Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Suppression by Exogenous Phospholipid of Cyanide-Insensitive Respiration of Submitochondrial Particles from Sweet Potato Root Tissue

Submitochondrial particles from sweet potato root tissue retainedthe respiratory characteristics of the intact mitochondria withrespect to the sensitivity to cyanide and salicylhydroxamicacid. The activities of total, cyanide-insensitive, and salicylhydroxamate-sensitiverespiration of the submitochondrial particles yielded from adefinite weight of tissue slices incubated under aerobic conditions,particularly in ethylenecontaining air, were higher than thosefrom the same weight of intact tissue. The less phospholipidthe submitochondrial particles contained relative to protein,the higher the activities of cyanide-insensitive and salicylhydroxamate-sensitiverespiration tended to be relative to total respiratory activity.When the submitochondrial particles were incubated with phospholipidliposomes, the activities of cyanide-insensitive and salicylhydroxamate-sensitive,but not cyanide-sensitive, respiration became extremely low.All phospholipids showed this effect. Such incubation of thesubmitochondrial particles with phospholipid liposomes yieldedlighter particles, indicating close association of exogenouslyadded phospholipid with the particles. Phospholipid moleculesseemed to enter the membrane of the particles. We propose thatphospholipid deficiency in the mitochondrial inner membranefacilitates operation of the cyanide-insensitive electron transportpath. (Received March 30, 1984; Accepted June 15, 1984)     

Effect of aging on the composition of mitochondrial membranes from potato slices

The phospholipid content of mitochondrial membranes from slices of potato tuber (Solanum tuberosum) remains stable during aging. The phospholipid compositions of whole mitochondria and inner membranes do not vary during aging whereas the concentrations of phosphatidylinositol and phosphatidyl-glycerol in outer membranes are slightly amplified. The saturation of outer membrane fatty acids is slightly increased during aging. Gel electrophoresis of mitochondrial membrane proteins show slight variations of one polypeptide in outer membranes and of three polypeptides in inner membranes. These results suggest parallel variations of lipids and proteins in membranes during aging, in marked contrast with the large modifications observed in mitochondrial activities.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Investigation of respiratory and ion transport properties of aging bean stem slices

Ion and oxygen uptake were studied on aging bean stem slices. Oxygen uptake was high immediately after slicing, decreased to a minimum at 100 minutes, and then increased again. Ion uptake per unit of O₂ uptake data suggested that metabolic energy was utilized almost exclusively for sodium transport in fresh tissue but was diverted to potassium transport as the slices aged. Oxygen and ion uptake in fresh slices was less sensitive to 2,4-dinitrophenol as compared to the aged slices, indicating major metabolic and physiological changes occurred during aging. This was further substantiated by the tissue response to cyanide and antimycin A. Oxygen uptake was decreased by cyanide (22% by 1 mm) and antimycin A (14% by 1 microgram per milliliter) in fresh slices but not in aged slices. Potassium uptake that developed during aging was sensitive to cyanide and antimycin A. The results are pertinent to understanding the role of the stem in regulating ion transport in plants.     

Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.     

Presence of an inactive protein immunologically analogous to cytochrome c oxidase in the inner membrane of sweet potato root mitochondria

A protein, which was immunoreactive to antibody against cytochrome c oxidase, was found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner membrane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme a. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57,000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.     

Control of Changes in Mitochondrial Activities during Aging of Potato Slices

Aging of slices of potato tuber (Solanum tuberosum L.) in an aerated liquid medium induces a number of changes in mitochondrial activities. A nonphosphorylative, cyanide-insensitive electron transport pathway (alternate pathway) is brought into operation. The rate of oxidation of exogenous NADH increases markedly and the efficiency of phosphorylation with this substrate remains the same as it is in mitochondria isolated from fresh tissue slices. On the contrary, the rates of oxidation of succinate and malate do not increase while lower phosphorylative efficiencies indicate that a fraction of their electrons reaches oxygen through the alternate pathway. Chloramphenicol, a specific inhibitor of the mitochondrial protein-synthesizing system, has no effect whatsoever on these events. However, cycloheximide, which acts on the corresponding cytoplasmic system, prevents both the development of the alternate pathway and the rise in the rate of oxidation of exogenous NADH. These effects are interpreted as showing a specific control of the cytoplasmic protein-synthesizing system on the changes in mitochondrial oxidations during aging.     

Membrane Lipid Breakdown in Relation to the Wound-induced and Cyanide-resistant Respiration in Tissue Slices: A COMPARATIVE STUDY

A study of a variety of bulky storage organs and fruits reveals that fresh slices fall into two categories with respect to their sensitivity to CN. Fresh slices in the first class are CN-sensitive, whereas slices of the second class are resistant to, and often stimulated by, CN. In tissue slices which are initially CN-sensitive, cutting initiates a burst of lipolytic activity. In CN-resistant fresh slices, there is no measurable lipid breakdown.Slicing evokes the wound-respiration which is 5- to 10-fold that of the parent organ. Slice aging, in turn, evokes a further 2- to 3-fold respiratory increase, the wound-induced respiration, whether fresh slice respiration is CN-sensitive or -resistant. Estimation of the contribution by the cytochrome and alternative paths shows that the wound respiration in both groups is mediated by the cytochrome path. On the other hand, the wound-induced respiration in the first class is cytochrome path mediated, whereas, in some members of the second group, both pathways are utilized. Uncouplers of oxidative phosphorylation elicit a CN-sensitive increment in fresh slices as great or greater than the wound-induced respiration. Accordingly, de novo synthesis of mitochondria is ruled out as an explanation of the latter.The integrity of endomembranes, perhaps including mitochondrial membranes, is seemingly a prerequisite for the operation of the alternative path, that is, alternative path activity is lost concomitantly with membrane lipid breakdown. The development of the wound-induced respiration is not co-extensive with the development of the CN-resistant path in all tissue slices. The fundamental process of aging appears to involve activation of pre-existing respiratory capacity.Fresh slices from whatever source fail to utilize exogenous (14)C-labeled glucose, whereas aged slices do so readily. A transport lesion is indicated, the healing of which does not depend on the development of the wound-induced respiration but does depend on fatty acid, and presumably membrane lipid, biosynthesis.     

The effect of aging and acetyl-L-carnitine on the activity of the phosphate carrier and on the phospholipid composition in rat heart mitochondria.

The effect of aging and treatment with acetyl-L-carnitine on the activity of the phosphate carrier and on the phospholipid composition in rat heart mitochondria was studied. It was found that the activity of the phosphate carrier was reduced by aging. Treatment of aged rats with acetyl-L-carnitine reversed this effect. The mitochondrial level of cardiolipin was decreased with aging. Treatment of aged rats with acetyl-L-carnitine restored the level of cardiolipin to that of young rats. It is proposed that acetyl-L-carnitine may restore the correct phospholipid composition (cardiolipin level) of the mitochondrial membrane, altered by aging, thereby restoring the activity of the phosphate carrier.     

The mitochondrial energy transduction system and the aging process

Aged mammalian tissues show a decreased capacity to produce ATP by oxidative phosphorylation due to dysfunctional mitochondria. The mitochondrial content of rat brain and liver is not reduced in aging and the impairment of mitochondrial function is due to decreased rates of electron transfer by the selectively diminished activities of complexes I and IV. Inner membrane H⁺ impermeability and F₁-ATP synthase activity are only slightly affected by aging. Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O₂ uptake, by an increased content of oxidation products of phospholipids, proteins and DNA, a decreased membrane potential, and increased size and fragility. Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Conditions that increase survival, as vitamin E dietary supplementation, caloric restriction, high spontaneous neurological activity, and moderate physical exercise, ameliorate mitochondrial dysfunction in aged brain and liver. The pleiotropic signaling of mitochondrial H₂O₂ and nitric oxide diffusion to the cytosol seems modified in aged animals and to contribute to the decreased mitochondrial biogenesis in old animals. oxidative damage; survival; complexes I and IV; nitric oxide synthase     

Isolation and characterization of a mitochondrially synthesized polypeptide from Neurospora crassa cni-1 mutant.

A polypeptide with a molecular weight of 8 500 (HP 8 500) was isolated from the mitochondrial membrane of the nuclear mutant cni-1 of Neurospora crassa. This mutant is characterized by a cyanide-insensitive respiration and by a deficiency in the cytochromes aa3 and b. The polypeptide is synthesized on mitochondrial ribosomes. It has an extremely hydrophobic character; it is insoluble in aqueous media in the absence of sodium dodecylsulfate and is soluble in acid chloroform/methanol. It lacks histidine. The polar amino acids lysine, arginine, aspartic acid, glutamic acid, serine and threonine make up only 25% of the total amino acids on a mole-percent basis. The N-terminal amino acid is tyrosine. The possible function of this polypeptide in the mitochondrial membrane is discussed.     

Properties of mitochondrial porin from cattle heart]

Porin, a protein able to form ionic channels in model phospholipid membranes, has been isolated for the first time from bovine heart mitochondria. One-dimensional electrophoresis in the presence of sodium dodecyl sulfate revealed a major band with Mr of 32-34 kDa. On two-dimensional electrophoregrams this protein is represented by four components with pI ranging from 6.5 to 7.1. Porin spots were identified on two-dimensional electrophoregrams in a complete mixture of mitochondrial proteins. The presence of porin in bovine heart submitochondrial particles was demonstrated by two-dimensional electrophoresis.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins.     

Influence of calcium on sodium and potassium absorption by fresh and aged bean stem slices

The influence of Ca on the aging processes of bean stem (Phaseolus vulgaris) slices and on the absorption of K and Na by fresh and aged slices was investigated. In the presence of Ca, fresh tissue showed a preferential Na uptake. The preference for Na over K resulted from a differential depressive effect of Ca on absorption of these two ions. In aged tissue Na uptake was also depressed, but K absorption was accelerated, with a net result of a much greater absorption of K than Na.     

Cyanide-resistant Respiration in Fresh and Aged Sweet Potato Slices

The respiration of fresh sweet potato (Ipomoea batatas) slices is resistant to, and often stimulated by, cyanide and antimycin A. m-Chlorobenzhydroxamic acid (CLAM), a selective inhibitor of the alternate path, inhibits respiration in the presence of cyanide and has a limited inhibitory effect in the presence of antimycin A. Thus, a partial bypass of the antimycinsensitive site is indicated. Respiration rises 2-fold at best with slice aging, the increment being cytochrome-mediated. The cyanide-resistant pathway contributes neither to coupled fresh slice respiration nor to the induced respiration in the absence of inhibitors of the cytochrome path. In the presence of uncoupler, however, the alternate path is engaged both in fresh and aged slices. V_cyt, the maximal capacity of the cytochrome path, remains essentially the same with slice aging, whereas V_alt decreases from 20 to 60 per cent. The induced respiration is readily accommodated by the potential cytochrome path capacity of fresh slices, which is realized on aging. Accordingly, there is no need to invoke mitochondrial proliferation in explanation of the development of the induced respiration. The engagement of the alternate path in response to uncoupler reflects substrate mobilization to a degree that substrate oxidation exceeds the electron transport capacity of the cytochrome path.

Fresh slices do not utilize exogenous substrates, whereas aged slices do so readily. Cerulenin, a specific inhibitor of fatty acid synthesis, prevents the development of the induced respiration as well as the capacity to oxidize exogenous substrates. It is suggested that lipid, and ultimately membrane, biosynthesis is central to the development of the induced respiration and the ability to use exogenous substrates, much as in potato.

     

Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.     

Influx and efflux of inorganic phosphate in sweet potato slices. effects of abscisic acid on bi-phasic kinetics

Influx and efflux of inorganic phosphate was determined in freshly cut and “aged” slices of sweet potato tubers, in the presence or absence of abscisic acid. Slices aged in the phytohormone took up considerably less phosphate than did the control slices. No difference in the rates of uptake of phosphate by the fresh tissue was observed. Kinetics of uptake of the aged untreated slices, proved to be bi-phasic. This was not the case for the treated slices. Neither the treated nor the untreated uptake curves for the fresh slices were bi-phasic. Efflux of phosphate from the slices aged in abscisic acid was greater than from the untreated slices. The results strongly suggest an effect of abscisic acid on the cell membrane.