Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

Large-scale purification procedure of spinach leaf mitochondria —isolation and immunological studies of the F1–ATPase

A large–scale purification procedure for mitochondria from spinach ( Spinacia oteracea L, cv Medania) leaves is described. It involves differential centrifugation and density gradient centrifugation on a self–generating gradient of Percoll, From 3 kg of spinach leaves, 150 mg mitochondrial protein are obtained. The thylakoid contamination is lower than 0.2% on a chlorophyll basis. The mitochondria oxidize malate and glycine with state 3 rates of 108 and 140 nmol (mg protein)^-1 min^-1, with respiratory control ratios of 2,7 and 3,8 and ADP/O ratios of 2,0 and 2.1, respectively. The present large–scale purification procedure will facilitate further biochemical and molecular biological studies of leaf mitochondrial proteins.
A pure and active catalytic moiety of the F₁–ATPase (EC 3,6,1,3) was purified from the isolated mitochondria. The yield was 5 mg of F₁–ATPase from 150 mg mitochondria. The F₁–ATPase contained five polypeptides of apparent molecular mass 54 kDa (α), 52 kDa (β), 33 kDa (γ), 22 kDa (ω) and 11 kDa (ɛ). An additional component at 24 kDa was present in variable amounts in some preparations and was therefore not ascribed to the ATPase complex. The enzyme catalyzed ATP hydrolysis at a rate of 12.5 nmol (mg protein)^-1 min^-1. Antibodies against the spinach mitochondrial F₁–ATPase cross–reacted only with the a and β subunits of F₁–ATPases of spinach chloroplasts, photosynthetic bacteria Rhodospirillum rubrum and beef heart mitochondria.     

SUBCELLULAR FRACTIONS OF NORMAL HUMAN SUBSTANTIA NIGRA AND CAUDATE NUCLEUS; A STUDY OF THEIR MORPHOLOGY AND SOME ENZYMES INCLUDING GLUTAMATE DECARBOXYLASE AND CHOLINE ACETYLTRANSFERASE

Abstract— Subcellular fractions have been prepared from normal human caudate nucleus and substantia nigra by a standard fractionation technique and the fractions assayed for the following enzymes, which were studied because of their relevance to neurotransmission and pathological change: glutamate decarboxylase (EC 4.1.1.15), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), acid phosphatase (EC 3.1.3.2) and succinate dehydrogenase (EC 1.3.99.1). The distribution of these enzymes was assessed in relation to the morphology of the fractions as observed by electron microscopy. As with preparations from animal cerebral cortex, acetylcholinesterase and acid phosphatase were found mainly in fractions known to contain plasma membranes, synaptosomal membranes and microsomes. The levels of choline acetyltransferase in fractions from the substantia nigra were too low to measure but, in the caudate nucleus, the enzyme was concentrated in the crude mitochondrial fraction (P₂), especially in the P₂B and P₂C subfractions. A high proportion of the glutamate decarboxylase activity was present in the P₂ fractions of the substantia nigra and caudate nucleus and, although the synaptosomal (P₂B) fraction contained the enzyme, significant amounts were found in the mitochondrial (P₂C) fraction. This may have been due to some contamination of the mitochondria with small synaptosomes. Succinate dehydrogenase showed a conventional bimodal distribution between synaptosomes and mitochondria with a concentration in the latter.     

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons

This study examines the role of c- jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H₂O₂ resulted in activation (phosphorylation) of JNK (mostly p46^JNK1) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E_1α subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Alternative oxidase from Arum and soybean: Its stabilization during purification

Complete purification of the alternative oxidase from plant mitochondria has not been achieved successfully, because of its instability on solubilization. We report here that the addition of pyruvate to the isolation medium stabilizes the activity of the solubilized enzyme. A procedure is described for the rapid isolation and partial purification of the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon ( Glycine max ) mitochondria. The degree of purification was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen consumed mg⁻¹ protein min⁻¹ (using duroquinol as substrate) after purification for the Arum erizyme and from 6 to 445 nmol oxygen consumed mg⁻¹ protein min⁻¹ for the soybean enzyme. A turnover for the partially purified Arum enzyme was estimated to be 47 electrons s⁻¹.
The partially purified enzyme from both Arum and soybean cotyledon mitochondria was sensitive to alternative oxidase inhibitors such as salicylhydroxamic acid, n -propyl gallate and octyl gallate, but not to myxottriazol, KCN or antimycin A. The activity of the enzyme could be stimulated by pyruvate, but not by malate and suceinate. The stability of the purified enzyme was also dependent on the continued presence of pyruvate. In the absence of pyruvace, the enzyme activity was lost in a time-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Effect of alcohols on the association of photosynthetic fructose-1,6-bisphosphatase to thylakoid membranes

Aliphatic alcohols have a positive effect on the assoociation of pea ( Pisum sativum L. cv. Lincoln) chloroplast fructose- 1,6-bisphosphatase (FBPase; EC 3.1.3.11) with thylakoid membranes. The alcohol concentration needed to obtain a fixed percentage of enzyme association decreased with increased length of the aliphatic chain of the alcohol; maximum binding was obtained when the lysis medium contained, in molar fractions (or v/v percentages): 48×10^-4(T4 (2.4%), 26×10^-3 (10%), 40×10^-3 (15%), 76×10^-3 (21%), and 13×10^-2 (24%), of 1-butanol, 1-propanol, 2-propanol, ethanol, and methanol, respectively. A good correlation of binding with the octanol/water partition coefficient was observed. Since this coefficient constitutes a measure of hydrophobicity, we suggest that the binding of FBPase to the membranes occurs via hydrophobic clusters of both components.     

Effects of Aluminum and Calcium on Acetyl-CoA Metabolism in Rat Brain Mitochondria

Abstract: Al complexes are known to accumulate in extra- and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca²⁺_total effects were 10–20 times stronger than those of Al. Al decreased mitochondrial acetyl-CoA content in a concentration-dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of P_i in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl-CoA content in Ca-free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca-evoked suppression of mitochondrial metabolism. On the other hand, in Ca-supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil-evoked rise of Ca²⁺_total level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO₄)OH⁻ complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca²⁺]_total resulting from the Al-evoked rise of cytoplasmic [Ca²⁺]_free, as well as from inhibitory interference with the verapamil binding site on the Na⁺/Ca²⁺ antiporter.     

Glycolate metabolism in algal chloroplasts: inhibition by salicylhydroxamic acid (SHAM)

Unicellular green algae such as Chlamydomonas and Dunaliella excrete small amounts of glycolate during active photosynthesis. This phenomenon has been explained by the fact that these algae do not have leaf-type peroxisomes and glycolate oxidase; instead, they have a limited capacity to metabolise glycolate in their mitochondria by a membrane-associated glycolate dehydrogenase. Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase in plant and algal mitochondria, stimulates glycolate excretion by the algae or their isolated chloroplasts 5-fold. In the presence of SHAM, cells of Chlamydomonas or Dunaliella grown with high-CO₂ (5% CO₂ in air, v/v) or adapted with air levels of CO₂ excreted glycolate at a rate of about 14 µmol glycolate mg⁻¹ Chl h⁻¹. Aminooxyacetate (AOA), an inhibitor of aminotransferases, also increases glycolate excretion by the algal cells or chloroplasts but at a lower rate (about 50%) than SHAM. The algal, light dependent, SHAM-sensitive glycolate oxidizing system in the chloroplasts appears to be the primary site for glycolate oxidation, and it is different and more active then the minor mitochondrial glycolate dehydrogenase.     

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber ( Cucumis sativus L.) and tomato ( Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate–glutathione (AsA–GSH) cycle enzymes and the production of H₂O₂ in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA–GSH cycle enzymes followed by an increased H₂O₂ accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.

Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.

     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Alterations in the pyruvate dehydrogenase complex during adaptation to glucose by Neurospora

A 20-fold induction of the pyruvate dehydrogenase complex, pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoate S-acetyltransferase, (lipoyltransacetylase) (EC 2.3.1.12) plus dihydrolipoyl dehydrogenase, NADH : lipoamide oxidoreductase, (EC 1.6.4.3), from a specific activity of 3.5–65.0 was observed in mitochondrial extracts during adaptation of Neurospora to glucose from acetate media. The extent of ATP-dependent, time-dependent inactivation of the pyruvate dehydrogenase complex was approximately the same in both acetate- and glucose-grown cells, thereby indicating that the low pyruvate dehydrogenerase complex activities in acetate-grown cells did not represent phosphorylated pyruvate dehydrogenase complex molecules. High levels of dihydrolipoyl transacetylase (EC 2.3.1.12) were observed in mitochondrial extracts from acetate-grown cells; this lipoyltransacetylase was analyzed on sucrose density gradients and found to be associated with the pyruvate dehydrogenase complex. Digitonin fractionation of mitochondria revealed that both the pyruvate dehydrogenase complex and lipoyltransacetylase were primarily associated with the mitochondrial outer membrane.     

Distribution of ATPases in wheat root membranes separated by phase partition

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots ( Tritium aestivum L . cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g , 10,000 g , 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two-phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter-current distribution id a 6.3/6.3% two-phase system. Protein and activities of Ca²⁺, Mg²⁺, and Mn²⁺ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca²⁺, Mg²⁺ or Mn²⁺ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3-system was selected for further separation of the membranes in the 30 KP fraction by counter-current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.     

Changes in Enzyme Regulation during Growth of Maize: III. Intracellular Localization of Homoserine Dehydrogenase in Chloroplasts

Extracts of leaf tissue of Zea mays L. seedlings were fractionated on nonlinear sucrose gradients to separate subcellular organelles. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact chloroplasts, as judged by the presence of chlorophyll and triosephosphate isomerase activity. Neither enzyme activity was detected in fractions containing ruptured chloroplasts, mitochondria, or microbodies. Quantitative measurements of enzyme activity and chlorophyll, and electron microscopic analysis of plastid preparations support the conclusion that maize mesophyll chloroplasts contain a significant fraction of the total cellular content of homoserine dehydrogenase.     

Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures

By reducing the concentration of nitrogen (from 5.0 to 2.5 mmol 1^-1), batch cultures of Xanthomonas campestris induced the enzyme UDP-glucose dehydrogenase and stimulated the Entner-Doudoroff pathway enzyme glucose-6-P dehydrogenase. The surplus energy generation was directed to xanthan biosynthesis resulting in a 10% polysaccharide increase. The nitrogen restriction led to a higher consumption of nitrogen (93%) whereas glucose consumption did not surpass 75% utilization. Low concentrations of both magnesium and sulphur exerted a negative effect on xanthan formation. Both restrictions reduced the phosphomannose isomerase enzyme activity by 10-fold turning the mannose transference presumably into the rate-limiting step for xanthan biosynthesis. Conversely, the rate of synthesis of glucuronic acid residues did not affect the rate of xanthan biosynthesis. Polysaccharide synthesis in magnesium and sulphur cultures was negatively affected in comparison with cell formation as the cell volumetric production rate increased from 0.037 to 0.091 g 1^-1 h^-1 and the xanthan volumetric production rate dropped from 0.133 g 1^-1 h^-1 to the minimum obtained at 0.083 g 1^-1 h^-1. The efficiency of the carbon substrate conversion was also greatly changed.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.