Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

A possible role for acid phosphatase with thiamin-binding activity encoded by PHO3 in yeast

Periplasmic soluble thiamin-binding protein in Saccharomyces cerevisiae (Iwashima, A. et al. (1979) Biochim. Biophys. Acta 577, 217-220) was demonstrated to be encoded by PHO3 gene that codes for thiamin repressible acid phosphatase (Schweingruber, M.E. et al. (1986) J. Biol. Chem. 261, 15877-15882) by genetic analysis. The pho3 mutant cells of S. cerevisiae in contrast to the parent cells have markedly reduced activity of the uptake of [14C]thiamin phosphates, suggesting that thiamin repressible acid phosphatase plays a role in the hydrolysis of thiamin phosphates in the periplasmic space prior to the uptake of their thiamin moieties by S. cerevisiae.     

硫胺素通过提高线粒体氧化状态促进植物快速响应外界胁迫的研究

硫胺素在植物应对环境胁迫刺激及植物免疫方面发挥的作用越来越受到人们的关注。本文主要研究了硫胺素对线粒体氧化还原状态的调节作用。研究发现硫胺素可以提高线粒体的氧化状态,这一效应具有浓度依赖性,当硫胺素浓度高于1mmol／L时较为明显。此外,硫胺素还可增强植物细胞线粒体丙酮酸脱氢酶（PDH）的活性。并且在应对铝、镉胁迫时,硫胺素预处理组能较快促进活性氧的进发。线粒体氧化状态的提高对细胞应对胁迫因子刺激,较快释放活性氧从而激活下游信号具有重要意义。     

Photoinactivation of the thiamin transport system in Saccharomyces cerevisiae with azidobenzoyl derivatives of thiamin

In an attempt to obtain a potent inhibitor for thiamin transport of Saccharomyces cerivisiae three novel thiamin derivatives having an arylazido substituent in the thiazole moiety have been synthesized. The derivatives prepared were 4-azidobenzoylthiamin (ABT), 4-azidobenzoylthiamin disulfide (ABTD), and 4-azido-2-nitrobenzoylthiamin disulfide (ANBTD). Among the newly prepared photoreactive azidobenzoyl derivatives of thiamin, ANBTD showed the strongest competitive inhibition with an apparent Ki of 7.9 nM against thiamin uptake by S. cerevisiae IFO-2375. The Ki values for ABT, 4-azido-2-nitrobenzoylthiamin (ANBT), and ABTD were 187 nM, 83 nM, and 15 nM, respectively. When exposed to visible light, ANBTD inactivated in a time- and concentration-dependent manner the uptake of [14C]thiamin by yeast protoplasts as well as intact cells. Remaining activities of the thiamin uptake by the intact cells were 71.9%, 27.3%, 40.1%, and 15.0% after visible light irradiation for 15 min in the presence of 1 microM ABT, ANBT, ABTD, and ANBTD, respectively. The inactivation by ANBTD (0.05 microM) was partially prevented by previous addition of an excessive amount of thiamin (5 microM). Furthermore, it was found that ANBTD (0.5 microM) irreversibly inactivated 70.6% of the thiamin-binding activity of the membrane fraction from S. cerevisiae IFO-2375. These results suggest that ANBTD can inhibit yeast thiamin transport by photoinactivation of membrane-bound thiamin-binding protein in the plasma membrane which may be a functional component involved in the thiamin transport system of S. cerevisiae.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

Relationship between thiamin phosphorylation and intestinal transport in vitro: effect of chloroethylthiamin.

The effect of Chloroethylthiamin (CET), a structural analog of thiamin, on thiamin pyrophosphokinase (TPKase) activity of the supernatant of rat isolated enterocytes was compared with that on thiamin intestinal transport by rat everted jejunal sacs. Thiamin- thiazol-2(14C) was used as a substrate both for TPKase activity and for thiamin serosal transport and uptake. CET strongly inhibited TPKase activity of isolated enterocytes: at molar concentrations 10 or 100 times higher than labeled thiamin, the inhibition was 57 and 100% respectively. The inhibition was of the competitive type, with a Ki = 15 microM. At a molar concentration 10 times higher than labeled thiamin, CET lowered the thiamin serosal transport by 60%, and the sac wall content of free and phosphorylated thiamin by 54 and 42% respectively. At the 1:10 thiamin: CET molar ratio, the extent of the reductions of TPKase activity (57%) and of phosphorylated thiamin content of intestinal sac walls (42%) were of the same order. This indicates a relationship between the two events. Moreover, since TPKase activity inhibition alone resulted in the lowering of labeled thiamin serosal transport, thiamin phosphorylation and transport are probably two strictly related processes.     

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.     

Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine

The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.     

Mitochondrial Uptake of Thiamin Pyrophosphate: Physiological and Cell Biological Aspects

Mammalian cells obtain vitamin B1 (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. Most of TPP is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (MTPPT). Knowledge about the physiological parameters of the MTPP-mediated uptake process, MTPPT targeting and the impact of clinical mutations in MTPPT in patients with Amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made ³H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed ³H-TPP uptake by mouse liver mitochondria to be pH-independent, saturable (K_m =6.79±0.53 µM), and specific for TPP. MTPPT protein was expressed in mouse liver and HepG2 cells, and confocal images showed a human (h)MTPPT-GFP construct to be targeted to mitochondria of HepG2 cells. A serial truncation analysis revealed that all three modules of hMTPPT protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. Finally, the hMTPPT clinical mutants (G125S and G177A) showed proper mitochondrial targeting but displayed significant inhibition in ³H-TPP uptake and a decrease in level of expression of the MTPPT protein. These findings advance our knowledge of the physiology and cell biology of the mitochondrial TPP uptake process. The results also show that clinical mutations in the hMTPPT system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria.     

Relative contribution of THTR-1 and THTR-2 in thiamin uptake by pancreatic acinar cells: studies utilizing Slc19a2 and Slc19a3 knockout mouse models

Thiamin is essential for normal function of pancreatic acinar cells, and its deficiency leads to a reduction in pancreatic digestive enzymes. We have recently shown that thiamin uptake by rat pancreatic acinar cells is carrier-mediated and that both thiamin transporter (THTR)-1 and THTR-2 are expressed in these cells; little, however, is known about the relative contribution of these transporters toward total carrier-mediated thiamin uptake by these cells. We addressed this issue using a gene-specific silencing approach (siRNA) in mouse-derived pancreatic acinar 266-6 cells and Slc19a2 and Slc19a3 knockout mouse models. First we established that thiamin uptake by mouse pancreatic acinar cells is via a carrier-mediated process. We also established that these cells as well as native human pancreas express THTR-1 and THTR-2, with expression of the former (and activity of its promoter) being significantly higher than that of the latter. Using gene-specific siRNA against mouse THTR-1 and THTR-2, we observed a significant inhibition in carrier-mediated thiamin uptake by 266-6 cells in both cases. Similarly, thiamin uptake by freshly isolated primary pancreatic acinar cells of the Slc19a2 and Slc19a3 knockout mice was significantly lower than uptake by acinar cells of the respective littermates; the degree of inhibition observed in the former knockout model was greater than that of the latter. These findings demonstrate, for the first time, that both mTHTR-1 and mTHTR-2 are involved in carrier-mediated thiamin uptake by pancreatic acinar cells.     

Cellular and molecular aspects of thiamin uptake by human liver cells: studies with cultured HepG2 cells

The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them.     

Ornithine/phosphate antiport in rat kidney mitochondria. Some characteristics of the process

[14C]Ornithine uptake by rat kidney mitochondria has been investigated according to the stop inhibitor method by using praseodimium chloride as an inhibitor. The existence of an ornithine/Pi exchange was found occurring with 1:1 stoichiometry. Both uptake and efflux follow first-order kinetics with a k of 2.4 min-1. Uptake increases with increasing pH. The activation energy for the process is 58.6 kJ/mol and Q10 is 2.6. Ornithine/Pi exchange is electrical and energy-dependent, as suggested by the sensitivity of the process to the ionophores valinomycin and nigericin. Measurements both of proton movement across the mitochondrial membrane and of membrane potential strongly suggest that ornithine uptake into mitochondria is driven by the electrochemical proton gradient via the dependent ornithine/Pi translocator and delta pH-dependent Pi carrier.     

The uptake of oxalate by rat liver and kidney mitochondria

Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.     

Transport and metabolism of thiamin in isolated rat hepatocytes.

This study examines thiamin transport in isolated rat hepatocytes and its relationship to thiamin phosphorylation. In an Na+ medium, [35S]thiamin, 3 microM, was accumulated rapidly by the cells, and a near study state intra-/extracellular distribution ratio of 3 was attained in 1 min. However, the uptake of radioactivity continued to increase with time owing principally to the accumulation of [35S]thiamin pyrophosphate (TPP). In a choline, Li+ or K+ medium, the steady state intra-/extracellular distribution ratio of [35S]thiamin was decreased to less than or equal to 1.1. Accordingly, the rate of formation of [35S]TPP also decreased. Ouabain and uncouplers of oxidative phosphorylation significantly lowered the distribution ratio of intra-/extracellular [35S]thiamin. These data indicate that thiamin transport in liver is concentrative, Na+-dependent, and dependent on biological energy. Additionally, they suggest that thiamin transport plays a significant role in governing the rate of synthesis of TPP. Neither pyrithiamin, an inhibitor of thiamin pyrophosphokinase nor o-benzoylthiamin disulfide, a permeable thiamin analog, affected the distribution ratio of intra-/extracellular [35S]thiamin, but preferentially inhibited the phosphorylation of [35S]thiamin. By contrast, amprolium primarily inhibited uptake. These data suggest that thiamin transport and phosphorylation can be differentiated by the action of appropriate inhibitors.     

Thiamin uptake in Euglena gracilis

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.     

Anthranilic acid release in adenosine-inhibited cultures of Saccharomyces cerevisiae and its inhibition by thiamin

Adenosine, at 1 mM concentrations or above, was found to have a fungistatic effect on Saccharomyces cerevisiae. A substance with amethyst fluorescence was detected in the medium of adenosine-inhibited cultures of S. cerevisiae. This compound was isolated and physicochemically identified as anthranilic acid. Both the inhibition of growth and release of anthranilic acid induced by adenosine were abrogated by thiamin or by the pyrimidine portion of thiamin, 2-methyl-4-amino-5-hdroxymethyl-pyrimidine (hydroxymethyl-pyrimidine); the latter was found to restore intracellular thiamin content that had been reduced by adenosine. It was demonstrated that effects of thiamin and hydroxymethylpyrimidine on S. cerevisiae cultured with adenosine resulted from their inhibition of adenosine uptake by growing yeast cells.     

Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine in Saccharomyces cerevisiae

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) was studied in resting cells of Saccharomyces cerevisiae. Hydroxymethylpyrimidine uptake was an energy- and temperature-dependent process which has an optimal pH at 4.5. The apparent Km for hydroxymethylpyrimidine uptake was 0.37 microM, and the uptake was inhibited by 2-methyl-4-amino-5-aminomethylpyrimidine, thiamin and pyrithiamin. Furthermore, hydroxymethylpyrimidine uptake was inhibited by 4-azido-2-nitrobenzoylthiamin, a specific and irreversible inhibitor of the yeast thiamin transport system and it was greatly impaired in the thiamin transport mutant of S. cerevisiae. Thus, hydroxymethylpyrimidine is taken up by a common transport system with thiamin in S. cerevisiae, but in contrast to thiamin transport, accumulated hydroxymethylpyrimidine is released from yeast cells showing an overshoot phenomenon.