Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 l·l^-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 l·l^-1 increased it from 10 to 50 nmol O₂·min^-1·(mg protein)^-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.Abbreviations Cyt c cytochrome c - SHAM salicylhydroxamic acid     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

Respiration and Mitochondrial Membrane Potential Are not Required for Apoptosis and Anti-apoptotic Action of Bcl-2 in HeLa Cells

The release of cytochrome c from intermembrane space of mitochondria into cytosol is one of the critical events in apoptotic cell death. The important anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it was shown that apoptosis and release of cytochrome c induced by staurosporine or by tumor necrosis factor- in HeLa cells were not affected by inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers (CCCP, DNP) that decrease the membrane potential at the inner mitochondrial membrane. The inhibitors of respiration and the uncouplers did not affect also the anti-apoptotic activity of Bcl-2.     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Relation between growth rate and metabolic organization of white muscle,liver and digestive tract in cod,Gadus morhua

To determine whether the aerobic capacity of tissues required for growth specifically reflects growth rates, we monitored the activities of key enzymes of oxidative, glycolytic and amino acid metabolism in muscle, liver and intestine of Atlantic cod (Gadus morhua) growing at different rates. Fish were maintained individually in small tanks at 10°C and fed on rations that allowed growth rates ranging from-0.6 to 1.6% per day. The correlation between growth rate and muscle enzyme activity was pronounced for the glycolytic enzymes (LDH, PFK and PK). The activities of glycolytic enzymes were more than four times higher for fish having higher growth rates compared to those that did not grow. Mitochondrial enzyme (cytochrome c oxidase, citrate synthase and -hydroxyacyl-CoA dehydrogenase) activities remained unchanged in fish with positive growth. The liver seems to respond to requirements of growth by an increase in size. In the liver, the activities of the enzymes of amino acid metabolism expressed as units · g DNA^-1 specifically increases with growth rate. In contrast to the two other tissues, the specific activities of mitochondrial enzymes in the intestine increased with growth rate while the relative mass of the intestine remained constant. Intestinal cytochrome c oxidase activity increased from a minimum of about 2 to more than 8 units · g intestine^-1. Cytochrome c oxidase activity increased in parallel with the food conversion efficiency. This suggests that the aerobic capacity of the intestine may initially limit the rates of digestion and growth in this species.Abbreviations AA amino acid(s) - BM body mass - CCO cytochrome c oxydase - CS citrate synthase - DTNB 5,5 dithiobis-2-nitrobenzoic acid - GDH glutamate dehydrogenase - GOT glutamate oxalacetate transaminase - GPT glutamate pyruvate transaminase - GR growth rate(s) - HOAD -hydroxyacyl-CoA dehydrogenase - HSI hepatosomatic index - LDH lactate dehydrogenase - MR metabolic rate(s) - PCA perchloric acid - PFK phosphofructokinase - PK pyruvate kinase - PMSF phenylmethylsulphonyl fluoride; TRIS     

Functional mitochondria in snake Bothrops alternatus erythrocytes and modulation of HbO2 affinity by mitochondrial ATP

Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca²⁺ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca²⁺ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O₂ affinity, which suggests that ATP production by mitochondria may participate in modulation of O₂ transport by hemoglobin.Abbreviations membrane potential - BAE Bothrops alternatus erythrocytes - DNP 2,4-dinitrophenol - DPG 2,3-diphosphoglycerate - EGTA ethyleneglycol tetra-acetic acid - FCCP carbonylcyanide p-trifloromethoxyphenylhydrazone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TPP⁺ tetraphenylphosphonium - TRIS tris-(hydroxymethyl)aminomethane     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

Sensitivity to drying in vitro of enzymes in mitochondrial subfractions from Vicia faba seed

In order to investigate the persistence of membrane and matrix functions following desiccation, enzymic activities were studied in Vicia faba L. seed mitochondrial subfractions subjected to drying and rehydration in vitro. Mitochondria were prepared after 0, 12 and 24 h of seed imbibition. These were fractionated into inner membranes ("submitochondrial particles"), outer membranes (12 and 24 h only) and the soluble matrix. Enzyme activities associated with the inner membrane and matrix were found to increase several-fold during the first 12 h of imbibition. The two matrix enzymes examined, malate dehydrogenase and glutamate dehydrogenase, were insensitive to in vitro drying at all stages of imbibition. The membrane-bound activities from 12 h and 24 h imbibed material, antimycin A-sensitive NADH: cytochrome c oxidoreductase and (F_o-F₁)-ATPase of the inner membrane and antimycin A-insensitive NADH: cytochrome c oxidoreductase of the outer membrane, were moderately sensitive to dehydration. The F₁-ATPase solubilized from the inner membrane (F_o-F₁) complex was much less sensitive to drying, provided this was done at room temperature.
Mitochondria posessing their outer membranes could not be prepared from dry seed. The antimycin A-sensitive NADH: cytochrome c oxidoreductase from inner mitochondrial membranes of unimbibed seed was extremely sensitive to desiccation in vitro, about 75 to 80% of the activity being lost. This loss could be somewhat reduced by addition of glycerol or sucrose before drying.
It is concluded that uncontrolled desiccation results in major damage to some of the membrane-bound enzymic systems in mitochondria, whereas activities in the soluble fraction are remarkably tolerant of desiccation.     

A Ca2+, Calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane

NAD kinase activity from dark grown corn coleoptiles is shown to be almost totally dependent on Ca2+ and calmodulin. Nearly all of the enzyme activity is found in a particulate fraction. Upon differential and density gradient centrifugation the NAD kinase activity co-migrates with the mitochondrial cytochrome c oxidase whereas marker activities for nuclei, etioplasts, endoplasmic reticulum, and microbodies could well be separated, indicating that the NAD kinase is associated with mitochondria. This NAD kinase, associated with intact mitochondria, can be activated by exogenously added Ca2+ and calmodulin. In order to investigate the submitochondrial localization of the NAD kinase, the organelles were ruptured by osmotic treatment and sonication and the submitochondrial fractions were separated by density gradient centrifugation. The NAD kinase activity exhibits the same density pattern as the antimycin A-insensitive NADH-dependent cytochrome c reductase, a marker enzyme of the outer mitochondrial membrane. Marker enzymes for the mitochondrial matrix and the inner mitochondrial membrane reveal different density profiles. These results indicate that the Ca2+, calmodulin-dependent NAD kinase from coleoptiles of dark grown corn seedlings is located at the outer mitochondrial membrane. The physiological relevance of the location and the Ca2+, calmodulin-dependence of the NAD kinase will be discussed.     

Cytochrome c oxidase activity in T/t 6 (balanced lethal) mutant mice

R-1 (1450g) and R-2 (25,000g) liver fractions from T/t ⁶ and B6CBAF1 hybrid mice were analyzed for their protein content, mitochondria concentrations, and activities of three respiratory-chain enzymes of the mitochondrial inner membrane: cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, E.C. 1.9.3.1), -glycerophosphate dehydrogenase [l-glycerol-3-phosphate: (acceptor) oxidoreductase, E.C. 1.1.99.5], and succinate-cytochrome c reductase. Only cytochrome c oxidase activity, calculated as units per 1010 mitochondria, was significantly lower in both R-1 and R-2 fractions of T/t ⁶ mice. Cytochrome c oxidase activity varied greatly among T/t ⁶ mice, as did their liver mitochondria concentrations and body weights. Cytochrome c oxidase activity in the R-1 fraction of T/t ⁶ mice, calculated as units per 1010 mitochondria per gram of body weight, averaged about 40% lower than in B6CBAF1 mice. -Glycerophosphate dehydrogenase activity was often elevated in T/t ⁶ mice, particularly in the R-2 fraction. The T/t locus, a complex genetic locus on chromosome 17, may contain genes important to the function and biogenesis of mitochondria.This investigation was supported by institutional funds from the Jackson Laboratory and by an allocation from NIH Biomedical Research Support Grant (RR-05545). The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Participation of a cytochrome b5-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in NADH-semidehydroascorbic acid reductase activity of rat liver

The participation of a cytochrome b₅-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in the NADH-semidehydroascorbate (SDA) reductase activity of rat liver was studied. NADH-SDA reductase activity was strongly inhibited by antibodies against OM cytochrome b and NADH-cytochrome b₅ reductase, whereas no inhibition was caused by anti-cytochrome b₅ antibody. NADH-SDA reductase exhibited the same distribution pattern as OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity among various subcellular fractions and submitochondrial fractions. Both activities were localized in outer mitochondrial membrane. These observations suggest that OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase system participates in the NADH-SDA reductase activity of rat liver.     

ELECTRON MICROSCOPIC EXAMINATION OF SUBCELLULAR FRACTIONS : II. Quantitative Analysis of the Mitochondrial Population Isolated from Rat Liver

Large granule fractions, containing about 80% of the cytochrome oxidase of the tissue, were isolated from rat liver and used to prepare thin pellicles of packed particles which were submitted to quantitative electron microscopic examination. Various parameters describing the mitochondrial population were determined by measuring the size and number of mitochondrial profiles in sections, and the ratio of the inner to the outer membrane area. The mean particle radius and volume were found to be respectively 0.38 µ and 0.29 µ³; the average areas per mitochondrion were 2 and 5 µ² for the outer and inner membranes respectively. On the basis of the cytochrome oxidase activity recovered in the particulate fractions, the results were extrapolated to the whole liver, and it was concluded that rat liver contains about 5.10¹¹ mitochondria per gram; this corresponds to a volume of 0.14 ml/g and to an area of 2.5 and 1 m²/g for the inner and outer membranes respectively. The validity and the accuracy of these determinations is discussed and the results are compared to the information which has been obtained by independent methods or by other investigators.     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

Summary The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).Deceased June 5, 1988     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both pro-mitochondria and mitochondria obtained from O₂-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N₂) to elaidic (O₂)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O₂-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10^-8 to 5.5·10^-8 m·s^-1·MPa^-1 in wheat and from 0.9·10^-8 to 4.8·10^-8 m·s^-1·MPa^-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10^-7 m·s^-1·MPa^-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10^-7 m·s^-1·MPa^-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.Symbols L _{p, cell} cell hydraulic conductivity - L _{p, root} root hydraulic conductivity - L _{p, root} calculated root hydraulic conductivity - root reflection coefficient     

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg²⁺-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Con A concanavalin A - Cyt cytochrome - LysoPC lysophosphatidylcholine - Pi orthophoshate - PP_iase pyrophosphatase - IDPase inosine-5-diphosphatase We thank Pr. Robert Dargent and André Moisan (Laboratoire de Cryptogamie, Toulouse, France) for use of their electron-microscope facilities. This work was supported by the Centre National de la Recherche Scientifique and by a grant Dynamique du fonctionnement de la vacuole from the Ministère de la Recherche et de la Technologie.     

Enzyme der Elektronentransportpartikel aus Rhodospirillum rubrum: Eigenschaften von NADH- und Succinat-Cytochrom c-Reduktase

Zusammenfassung Es werden einige Eigenschaften von NADH- und Succinat-Cytochrom c-Reduktase aus R. rubrum beschrieben. Beide Aktivitäten sind ausschließlich in der durch Ultrazentrifugation erhaltenen Partikelfraktion lokalisiert. Aerob im Dunkeln und anaerob im Licht gezüchtete Zellen zeigen keine Unterschiede in ihren spezifischen Aktivitäten.Das Optimum der Reaktion liegt für beide Aktivitäten bei 8,3; das von Succinatdehydrogenase bei 7,7. K _Mfür NADH ist 7,1·10^-6 m, für succinat 1,1·10^-4 m. NADPH wird nicht umgesetzt. Die Partikel zeigen unterschiedliche Affinität zum Elektronenacceptor Cytochrom c. K _Mfür Cytochrom c bei NADH 9,5·10^-6 m, bei Succinat 4,5·10^-6 m.NADH-Cytochrom c-Reduktase wird durch Verdünnung inaktiviert. Durch Serumalbumin kann das Enzym nur begrenzt geschützt werden. Das Substrat und der Acceptor sind wirkungslos.Succinat und Fumarat, jedoch nicht Malonat inaktivieren Succinat-Cytochrom c-Reduktase, wenn das Enzym in verdünntem Zustand vorliegt. Die beiden Substanzen zeigen unterschiedliche Inaktivierungskinetik. Serumalbumin bewirkt hier weitgehenden Schutz des Enzyms.Succinat-Cytochrom c-Reduktase wird durch Phosphat aktiviert.NADH- und Succinat-Cytochrom c-Reduktase werden durch steigende Phosphatkonzentrationen verschieden stark gehemmt.Detergentien (Triton X-100) inaktivieren beide Aktivitäten; NADH-Cytochrom c-Reduktase ist sehr viel empfindlicher als die Succinat-oxydierende Aktivität.Inhibitoren des Elektronentransports wirken auf beide Aktivitäten. NADH-Cytochrom c-Reduktase wird besonders durch Rotenon stark gehemmt.Succinat-Cytochrom c-Reduktase wird durch Malonat, Fumarat und durch Oxalacetat gehemmt. Oxalacetat ist bei R. rubrum ein sehr starker Inhibitor.

---

Enzymes of the electron transport particles of Rhodospirillum rubrum: Properties of NADH- and succinate-cytochrome c-reductase

Summary Some properties of NADH- and succinate-cytochrome c-reductase from R. rubrum are described. Both activities are predominantly located in the particlefraction obtained by ultracentrifugation. No differencies in specific activities of the enzymes are observed between aerobic darkgrown and anaerobic lightgrown cells.The optimum pH of both reactions is at 8,3, the optimum for Succinicdehydrogenase at 7.7. K _M for NADH is 7.1×10^-6 m, for succinate 1.1×10^-4 m. NADPH is not oxidized. The particles also show different affinities for the electronacceptor cytochrome c. K _Mfor cytochrome c with NADH 9.5×10^-6 m and with succinate 4.5×10^-6 m.NADH-cytochrome c-reductase is inactivated by dilution. Serumalbumine can protect the activity to a limited extent. Substrate and acceptor are without effect.Succinate and fumarate but not malonate inactivate succinate-cytochrome c-reductase, when the enzyme is in a diluted form. Succinate and fumarate exhibit different kinetics of inactivation. There is a large protection by serumalbumine against the inactivation.Succinate-cytochrome c-reductase is activated by phosphate.Increasing concentrations of phosphate inhibit NADH- and succinate-cytochrome c-reductase to a different degree.The activities also show different sensitivities to inactivation by treatment with detergents (Triton X-100), NADH-cytochrome c-reductase being much more sensitive than succinate-cytochrome c-reductase.Inhibition of both activities by inhibitors of the electron transport chain is observed, notably a strong inhibition of the NADH-enzyme by low concentrations of rotenone.Succinate-cytochrome c-reductase is inhibited by malonate, fumarate and also by oxalacetate. In R. rubrum the latter substance is a potent inhibitor.

---

Verwendete Abkürzungen NADH reduziertes Nicotinamid-Adenin-Dinucleotid - DCPIP 2,6-Dichlorphenol-indophenol