Some kinetic and regulatory properties of the pea mitochondrial pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg²⁺. Reaction velocity was maximal at 40°C. Pyruvate was a better substrate than 2-oxo-butyrate; other 2-oxo-acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar; NAD, 122 micromolar; Coenzyme-A, 5 micromolar; Mg²⁺, 0.36 millimolar; Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Intracellular Localization and Properties of NADH-Glutamate Dehydrogenase from Vitis vinifera L.: Purification and Characterization of the Major Leaf Isoenzyme

Glutamate dehydrogenase was partially purified from grapevine(Vitis vinifera L. cv. Soultanina) tissues and its activityand isoenzymic pattern were studied. Seven anodal migratingisoenzymes were revealed after PAGE. Leaf protoplasts were isolatedfrom in vitro-grown axenic shoot cultures and used to studythe intracellular localization of GDH. Results revealed thatthe enzyme was associated with the mitochondrial fraction. Theisoenzyme with the lowest electrophoretic mobility, which accountedfor 35 to 40% of total activity, was purified 2050-fold to homogeneityfrom leaves. The purification method included ammonium sulphatefractionation, DEAE-cellulose chromatography, Sephadex G-200gel filtration and NAD-sepharose affinity chromatography. Themolecular weight of the native enzyme was estimated to be 252kDa and it consisted of identical 42.5 kDa subunits. pH optimumfor the aminating reaction was 8.0 and for the deaminating reaction9.3. At optimum pH conditions the apparent K_m values for ammonium,as ammonium chloride and ammonium sulphate, -ketoglutarate,NADH, glutamate, and NAD⁺ were 45.0, 13.0, 2.1, 0.069, 18.0,and 0.195 mM, respectively. The amination reaction of GDH wasfully activated with about 100 µM Ca²⁺ while the deaminationreaction was not affected by the addition of Ca²⁺. The isoenzymesof GDH showed different magnitude of their activating responseto calcium ions. Key words: Vitis vinifera L., glutamate dehydrogenase     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)     

Purification and Properties of Phosphoenolpyruvate Carboxylase from Immature Pods of Chickpea (Cicer arietinum L.)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg²⁺ ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a K_m of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO₃⁻ with S_0.5 value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, α-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca²⁺ and Mn²⁺. Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Metabolism of calcium and effect of divalent cations on respiratory activity of yeast mitochondria

The adsorption of Ca²⁺ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca²⁺ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca²⁺ and Mg²⁺ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca²⁺ in the mitochondria ofTorulopsis utilis and with Co²⁺ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn²⁺ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd²⁺ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca²⁺ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.     

Soluble,Prefibrillar α-Synuclein Oligomers Promote Complex I-dependent,Ca2+-induced Mitochondrial Dysfunction

α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca²⁺ oscillations that burden mitochondria, we examined mitochondrial Ca²⁺ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca²⁺, promoted Ca²⁺-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca²⁺. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca²⁺ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca²⁺-handling requirements.     

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)⁺-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO⁻ aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd²⁺, Co²⁺, Hg²⁺, Mg²⁺, Pb²⁺ and Zn²⁺ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD⁺-GDH activity was negligible. Among the applied ions, only Ca²⁺ activated NADH-GDH.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Polyamine uptake in carrot cell cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca²⁺. The effects of Ca²⁺ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca²⁺ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca²⁺ concentration range between 10 micromolar and 1 millimolar. La³⁺ nullified the stimulatory effect of 10 micromolar Ca²⁺ on putrescine uptake and that of 1 millimolar Ca²⁺ on spermidine uptake. La³⁺ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.     

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca²⁺ or Mg²⁺. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca²⁺ and Mg²⁺. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca²⁺ and Mg²⁺, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg²⁺. Kinetic constants (apparent K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO₃⁻, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.     

Purification of the channel component of the mitochondrial calcium uniporter and its reconstitution into planar lipid bilayers

The purification of the channel-forming component of the mitochondrial calcium uniporter and its channel properties are described. After ethanol and 50% ethanol-water extraction of mitochondria from beef heart or perfused rat liver, the extract was passed through thiopropyl-Sepharose 6B column, and absorbed components were eluted with 2-mercaptoethanol, followed by gel-filtration on Sephadex G-15. The last fraction eluted (M _r about 2000) was then subjected to reverse-phase high-performance liquid chromatography. Of the more than 10 distinct peaks, only one showed specific Ca²⁺-channel activity in BLM with properties similar to earlier, less extensively purified preparations, i.e., conductance of 20 pS and multiples thereof, clustering of channels, participation of 2 or more subunits in channel formation, and sensitivity to 1 µM ruthenium red. Voltage sensitivity and cooperativity between channels are described. The Ca²⁺-binding glycoprotein with which the peptide was associated was found to have high homology with human acid ₁-glycoprotein (orosomucoid) and to show identity with beef plasma orosomucoid in the Ouchterlony immunodiffusion test.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.