Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates.
(1/865)
1. The activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenases were measured in nervous tissue from different animals in an attempt to provide more information about the citric acid cycle in this tissue. In higher animals the activities of citrate synthase are greater than the sum of activities of the isocitrate dehydrogenases, whereas they are similar in nervous tissues from the lower animals. This suggests that in higher animals the isocitrate dehydrogenase reaction is far-removed from equilibrium. If it is assumed that isocitrate dehydrogenase activities provide an indication of the maximum flux through the citric acid cycle, the maximum glycolytic capacity in nervous tissue is considerably greater than that of the cycle. This suggest that glycolysis can provide energy in excess of the aerobic capacity of the tissue. 2. The activities of glutamate dehydrogenase are high in most nervous tissues and the activities of aspartate aminotransferase are high in all nervous tissue investigated. However, the activities of alanine aminotransferase are low in all tissues except the ganglia of the waterbug and cockroach. In these insect tissues, anaerobic glycolysis may result in the formation of alanine rather than lactate. (+info)
cis-Acting elements responsible for low-temperature-inducible expression of the gene coding for the thermolabile isocitrate dehydrogenase isozyme of a psychrophilic bacterium, Vibrio sp. strain ABE-1.
(2/865)
Transcriptional control of the low-temperature-inducible icdII gene, encoding the thermolabile isocitrate dehydrogenase of a psychrophilic bacterium, Vibrio sp. strain ABE-1, was found to be mediated in part by a transcriptional silencer locating at nucleotide positions -560 to -526 upstream from the transcription start site of icdII. Deletion of the silencer resulted in a 20-fold-increased level of expression of the gene at low temperature (15 degrees C) but not at high temperature (37 degrees C). In addition, a CCAAT sequence located 2 bases upstream of the -35 region was found to be essential for the low-temperature-inducible expression of the gene. By deletion of this sequence, low-temperature-dependent expression of the gene was completely abolished. The ability of the icdII promoter to control the expression of other genes was confirmed by using a fusion gene containing the icdII promoter region and the promoterless icdI open reading frame, which encodes the non-cold-inducible isocitrate dehydrogenase isozyme of Vibrio sp. strain ABE-1. Escherichia coli transformants harboring icdII acquired an ability to grow rapidly at low temperature. (+info)
Purification, properties and enhanced expression under nitrogen starvation of the NADP+-isocitrate dehydrogenase from the cyanobacterium Phormidium laminosum.
(3/865)
Nitrogen starvation enhances up to 8-fold the cellular level of the NADP+-dependent isocitrate dehydrogenase activity (isocitrate:NADP+ oxidoreductase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous non-N2-fixing cyanobacterium Phormidium laminosum. The enzyme was purified 650-fold to electrophoretic homogeneity from nitrogen-starved cells with an activity yield of 25% and a specific activity of 500 U (mg protein)-1. The native enzyme showed a pI of 5.9 and it was a dimer of 107 kDa consisting of two identical subunits of 53 kDa. The activity required the presence of a divalent metal cation as an essential activator, Mn2+ or Mg2+ being the most effective. The optimum temperature for activity was 55 degrees C and the Ea for catalysis was 39.7 kJ mol-1. An optimum pH for activity of 8.5 was found and the calculated pKE1, pKE2 and pKES1 of enzyme ionisation groups were 6.0, 8.9 and 6.3, respectively. Km values of 22, 50 and 24 microM were calculated for d,l-isocitrate, NADP and Mn2+, respectively, in the Mn2+-dependent reaction and 70, 32 and 159 microM for d,l-isocitrate, NADP and Mg2+, respectively, in the Mg2+-dependent reaction. The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutarate and NADPH2. Polyclonal antibodies raised against the pure IDH were used to assess the presence of the enzyme in cells subjected to nitrogen starvation. (+info)
Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.
(4/865)
NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis. (+info)
Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis.
(5/865)
A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation. (+info)
Role of SpoVG in asymmetric septation in Bacillus subtilis.
(6/865)
Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF. However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation. (+info)
Identification of a cytosolic NADP+-dependent isocitrate dehydrogenase that is preferentially expressed in bovine corneal epithelium. A corneal epithelial crystallin.
(7/865)
Recently, metabolic enzymes have been observed in both the lens and corneal epithelium at levels greatly exceeding what is necessary for normal metabolic functions. These proteins have been termed taxon-specific crystallins and are thought to play a role in maintaining tissue transparency. We report here that cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) represents a new corneal crystallin. Using suppression subtractive hybridization, we identified a gene (with a deduced amino acid sequence that showed 94% identity to rat cytosolic NADP+-dependent ICDH) that is preferentially expressed in bovine corneal epithelium. Northern blots established that its mRNA level in the corneal epithelium was 31-, 39-, 133-, 230-, and 929-fold more than in the liver, bladder epithelium, stomach epithelium, brain, and heart, respectively. This mRNA was detected primarily in corneal epithelial basal cells by in situ hybridization. SDS-polyacrylamide gel electrophoresis, two-dimensional gel analysis, and Western blotting showed that this protein was overexpressed in the corneal epithelium, constituting approximately 13% of the total soluble bovine corneal epithelial proteins. Enzyme assays showed a corresponding overabundance of this protein in bovine corneal epithelium. Taken together, these data indicate that bovine cytosolic ICDH fulfills the criteria for a corneal epithelial crystallin and may be involved in maintaining corneal epithelial transparency. (+info)
Population structure and genetic divergence in Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia.
(8/865)
Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America. (+info)