Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria

Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases.     

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.     

Thermostable NAD+-dependent (R)-specific secondary alcohol dehydrogenase from cholesterol-utilizing Burkholderia sp. AIU 652

An alcohol dehydrogenase produced by Burkholderia sp. AIU 652, which was isolated with a cholesterol medium, was purified to homogeneity and characterized. The enzyme had broad substrate specificity, and the best reaction was the reversible oxidation of 2-propanol to acetone and 2-butanol to 2-butanone. The K(m) values for secondary alcohols and ketones were much lower than those for primary alcohols or diols. In addition, the enzyme oxidized R-(-)-alcohols in preference to S-(+)-alcohols, and utilized NAD+, but not NADP+ as the cofactor. The molecular mass was 150 kDa with four identical subunits, and the activity was inhibited by o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha'-dipyridyl. Thus, this enzyme was classified into a group of NAD+-dependent R-(-)-specific secondary alcohol dehydrogenases. However, this enzyme was better than the previously reported NAD+-dependent R-(-)-specific secondary alcohol dehydrogenases for chiral chemical synthesis in terms of substrate specificity, stereospecificity, and thermostability. This enzyme might be applicable as an effective biocatalyst for the production of chiral alcohols and related compounds.     

Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)‐isocitrate dehydrogenase subunit I‐deficient mutant of Saccharomyces cerevisiae

A cDNA library was prepared from Histoplasma capsulatum strain G‐217B yeast cells and an apparently full‐length cDNA for a subunit of the citric acid cycle enzyme NAD(+)‐isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)‐isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)‐isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle. The HcIDH1 cDNA sequence is available in GenBank with Accession No. AF009036. Copyright © 1999 John Wiley & Sons, Ltd.     

柠檬醛对酸腐菌线粒体形态和功能的影响

本研究探讨了柠檬醛对酸腐菌线粒体形态、三磷酸腺苷（adenosine triphosphate,ATP）合成和三羧酸（tricarboxylic acid cycle,TCA）循环的影响。扫描电子显微镜结果显示,柠檬醛处理后,酸腐菌线粒体出现扭曲、坍塌甚至破裂的现象。酸腐菌线粒体结构的破坏导致其胞内ATP流失,胞外ATP含量增加。经最小抑菌浓度和最小杀菌浓度的柠檬醛处理后,酸腐菌TCA循环中柠檬酸合酶、α-酮戊二酸脱氢酶、异柠檬酸脱氢酶、琥珀酸脱氢酶和苹果酸脱氢酶活力以及柠檬酸含量都呈下降趋势。结论：柠檬醛处理影响了酸腐菌线粒体的形态和功能,从而抑制其生长。     

Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii

In the genome data base of the hyperthermophilic archaeon Pyrococcus horikoshii, an open reading frame with sequence homology to a gene encoding alcohol dehydrogenase was found. It was demonstrated that the encoded enzyme was a thermostable L-threonine dehydrogenase which can oxidize the hydroxy alkyl residue of L-threonine associated with the reduction of NAD+ or NADP+. This enzyme is a member of the zinc-containing L-threonine dehydrogenase family. One enzyme molecule contained one zinc atom, and this metal was considered to contribute to the hyperthermostablility of the enzyme. The reaction of the enzyme proceeded via a sequential mechanism. The Michaelis constants (Km) for L-threonine and NAD+ were 0.013 and 0.010 mM, respectively, and the maximum reaction rate (Vmax) was 1.75 mmol NADH formed/min/mg-protein at 65 degrees C. The Km values for both L-threonine and NADP+ were larger than those for L-threonine and NAD+ with a similar Vmax value. These results indicate that the enzyme has lower affinity to NADP+ than to NAD+, and the binding affinity for L-threonine depends on the coenzymes.     

酿酒酵母线粒体NADH激酶功能相关表型研究

NADH激酶是辅酶NADP(H)从头合成的关键途径。酿酒酵母中由POS5基因编码的线粒体NADH激酶是线粒体NADPH供应的重要来源之一。由IDP1基因编码的一种关键的NADP+依赖性脱氢酶也能够供应线粒体NADPH。通过对POS5单缺失体、IDP1单缺失体、POS5IDP1双缺失体关键的表型研究,包括它们的生长性能、温度敏感性、对非发酵性碳源的利用能力、线粒体及细胞质内代表性氨基酸的合成性能,初步解析酿酒酵母线粒体中NADPH的主要供应方式及NADH激酶在线粒体中的功能。     

NADP-linked dehydrogenases in secreted milk

The activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malic enzyme, lactate dehydrogenase and malate dehydrogenase have been determined in secreted milk from sows, rats and rabbits. Within each species, although there was considerable variation in the absolute activities of these enzymes, the relative activities were similar to those observed for, or previously published for mammary homogenates. The only exception was milk glucose 6-phosphate dehydrogenase which tended to lose activity upon prolonged storage in the mammary gland. These results suggest that the pattern of milk enzymes can be an accurate reflection of that occurring in the mammary gland.     

Purification and properties of betaine aldehyde dehydrogenase with high affinity for NADP from Arthrobacter globiformis

Betaine aldehyde dehydrogenase from Arthrobacter globiformis was purified to apparent homogeneity by ammonium sulfate fractionation, followed by ion-exchange, butyl-Toyopearl and gel filtration chromatography. The enzyme was found to be a tetramer with identical 55 kDa subunits. Both NAD+ and NADP+ could be used as a cofactor for the enzyme and Michaelis constants (K(m) value) for NAD+ and NADP+ were 1075 microM and 48 microM, respectively. The enzyme was highly specific for betaine aldehyde and the K(m) value for betaine aldehyde was 36 microM.     

Cytosolic redox metabolism in aerobic chemostat cultures of Saccharomyces cerevisiae

Cytosolic redox balance has to be maintained in order to allow an enduring cellular metabolism. In other words, NADH generated in the cytosol has to be re-oxidized back to NAD(+). Aerobically this can be done by respiratory oxidation of cytosolic NADH. However, NADH is unable to cross the mitochondrial inner membrane and mechanisms are required for conveying cytosolic NADH to the mitochondrial electron transport chain. At least two such systems have proved to be functional in S. cerevisiae, the external NADH dehydrogenase (Luttik et al., 1998; Small and McAlister-Henn, 1998) and the G3P shuttle (Larsson et al., 1998). The aim of this investigation was to study the regulation and performance of these two systems in a wild-type strain of S. cerevisiae using aerobic glucose- and nitrogen-limited chemostat cultures. The rate of cytosolic NADH formation was calculated and as expected there was a continuous increase with increasing dilution rate. However, measurements of enzyme activities and respiratory activity on isolated mitochondria revealed a diminishing capacity at elevated dilution rates for both the external NADH dehydrogenase and the G3P shuttle. This suggests that adjustment of in vivo activities of these systems to proper levels is not achieved by changes in amount of protein but rather by, for example, activation/inhibition of existing enzymes. Adenine nucleotides are well-known allosteric regulators and both the external NADH and the G3P shuttle were sensitive to inhibition by ATP. The most severe inhibition was probably on the G3P shuttle, since one of its member proteins, Gpdp, turned out to be exceptionally sensitive to ATP. The external NADH dehydrogenase is suggested as the main system employed for oxidation of cytosolic NADH. The G3P shuttle is proposed to be of some importance at low growth rates and perhaps its real significance is only expressed during starvation conditions.     

Effects of polyunsaturated fatty acids on Krebs cycle in the rat kidney in chronic phosphorus intoxication]

The investigation of Krebs cycle state in kidney homogenates of August rats subjected to oral intoxication with oil solution of yellow phosphorus in a dose of 0.3 mg/kg, has shown that under conditions of balanced nutrition the activity of NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase and accumulation of the substrate fund of the cycle decreased 3.5-fold as compared to the control. The addition of polyunsaturated fatty acids to the ration produced a positive effect on Krebs cycle state: dehydrogenase activity was not significantly changed, accumulation of Krebs cycle substrate was two-fold lower. However, this ration did not completely abolish the toxic action of yellow phosphorus on Krebs cycle.     

Fluorometric determination of free and total isocitrate in bovine milk

Isocitrate is an intermediate metabolite in the citric acid cycle found both inside the mitochondria as well as outside in the cytosolic shunt. Oxidation of isocitrate is believed to deliver large fractions of energy [i.e., reducing equivalents (NADPH) in the bovine udder] used for fatty acid and cholesterol synthesis. This study describes a new analytical method for determination of free and total isocitrate in bovine milk where time-consuming pretreatment of the sample is not necessary. Methods for estimation of both total isocitrate and free isocitrate are described, the difference being the esterified or even lactonized isocitrate. On average, 20% (6–27%) of cow milk isocitrate was esterified and free isocitrate correlated significantly with total isocitrate (r = 0.98). The present fluorometric determination correlated well with the traditional spectrophotometric determination of isocitrate. Milk samples from Danish Holstein cows (984) contained significantly less isocitrate than milk from Danish Jersey cows (760; i.e., 0.134 vs. 0.211 mmol/L). Isocitrate in milk is correlated to milk protein, fat, and citrate, and it is speculated, based on biochemistry, former studies, and the present, that isocitrate may reflect the energy situation in the mammary gland. The use of isocitrate as a biomarker of the energy status in the dairy cow is warranted.     

茶树精油对扩展青霉线粒体功能的影响

为了揭示茶树精油(tea tree oil,TTO)对果蔬采后病原真菌扩展青霉(Penicillium expansum)的抑菌作用机制,以TTO处理的P.expansum孢子、菌丝和线粒体为研究对象,测定活性氧(reactive oxygen species,ROS)积累及6种线粒体功能相关酶类的活力变化,并采用扫描...     

Purification and characterization of the alcohol dehydrogenase with a broad substrate specificity originated from 2-phenylethanol-assimilating Brevibacterium sp. KU 1309

A novel 2-phenylethanol dehydrogenase has been purified from a soil bacterium Brevibacterium sp. KU 1309. The enzyme was purified about 1400-fold to homogeneity, and found to be a monomeric enzyme of apparent 39 kDa. The enzyme had broad substrate specificity and catalyzes a reversible oxidation of various primary alcohols to aldehydes. The enzyme required NAD+, but not NADP+ as a cofactor. Thus, the enzyme was classified into a group of NAD+-dependent primary alcohol dehydrogenase. The activity was inhibited by Cu2+, Ni2+, Ba2+, Hg2+ and p-chloromercuribenzoate. The enzyme is expected to be applicable as an effective biocatalyst in the oxidation of various alcohols.     

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.     

Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans

3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.     

Quantification of progesterone binding in mammary tissue of pregnant ewes

Progestin-binding sites in mammary tissue from 14 prepartum, multiparous ewes at 50, 80, 115, and 140 d of gestation were demonstrated by the binding of [3H] R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) to ovine mammary cytosol in the presence of sodium molybdate and excess cortisol. Homogenization extracted 89% of total mammary receptors (nuclear) into cytosol. Binding was specific for progestins and was of high affinity. The average dissociation constant for [3H] R5020 specifically bound to receptors extracted into mammary cytosol was 1.9 (+/- .4) X 10(-9) M (n = 14) and did not change significantly over the test period. However, binding capacities (fmol/mg cytosolic protein) differed according to stage of gestation with averages of 125 +/- 53, 149 +/- 26, 656 +/- 216, 57 +/- 22 at 50, 80, 115, and 140 d of pregnancy, respectively. Increased number of progestin-binding sites at 115 d of gestation (whether data are expressed per unit of tissue weight, DNA, or cytosolic protein) suggests that an increase per mammary epithelial cell may be necessary to produce the full lobuloaveolar proliferation observed at this stage of gestation.     

Graduate Student Literature Review: Mitochondrial adaptations across lactation and their molecular regulation in dairy cattle*

As milk production in dairy cattle continues to increase, so do the energetic and nutrient demands on the dairy cow. Difficulties making the necessary metabolic adjustments for lactation can impair lactation performance and increase the risk of metabolic disorders. The physiological adaptations to lactation involve the mammary gland and extramammary tissues that coordinately enhance the availability of precursors for milk synthesis. Changes in whole-body metabolism and nutrient partitioning are accomplished, in part, through the bioenergetic and biosynthetic capacity of the mitochondria, providing energy and diverting important substrates, such as AA and fatty acids, to the mammary gland in support of lactation. With increased oxidative capacity and ATP production, reactive oxygen species production in mitochondria may be altered. Imbalances between oxidant production and antioxidant activity can lead to oxidative damage to cellular structures and contribute to disease. Thus, mitochondria are tasked with meeting the energy needs of the cell and minimizing oxidative stress. Mitochondrial function is regulated in concert with cellular metabolism by the nucleus. With only a small number of genes present within the mitochondrial genome, many genes regulating mitochondrial function are housed in nuclear DNA. This review describes the involvement of mitochondria in coordinating tissue-specific metabolic adaptations across lactation in dairy cattle and the current state of knowledge regarding mitochondrial-nuclear signaling pathways that regulate mitochondrial proliferation and function in response to shifting cellular energy need.     

Glucocorticoid metabolism and reproduction: a tale of two enzymes

Within potential target cells, the actions of physiological glucocorticoids (cortisol and corticosterone) are modulated by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To date, two isoforms of 11 beta HSD have been cloned: 11 beta HSD1 acts predominantly as an NADP(H)-dependent reductase to generate active cortisol or corticosterone, and 11 beta HSD2 is a high affinity NAD(+)-dependent enzyme that catalyses the enzymatic inactivation of glucocorticoids. Whereas the regeneration of active glucocorticoids by 11 beta HSD1 has been implicated in the cellular mechanisms of pituitary function, ovulation and parturition, the enzymatic inactivation of cortisol and corticosterone by 11 beta HSD enzymes appears to be central to the protection of gonadal steroidogenesis, prevention of intra-uterine growth retardation, and lactation. Recent evidence indicates that follicular fluid contains endogenous modulators of cortisol metabolism by 11 beta HSD1, the concentrations of which are associated with the clinical outcome of assisted conception cycles and are altered in cystic ovarian disease. In conclusion, the two cloned isoforms of 11 beta HSD fulfil diverse roles in a wide range of reproductive processes from conception to lactation.     

Alterations in placental 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) activities and fetal cortisol:cortisone ratios induced by nutritional restriction prior to conception and at defined stages of gestation in ewes

In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD(+)-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.