Properties of Mitochondria Prepared by Two Different Isolation Procedures From the Cotyledons of Germinating Bean Seedlings

Mitochondria from 1-day-old bean cotyledons were isolated bya ‘slow’ isolation procedure involving a wash andby a ‘rapid’ procedure. The mitochondria isolatedby the ‘rapid’ procedure were more tightly coupledthan those isolated by the ‘slow’ procedure. Anexogenous supply of cytochrome c or NAD was shown to improvethe activity of mitochondria isolated by the ‘slow’procedure, but not those isolated by the ‘rapid’procedure. The phosphorylative abilities of the latter weremuch greater than the former and were retained for longer periods.It was found that there was a leakage of NAD out of the mitochondria.     

The Oxidation of Malate by Isolated Turnip (Brassica napus L.) Mitochondria: I. ULTRASTRUCTURAL OBSERVATIONS AND THE PRODUCTS OF MALATE OXIDATION

Washed and purified turnip mitochondria oxidize malate withrespiratory control and ADP: O values approaching 3.0 and producepyruvate as the principal product. Oxaloacetate is also producedin significant amounts but is removed by an endogenous mechanism.Malate dehydrogenase appears to be important to the oxidationof malate but requires the removal of oxaloacetate. During malateoxidation the mitochondria undergo configurational changes similarto those observed in animal mitochondria. Both ‘rightside-out’ and ‘inside-out’ submitochondrialparticles have been prepared. Right side-out particles oxidizemalate in the same way as intact mitochondria, whereas the inside-outparticles have a biphasic oxidation, the first phase producingoxaloacetate and the second, NAD-requiring phase producing pyruvate.It is concluded that malate oxidation is a complex process usingseveral enzymes located in the matrix compartment with a minorcomponent possibly on the outer face of the inner membrane.     

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA)

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated (“light”) and a hexadeuterated (“heavy”). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the “heavy” labelled standard with the “light” acetic anhydride and the part containing the “light” labelled standard with the “heavy” acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the “light” and the “heavy” labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: The Italian Ministry of Health     

Study of soluble lipoprotein in rat liver mitochondria

1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at -18 degrees C to -20 degrees C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4 degrees C for 4 days or repeated freezing and thawing results in 15-30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-(14)C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria, suggesting that the soluble lipoprotein could be a distinct entity of mitochondrial origin.     

Purification and Properties of α-Aminoadipate Aminotransferase from Rat Liver and Kidney Mitochondria

Abstract

Recently we reported an affinity chromatography method to purify α-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

Coenzyme Q,peroxidation and cytochrome oxidase features after Parkinson's-like disease by MPTP toxicity in intra-synaptic and non-synaptic mitochondria fromMacaca Fascicularis cerebral cortex and hippocampus: action of dihydroergocriptine

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on respiratory chain features were studied in synaptic and non-synaptic mitochondrial populations from cerebral cortex andhippocampus ofMacaca Fascicularis (Cynomolgus monkey). Enzymatic activity, cytochromea+a ₃ content and turnover numbers of Complex IV, contents of Coenzyme Q₁₀, of hydroperoxides and membrane fluidity were assessed in non-synaptic “perikaryal” and intra-synaptic “light” and “heavy” mitochondria isolated: (a) from the dopaminergic ascending terminal areas of cerebral cortex of monkeys treatedp.o. with dihydroergocriptine at the dose of 2, 6 or 20 mg/kg/day for 52 weeks; (b) from the dopaminergic terminal areas ofhippocampus of monkeys treatedp.o. with dihydroergocriptine at the dose of 12 mg/kg/day before and during the induction of a Parkinson's-like syndrome by MPTP administration (i.v., 0.3 mg/kg/day for 5 days). Dihydroergocriptine administration moderately increased both cytochrome oxidase activity and cytochromea+a ₃ content in “light” intra-synaptic mitochondria and hydroperoxides/CoQ₁₀ ratio in all the types of mitochondria, as a consequence of the enhanced energy metabolism. The Parkinson's-like syndrome by MPTP changed the biochemical investigated parameters, affecting both directly the respiratory chain structures,i.e. by respiratory chain complexes inhibition and indirectly,i.e. by free radical mediated damages. MPTP administration negatively influenced Complex IV activity and Turnover Number of intra-synaptic mitochondria, without affecting the total cytochromea+a ₃ amount. In all types of mitochondria and particularly on the “light” intra-synaptic ones, MPTP-induced lesion enhanced hydroperoxides/Coenzyme Q₁₀ molar ratio due to the fall in Coenzyme Q₁₀ levels and the concomitant increase in hydroperoxides. Dihydroergocriptine treatment appeared to be effective in MPTP-treated animals in improving those mitochondrial features that probably suffered free radical insults.     

The Developmental Anatomy of the Root System in Yam Bean, Pachyrhizus erosus Urban

Investigations revealed that the anatomy of the primary radicularroot of yam bean (Pachyrhizus erosus L.) was typically dicotyledonousexcept that the xylem was not completely developed centripetally.Most of the roots had tetrarch xylem, although a few triarchand pentarch roots were also observed. In both tuberous andnon-tuberous roots, secondary thickening occurred by the formationof the meristematic vascular cambium which formed secondarytissues in a normal fashion. Subsequently, tuberization wasinitiated in the secondary xylem by the development of anomalous‘secondary’ cambia from parenchyma cells surroundingvessel elements. Anomalous ‘secondary’ cambia alsodeveloped from parenchyma cells not associated with vessels.Subsequently, anomalous ‘tertiary’ cambia differentiatedfrom tissues produced by the anomalous ‘secondary’cambia. Activities of these anomalous cambia resulted in theproduction of parenchyma storage cells and were chiefly responsiblefor the growth of the mature tuber. Pachyrhizus erosus L., yam bean, tuberous root, anatomy, anomalous ‘secondary’ cambia, anomalous ‘tertiary’ cambia, centripetal xylem development     

Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C

Surfactants and hydrolytic enzymes were used to probe the natureof the constituents) to which phytochrome binds in paniculatefractions from red-irradiated Cucurbita. [¹⁴C]-choline and [³H]-uridinepre-labelled tissue was used to monitor the release of phospholipidsand RNA by these agents. Ribonuclease (RNase) digestion of 20,000xgpellets eliminates both the phytochrome and ribonucleoprotein(RNP) which cosediment at 31S. Little [¹⁴C]-choline occurs inthe 31S fraction and the amount is not changed by RNase digestion.This is further evidence that phytochrome binds directly tothe RNP in the 31S fraction rather than to any membranous materialpresent. The distribution profile of the RNA in a second ( =‘heavy’)phytochrome fraction does not correlate with that of the pigment.This suggests that the phytochrome in this fraction is not boundto RNP. The RNA is of ribosomal origin but much less degradedthan that of the 31S RNP and is resistant to RNase digestion.Phospholipase C releases>80% of the [¹⁴C]-choline from the‘heavy’ fraction without freeing phytochrome. Thisindicates that the pigment does not bind to the polar head groupsof the membrane phospholipids present. Low concentrations ofdeoxycholate dissociate phytochrome from this fraction withoutreleasing substantial quantities of integral membrane proteinsor phospholipids. Some RNP is dislodged by the surfactant butthe phytochrome and RNP are not released as a complex. The datasuggest that the pigment in the ‘heavy’ fractionmay be loosely bound to a protein constituent rather than toRNP or polar phospholipids. ¹This work was done while on sabbatical leave from the WeizmannInstitute of Science, Rehovot, Israel. (Received April 1, 1976; )     

Effect of D-amino acids on some mitochondrial functions in rat liver

Summary.  We studied the role of the D-amino acids (D-aa) D-serine, D-alanine, D-methionine, D-aspartate, D-tyrosine and D-arginine on rat liver mitochondria. The stability of D-amino acids, mitochondrial swelling, transmembrane potential and oxygen consumption were studied under oxidative stress conditions in rat liver mitochondria. In the presence of glutamate-malate all D-aas salts increased mitochondrial swelling, while in the presence of succinate plus rotenone only D-ala, D-arg and D-ser, induced mitochondrial swelling. The transmembrane potential (ΔΨ) was decreased in the presence of 1 μM Ca²⁺. The D-aas inhibited oxygen consumption in state 3. The D-aa studied exerted effects on mitochondria via an increase of free radicals production. Received January 15, 2002 Accepted April 14, 2002 Published online September 4, 2002 Acknowledgements The authors appreciated the partial economical support from Mexican grants of CONACYT (to A.S.-M. during its sabbatical) and CIC-UMSNH (2.5) and critical readings from Rafael álvarez-González. Authors' address: Alfredo Saavedra-Molina, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3. C.U., Morelia, Mich. 58030. México, Fax: 52-443-326-5788, E-mail: saavedra@zeus.umich.mx     

Cultivar Differences in the Performance of Bean Seedlings at Suboptimal Temperatures

Growth analysis of plants raised under controlled environments(10–5, 12, 15, 18 and 20 °C, and 21 h photoperiod)was used to examine whether varietal differences in the minimumgermination temperature of four bean cultivars persist duringgrowth at suboptimal temperatures. A method to estimate theminimum vegetative growth temperature, based on axis relativegrowth rate, was developed. In order to compensate for ontogeneticdrift, the harvests were conducted at the same stage of developmentof the plants. Axis relative growth rates, reduction rates ofthe cotyledons and other growth parameters were calculated inorder to compare the cultivars. Cultivar ‘Marschall’showed better growth potential at 12 °C than the others,‘Pergousa‘ at 15 °C, and ‘Marschall’,‘Olsok’ and ‘Pergousa’ at 18 and 20°C. The effect of temperature on axis RGR was similar for‘Marschall’, ‘Olsok’ and ‘Pergousa’(Q₁₀ = 2·1) and more pronounced than for ‘Processor’(Q₁₀ = 1·3). Although there were significant differencesin the growth parameters among the cultivars within each temperatureused, the differences did not correspond with the differencesduring germination at low temperatures. The minimum vegetativegrowth temperature was close to 10 °C for all the cultivarstested. Phaseolus vulgaris L., beans, suboptimum temperature, growth analysis, minimum germination temperature, minimum vegetative growth temperature     

Effect of Source of Nitrogen on the Growth of Fiskeby Soya Bean: the Carbon Economy of Whole Plants

Fiskeby V soya bean was grown from seed germination to seedmaturation with two contrasting patterns of nitrogen metabolism:either wholly dependent on dinitrogen fixation, or with an abundantsupply of nitrate nitrogen, but lacking root nodules. The carbonand nitrogen economies of the plants were assessed at frequentintervals by measurements of photosynthesis, shoot and rootrespiration, and organic and inorganic nitrogen contents. Plantsfixing atmospheric nitrogen assimilated only 25–30 percent as much nitrogen as equivalent plants given nitrate nitrogen:c. 40 per cent of the nitrogen of ‘nitrate’ plantswas assimilated after dinitrogen fixation had ceased in ‘nodulated’plants. The rates of photosynthesis and respiration of the shootsof soya bean were not markedly affected by source of nitrogen;in contrast, the roots of ‘nodulated’ plants respiredtwice as rapidly during intense dinitrogen fixation as thoseof ‘nitrate’ plants. The magnitude of this respiratoryburden was calculated to increase the daily whole-plant respiratory loss of assimilate by 10–15 per cent over thatof plants receiving abundant nitrate. It is concluded that ‘nodulated’plants grew more slowly than ‘nitrate’ plants inthese experiments for at least two reasons: firstly, the symbioticassociation fixed insufficient nitrogen for optimum growth and,secondly, the assimila tion of the nitrogen which was fixedin the root nodules was more energy-demanding in terms of assimilatethan that of plants which assimilated nitrogen by reducing nitratein their leaves.     

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Effect of Sclerin on Incorporation of Oleic Acid into Phospholipids and Activity of Phospholipase in Mitochondria

Sclerin (SCL) stimulated the oxidation and the incorporation into the phospholipids of Na-[1-¹⁴C]-oleate in mitochondria isolated from rat liver, preventing the depression of the phosphorylating functions and protecting 2,4-dinitrophenol (DNP)-activated ATPase in mitochondria during incubation with oleate. Also, SCL markedly enhanced the activity of phospholipase to hydrolyze endogenous substrates in mitochondria. The increase in the activity was due to reconstruction of phospholipids through esterification of oleate in mitochondrial membrane, but not to the de novo enzyme synthesis. It was concluded that the level of endogenous phospholipase in mitochondria during incubation reflects the energy- dependent reacylation of the lysophospholipids produced by the action of phospholipase in mitochondrial membrane.     

Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross-reacted with antibodies raised against Neurospora ACP. Purified ACP from Neurospora cross-reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross-reaction with any of the three anti-ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and beta-oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro-organisms.     

Effect of Cultivar, Temperature and Seed Size on the Germination and Emergence of Soya Beans (Glycine max (L.) Merr.)

The rapid and uniform establishment of soya bean [Glycine max(L.) Merr.] stands is conducive to higher yields. This studywas undertaken to determine the effects of cultivar, temperature,and seed size on the rate of germination and emergence. No cultivar effect on the germination rate was observed. However,in an emergence study from a sand-soil-peat mixture, cultivardifferences in emergence rates were noted(‘Chippewa 64’> ‘Wayne’ > ‘Amsoy 71’). In anotheremergence study (sand media) the cvs ‘Calland’ and‘Williams’ emerged faster than the cv. 'Wayne or‘Wells’. Time required for 50 per cent germination decreased (18.8–4.0days) as the temperature increased from 10 to 30 °C (5 °Cincrements). Emergence (50 per cent) from a sand-soil-peat mixturewas more rapid (19.8–6.3 days) as the simulated plantingdate (growth chamber set to simulate field temperatures) wasdelayed from 16 April to 15 June with an intermediate date of16 May. In addition, time required for 50 per cent emergence of thecultivars from sand decreased (793–76 h) as the temperaturewas increased from 10 to 30 °C with no decrease from 30to 35 °C. Seed size effects were apparent, with the very small seed germinatingslower than the three larger seed sizes. In the emergence studieswith both the sand and sand-soil-peat mixture there was a generaltrend toward more rapid emergence with the smaller seeds. However,the absolute differences were small. Significant cultivar x temperature interactions were observedfor the germination and emergence rates. In most cases the cultivarsmerged in terms of germination and emergence rates at temperaturesbetween 10 and 20 °C and at the higher temperatures thecultivar rankings were different from those observed at temperaturesbelow the merging point. Glycine max (L.) Merr, soya bean, seed germination, establishment of seedlings     

Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

The presence of electrical charges on the surface of an organelle is the source of the organelle's electrophoretic mobility. Recently, we reported that capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be used to determine the electrophoretic mobility of individual mitochondria. Here, we describe the use of CE-LIF to monitor changes in the electrophoretic mobility distributions of: (i). mitochondria isolated from cultured NS-1 mouse hybridoma cells disrupted by nitrogen cavitation or mechanical homogenization; (ii). mitochondria isolated from rat liver and purified by gradient centrifugation before and after being frozen in liquid nitrogen; and (iii). mitochondria chemically transformed into mitoplasts. These results indicate that the organelle electrophoretic mobility observed by researchers is affected by preparation procedures and that CE-LIF is a complementary technique for monitoring the quality of mitochondrial preparations.     

Protective Effects of the Synthetic Cannabinoids CP55,940 and JWH-015 on Rat Brain Mitochondria upon Paraquat Exposure

The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition (MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Δψ_m) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O₂ ^∙−) and hydrogen peroxide (H₂O₂), maintaining Δψ_m and by avoiding Ca²⁺-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights into more effective therapeutic approaches for oxidative stress related disorders.