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Query: UNIPROT:P06889 (Mol)
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Lactate dehydrogenase (LDH) from Plasmodium falciparum was partially purified by two different procedures. In the first procedure, parasitized erythrocytes (80% parasitemia) were lysed, and the soluble fraction was purified on DEAE-Sephadex to separate the parasite LDH(LDH-P) from the LDH isoenzymes present in the human erythrocytes. LDH-P was then purified by high-performance liquid chromatography (HPLC) on a TSK-G-3000 SW protein column. This two-step procedure gave LDH-P with specific activity 85 micromol/min/mg protein; this represented a 700-fold increase in specific activity relative to the starting lysate. Alternatively, parasites of P. falciparum were isolated by mechanical rupture of infected erythrocytes followed by differential centrifugation. The 100,000 X g supernatant obtained after lysis of these parasites showed LDH-P specific activity 3.6 micromol/min/mg protein. This activity was free of contaminating erythrocyte LDH as determined by electrophoresis and specific staining for LDH. Further purification of LDH-P by HPLC, as before, gave material with specific activity 98 micromol/min/mg protein. Recoveries of activity on HPLC were more than 90%, demonstrating the usefulness of this procedure for the partial purification of small quantities of parasite protein. The kinetic properties of LDH-P were compared with those of two of the human isozymes, LDH-H4 and LDH-M4 . LDH-P resembles LDH-H4 in its kinetic properties: KM (NADH) is 7, 8.3 and 1.3 microM for LDH-P, LDH-H4 and LDH-M4, respectively; KM (pyruvate) is 30, 60 and 180 microM for LDH-P, LDH-H4 and LDH-M4. LDH-P differs significantly from LDH-H4 and LDH-M4 in that LDH-P is not sensitive to inhibition by high pyruvate nor sensitive to inhibition by the complex between NAD+ and pyruvate. LDH-P is inactivated within seconds by sodium deoxycholate at concentrations that do not affect LDH-H4 and slowly inactivate LDH-M4.
Mol Biochem Parasitol 1981 Dec 31
PMID:Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum. 703 78

Bioluminescence photokinetic assay of NADP+ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. the analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP+ as measurable product and for nucleotide assay in cell samples of 0.5--10 microgram dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP+. Previously, FMN, NADH, NAD+ and NADH have been analysed with the bacterial luciferase system. Its applicability has not been extended by the assay of NADP+.
Mol Cell Biochem 1980 Aug 29
PMID:Photokinetic microanalysis of NADP+, using bacterial luciferase. 744 56

NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.
Mol Cell Biochem 1980 Nov 20
PMID:Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification. 746 25

We have cloned and characterized a homologue of the previously isolated GPD1 gene, encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae. This second gene, called GPD2, encodes a protein of 384 amino acids that shares 69% sequence identity with GPD1. Like GPD1 it has an amino-terminal extension of unknown function. GPD2 is located on chromosome VII and cross-hybridizes with GPD1 at chromosome IV as well as with an unknown homologue at chromosome XV. Disruption of the GPD2 gene did not reveal any observable phenotypic effects, whereas overexpression resulted in a slight, but significant, increase of GPD enzyme activity in wild-type cells. Analysis of gene transcription by a CAT-reporter gene fused to the GPD promoters revealed decreased transcriptional activity of the GPD2 promoter in cells grown on nonfermentable as opposed to fermentable carbon sources, and no induction in cells exposed to high osmolarity or heat shock. Similar analysis of GPD1 demonstrated an 8-17-fold higher basal level of transcription compared to GPD2. Furthermore, such analysis revealed that the GPD1 promoter was induced by increased osmolarity essentially independent of the type of stress solute used, the level of GPD1 transcription being increased about sevenfold in cells growing at 1.4 M NaCl.
Mol Microbiol 1995 Jul
PMID:Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. 747 12

The effect of nuclear Ca2+ uptake inhibitors on the Ca(2+)-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca2+ (40 microM) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca(2+)-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca2+ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 microM arachidonic acid, 2.0 mM NAD+, 10 microM zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca(2+)-ATPase activity and Ca2+ uptake in the nuclei, produced a significant inhibition of the Ca(2+)-activated DNA fragmentation in the nuclei. The results show that the Ca(2+)-activated DNA fragmentation is involved in the uptake of Ca2+ by the nuclei, suggesting a role of Ca2+ transport system in the regulation of liver nuclear functions.
Mol Cell Biochem 1995 Jul 05
PMID:Effect of nuclear Ca2+ uptake inhibitors on Ca(2+)-activated DNA fragmentation in rat liver nuclei. 747 31

In rat pancreatic islets, D-glucose in concentrations exceeding 5.6 mM caused a concentration-related decrease of the mitochondrial NADH/NAD+ ratio, as judged from the changes in the islet content of glutamate, NH4+, and 2-ketoglutarate, and assuming that the glutamate dehydrogenase reaction is near equilibrium with the mitochondrial NAD system. The concentration dependency of the response to D-glucose was vastly different in islet and parotid cells, respectively. L-Leucine, 2-ketoisocaproate, BCH (a nonmetabolized but insulinotropic analog of L-leucine) and 3-phenylpyruvate also lowered the mitochondrial NADH/NAD+ ratio. In the presence of D-glucose, the latter ratio was also decreased by NH4+ or the absence of extracellular Ca2+, but dramatically increased by aminooxyacetate. Taking into account prior metabolic findings, the nutrient-induced fall in the mitochondrial redox state is thought to reflect an increased clearance of mitochondrial NADH through both the respiratory chain and malate-aspartate shuttle. The nutrient-induced decrease in the mitochondrial NADH/NAD+ ratio might favor both the circulation of metabolites in the Krebs cycle and the exit of Ca2+ from the mitochondria.
Biochem Mol Med 1995 Jun
PMID:Hexose metabolism in pancreatic islets: regulation of the mitochondrial NADH/NAD+ ratio. 755 20

The classical form of the enzyme 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3 beta-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3 beta HSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3 beta HSD I and III function as 3 beta-hydroxysteroid dehydrogenases and 5-en-->4-en isomerases using NAD+ as a cofactor. The enzymatic function of 3 beta HSD II has not been completely characterized. Mouse 3 beta HSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3 beta HSD I is expressed in the gonads and adrenal glands of the adult mouse. 3 beta HSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.
J Steroid Biochem Mol Biol 1995 Jun
PMID:The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function and tissue-specific expression. 762 43

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus, a homotetramer with a molecular mass of 33 kDa per subunit, belongs to the protein family of the NAD(+)-dependent L-2-hydroxycarboxylate dehydrogenases. L-HicDH was crystallized with ammonium sulphate as precipitant in the presence of NAD+. The crystals belong to the trigonal space group P3(2)21, with a = 135.9 A and c = 205.9 A, and diffract X-rays to 2.2 A resolution. The crystal structure was solved by Patterson search and molecular replacement techniques and refined to an R-value of 21.4% (2.2 to 8 A). The final structure model contains one NAD+ molecule and one sulphate ion per subunit, with 309 water molecules. An unusual feature of this crystal structure is the deviation of the protein subunits from non-crystallographic symmetry, which is so strong that it can be detected globally by self-rotation calculations in reciprocal space. This asymmetry is especially pronounced in the environment of the active site; it is reflected also in the nicotinamide conformation of NAD+ and allows some conclusions to be drawn about the catalytic mechanism. In this context, an "inner active site loop" is identified as a structural element of fundamental functional importance. Furthermore, with knowledge of the crystal structure of L-HicDH the differences in substrate specificity between L-HicDH and the L-lactate dehydrogenases can be partly explained.
J Mol Biol 1995 Aug 11
PMID:Crystal structure of L-2-hydroxyisocaproate dehydrogenase from Lactobacillus confusus at 2.2 A resolution. An example of strong asymmetry between subunits. 764 2

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.
J Mol Evol 1995 Jun
PMID:Self-incorporation of coenzymes by ribozymes. 764 6

Glyceraldehyde-3-phosphate dehydrogenase in the archaeon Sulfolobus solfataricus (SsGAPD) has been purified 232 fold with an overall recovery of about 25%. The enzyme is a homomeric tetramer with an M(r) of 41 kDa/subunit. It utilizes either NAD+ or NADP+ as coenzyme but its affinity for the latter is about 50 fold higher. SsGAPD activity is maximum at 87 degrees C. In the range 45-87 degrees C the Arrhenius plot is linear and the activation energy is 55 kJ/mol. The enzyme is thermostable, with a half-life of 45 min at 87 degrees C. The primary structure of SsGAPD shows 35% identity with that of other archaeal GAPDs. Its N-domain shows sequence motifs typical of the dinucleotide binding proteins while the catalytic C-terminal region contains a cysteine residue (C140), required for catalysis, that is conserved in all the archaeal, eukaryal and bacterial GAPDs. These remarks suggest that archaeal GAPDs show a convergent molecular evolution to the eukaryal and eubacterial enzymes in the catalytic region.
Biochem Mol Biol Int 1995 May
PMID:Glyceraldehyde-3-phosphate dehydrogenase in the hyperthermophilic archaeon Sulfolobus solfataricus: characterization and significance in glucose metabolism. 766 6


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