Respiration and Carbohydrate Metabolisms of Potato Tuber

When a potato tuber is cut into slices, the activity of dehydrogenase system linked to DPN decreases but the activities of tyrosinase system and G-6-P dehydrogenase increase in the slices. Accompanying the changes of these enzymatic activities, the metabolic pathway in the slices turns from glycolytic path to pentose phosphate path. Tyrosinase oxidizes phenols which are formed from an intermediate of the pentose phosphate path, sedo-heptulose via shikimate as in microorganisms, resulting in increase in the oxygen uptake of the slices. It seems impossible to play a role as a terminal oxidase linked to diphosphopyridine nucleotide.     

Effect of Glutaraldehyde on Cell Viability, Triphenyltetrazolium Reduction, Oxygen Uptake, and β-Galactosidase Activity in Escherichia coli

Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.     

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.     

山嵛菜抗氧化与抑制酪氨酸酶的实验研究

为了探讨山嵛菜植物各部分的抗氧化与抑制酪氨酸酶能力,将新鲜山嵛菜的叶、叶柄、块茎、根须等,用20%乙醇水溶液提取后,分别检测各部分的总抗氧化能力、灭活超氧阴离子能力和抑制酪氨酸酶能力。结果表明,总抗氧化能力以山嵛菜叶最强(68.4单位,0.1 g/mL);灭活超氧阴离子能力也是山嵛菜叶最强(IC50值0.7mg/mL);抑制酪氨酸酶能力以山嵛菜块茎、根须最强(IC50值0.3或0.4 mg/mL)。上述资料为进一步开发利用山嵛菜提供了依据。     

The response of plant mitochondria to media of high solute content

The state-3 rate of respiration of potato tuber mitochondria is inhibited by concentrations of KCl or NaCl above 125 mM, and by concentrations of sucrose, lactose, or maltose above 500 mM, but not at all by mannitol, glucose, glycine, or proline up to a concentration of 1500 mM in the medium. Mitochondria from cauliflower, beetroot, cucumber, rock melon, and watermelon behave very similarly to those from potato tuber. The variable response to different solutes proves that the reduction in respiration is not a simple function of the chemical potential of water in the medium. Disruption of potato mitochondria by ultrasonic vibration does not relieve the inhibition of succinate oxidation caused by KCl or sucrose. However, treatment with detergent abolishes completely the inhibition of respiration by sucrose. Inhibition of succinate dehydrogenase [Succinate:PMS, oxidoreductase (EC.1.3.99.1)] and malate dehydrogenase [L-Malate:NAD oxidoreductase (EC.1.1.1.37)] activities by sucrose is less than the inhibition of succinate- and malate-dependent oxygen uptake by the potato mitochondria. Limited substrate uptake and, alternatively, reduced electron flow as a consequence of a direct effect of solute on the mitochondrial membrane are considered as possible mechanisms of inhibition.     

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.     

Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation,

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD(+) reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and (13)C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions.     

Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.     

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

Effect of Sublethal Heat on the Metabolic Activity of Staphylococcus aureus

Cells of Staphylococcus aureus MF-31 which have been heat-injured at 52 C have an altered metabolic activity. Analyses of whole-cell preparations by means of the Thunberg technique and Warburg manometry showed decreased dehydrogenase activity and oxygen uptake on a variety of substrates. In cell-free extracts prepared from injured cells, it was demonstrated that the specific activity of fructose diphosphate aldolase, lactate dehydrogenase, and butanediol dehydrogenase was less than that of extracts prepared from normal unheated cells. Recovery of the heat-injured cells in a suitable medium supported a return of the dehydrogenase activity and oxygen uptake, but the activity of the enzymes in cell-free extracts prepared from such partially recovered cells did not fully return to the level of normal (unheated) preparations. Addition of chloramphenicol or actinomycin D to the recovery medium, singly or in combination, retarded the return of the normal metabolic activity. Radiorespirometric experiments indicated that the percentage participation of the Embden-Meyerhoff Parnas and hexose monophosphate pathways remained the same for normal and heat-injured cells. The sublethal heat treatment decreased the catabolic capabilities of S. aureus and the production of selected end products associated with the metabolism of glucose.     

COMPARATIVE STUDIES ON VARIOUS CALLUSES AND CROWN GALL OF JERUSALEM ARTICHOKE II. SOME BIOCHEMICAL PROPERTIES

A callus isolated from Jerusalem artichoke tuber was found tohave much more cell wall material and tyrosinase activity thanthe original tuber, and its dry weight was dependent on thesucrose concentration in the culture medium. All calluses anda crown gall tested contained no detectable inulin. Even thoughdifferent cultures were widely different in their dry weight,this was closely related to their total hexose content, andthe latter in turn was proportional to their free hexose content. (Received January 18, 1967; )     

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.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Respiration and dehydrogenase activity of pigmentless variants of Staphylococcus aureus]

Respiration and dehydrogenase activity were studied in four pigmentless mutants of Staphylococcus aureus 209-P. The rate of oxygen uptake from the incubation medium was higher in the mutants than in the parent strain. The rate of respiration and the activity of dehydrogenase in the pigmentless mutants were found to depend on the permeability of the cell membrane for substrates of respiration; carotenoids are presumed to be involved in this permeability.     

Piante critiche delle Alpi Venete

Abstract

INHIBITION OF GLUCOSE-6-P DEYDROGENASE BY OXIDATION PRODUCTS OF NATURAL PHENOLS. — The inhibition of Gl-6-P dehydrogenase activity found in highly concentrated homogenates from potato tuber tissue, seems due to natural occurring orto-di-quinones, such those arising from polyphenoloxidase catalized oxidation of chlorogenic acid, caffeic acid, and tyrosine.

These compounds strongly inhibit Gl-6-P dehydrogenase activity of the potato tuber homogenate as well as that of purified Gl-6-P dehydrogenase from yeast. The inhibition effect appears much stronger when the diphenols are oxidized in the presence of the Gl-6-P dehydrogenase. Gl-6-P and TPN efficently protect the enzyme from the inhibition. Ascorbic acid prevents the inhibition by mantaining the diphenols in the reduced form, but is ineffective in reversing the inhibition.     

A Quantitative Cytochemical Study of Glutamate and Glucose-6-Phosphate Dehydrogenase Activities during Chilling Injury in Tubers of Dioscorea rotundata Poir.

Onyia, G. O. C. and Gahan, P. B. 1985. A quantitative cytochemicalstudy of glutamate and glucose-6-phosphate dehydrogenase activitiesduring chilling injury in tubers of Dioscorea rotundala Poir.—J.exp. Bot. 36: 1249–1256. The response of glucose-6-phosphate dehydrogenase and glutamatedehydrogenase activities in healthy Jamaican Dioscorea rotundalatubers and those chilled at 3 ?C for 1,2,3,4, and 7 d at 70%r.h. were assessed by quantitative cytochemical assays. Bothenzymes in chill-damaged tuber tissues showed a substantiallyhigher activity than did those of the healthy tubers. An early,sharp increase in the response of the NADP-tetrazolium reductasesystem of damaged tuber tissue was significantly higher (P =0.001) than that of healthy tubers or those chilled but ableto recover. This response may be used as an early marker ofchilling injury in the yam tuber. Key words: Dioscorea rotundata Poir, quantitative cytochemistry, yam tuber, glucose-6-phosphate, dehydrogenase, glutamate dehydrogenase, NADPitetrazolium reductase     

The effects of abscisic acid on growth and respiratory characteristics of potato tuber tissue discs

Incubation of potato tuber tissue discs on B5 medium supplemented with 1-naphtyl-acetic acid (NAA) led to callus formation, irrespective of the presence of kinetin; without NAA no callus formation occurred. Incubation in the presence of abscisic acid (ABA) reduced the increases in fresh weight and dry weight both in callus-forming and in non-callus-forming tissue. Mitochondrial respiration was lowered by ABA as well. The induction of the alternative, CN-resistant pathway was inhibited by the presence of ABA, especially in mitochondria from non-callus-forming tissue.The in vivo respiration of the callus-forming tissue was higher than that of the non-callus-forming tissue. Total respiration, cytochrome pathway activity and the capacity of the alternative pathway were all lowered in callus-forming tissue by treatment with ABA. The in vivo activity of the alternative pathway was low in all tissue types, especially after ABA-treatment. The slight stimulation by hydroxamates of the oxygen uptake of callus-forming tissue incubated on medium with NAA and ABA indicates the involvement of a hydroxamate-activated peroxidase in the oxygen uptake of this tissue; this peroxidase seemed not to participate in the oxygen uptake of the other tissues types.In non-callus-forming tissue the oxygen uptake of ABA-treated tissue was very low and almost completely resistant to the combined addition of inhibitors of both the cytochrome and the alternative pathway, indicating that the in vivo activity of the mitochondria in the oxygen uptake of the tissue was very low. The possible causes for this ABA-effect are discussed. In non-callus-forming tissue the treatment with ABA creates a situation which is comparable with that observed in intact potato tubers. This situation is characterized by a tissue respiration lower than that of the isolated mitochondria and an alternative pathway capacity that is low or absent.     

Ethanol breaks dormancy of the potato tuber apical bud

Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].     

Aerobic performance capacity in athletes

Maximal oxygen uptake (max VO2) in leg and arm work, succinate dehydrogenase activity (SDH) and percentage of slow twitch fibers (%ST fibers) in M. vastus lateralis (VL), M. gastrocnemius c.l. (GL) and M. deltoideus (D) were studied in 89 athletes practising 11 different sport events. It was found that maximal oxygen uptake correlated positively with %ST fibers and SDH activity in M. VL. The SDH activity and %ST fibers in M. VL correlated also with one another. The results suggest that oxidative capacity of the muscles is not the limiting factor for maximal oxygen uptake. The role of the oxidative capacity of the muscles might be important during submaximal work of long duration and when a relatively small muscle mass is activated (long-distance running). MaxVO2 might be the most important determinant of performance when large muscle mass is activated during maximal work of a duration from several minutes up to 1 h (cross-country skiing).