of corn mitochondria in swelling and contraction. The mitochondria swell spontaneously in KCl but not in sucrose. Aged mitochondria will swell rapidly in sucrose if treated with citrate or EDTA. Swelling does not impair oxidative phosphorylation if bovine serum albumin is present.

Contraction can be maintained or initiated with ATP + Mg or an oxidizable substrate, contraction being more rapid with the substrate. Magnesium is not required for substrate powered contraction. Contraction powered by ATP is accompanied by the release of phosphate. Oligomycin inhibits both ATP-powered contraction and the release of phosphate. However, it does not affect substrate-powered contraction. Substrate powered contraction is inhibited by electron-transport inhibitors. The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, accelerates swelling and inhibits both ATP-and substrate-powered contraction. However, the concentrations required are well in excess of those required to produce uncoupling and to accelerate adenosine triphosphatase; the concentrations required inhibit respiration in a phosphorylating medium.

Phosphate is a very effective inhibitor of succinate-powered contraction. Neither oligomycin nor Mg affects the phosphate inhibition. Phosphate is less inhibitory with the ATP-powered contraction.

The results are discussed in terms of a hypothesis that contraction is associated with a nonphosphorylated high energy intermediate of oxidative phosphorylation.

     

Swelling and contraction of phaseolus hypocotyl mitochondria

Isolated Phaseolus mitochondria will swell spontaneously in buffered KCl and contract with an oxidizable substrate or ATP + Mg²⁺. The conditions under which the mitochondria are swollen affect subsequent contraction, substrate oxidation and ion accumulation, but not their oxidative phosphorylation ability. Bovine serum albumin reduces the rate of swelling and promotes substrate oxidation, contraction and ion accumulation. Swelling of these mitochondria is associated with the release of malic dehydrogenase and a loss of membrane integrity. The beneficial effects of bovine serum albumin in preserving the energy linked functions of Phaseolus mitochondria is discussed.     

Swelling and contraction of heart mitochondria suspended in ammonium phosphate

Bovine heart mitochondria which have been allowed to swell in isotonic NH ₄ ⁺ phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from P_i uptake being slower than removal of internal P_i via phosphorylation of external ADP. Similar results are obtained when K⁺ + nigericin is substituted for NH ₄ ⁺ . Mersalyl inhibition of P_i transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH ₄ ⁺ phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of P_i anions driven by electrogenic H⁺ ejection.     

Swelling of mitochondria in crayfish gills

Summary In the gill processes ofAstacus leptodactylus Esch. fixed in the hypotonic glutaraldehyde fixative the mitochondria appear swollen to a different extent. The swelling occurs in a special way. Part of a single mitochondrion is swollen and the remaining part is frequently unchanged as in the control sections made of tissues fixed in the isotonic fixative. It is concluded that the mitochondrial matrix occurs in a semisolid state and osmotic swelling begins as a local liquefaction of the matrix which extends gradually over the total mitochondrion.     

Citrate and succinate uptake by potato mitochondria

The uptake of [¹⁴C]citrate and [¹⁴C]succinate was studied in potato mitochondria (Solanum tuberosum var. Russet Burbank) using cellulose pore filtration and was found to occur by the same mechanisms as described for mammalian mitochondria. Potato mitochondria, in the absence of respiration, have a very low capacity for uptake by exchange with endogenous anions, taking up only 2.4 nanomoles citrate and 2.0 nanomoles succinate per milligram protein. Maximum citrate uptake of over 17 nanomoles per milligram protein occurs in the presence of inorganic phosphate, a dicarboxylic acid, and an external energy source (NADH), conditions where net anion accumulation proceeds, mediated by the interlinking of the inorganic phosphate, dicarboxylate, and tricarboxylate carriers. Maximum succinate uptake in the absence of respiratory inhibitors requires only added inorganic phosphate.     

Uncoupling of respiration-linked contraction in corn mitochondria

Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentrations of dinitrophenol. However, if a contraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error.

The uncoupling of contraction is consistent with the concept that dinitrophenol attacks a primary non-phosphorylated high energy intermediate (I~X). It is proposed that this intermediate is linked to some contractile mechanism such that the degree of contraction reflects the level of intermediate.

     

Identification and characterization of respirasomes in potato mitochondria

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.     

L-lactate metabolism in potato tuber mitochondria

We investigated the metabolism of L-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti-lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of L-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an L-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium L-lactate in a stereospecific manner, thus showing that L-lactate enters mitochondria by a proton-compensated process. Externally added L-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on L-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent L-lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of L-lactate plays a previously unsuspected role in the response of potato to hypoxic stress.     

Phosphorylation of formate dehydrogenase in potato tuber mitochondria

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr76 and Thr333, whereas PDH was phosphorylated on Ser294. Both Thr76 and Thr333 in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.     

Purification and characterization of an a type phospholipase from potato and its effect on potato mitochondria

A potato (Solanum tuberosum) phospholipid acyl-hydrolase, which - in the pH range 7.5 to 8.5—is at least 10,000 times more effective with phospholipids than with galactolipids, has been purified and characterized. It is a soluble enzyme readily distinguished from a neutral lipid lipase and a third lipid acyl-hydrolase which, while acting on phospholipid, shows a decided preference for glyceryl monoolein. The phospholipase in question has a pH optimum of 8.5, is stimulated by Ca²⁺ at pH above 7.5 and inhibited by Ca²⁺ at lower pH, is not dependent on detergents although stimulated by Triton X-100 to a moderate extent, and remains very active at temperatures close to zero. The phospholipids of intact potato mitochondria are highly susceptible to degradation by potato phospholipase, and it is suggested that this enzyme is involved in the extensive lipid breakdown which occurs in fresh potato slices following cutting, and in the deterioration of mitochondria during their preparation and aging.