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1.
Kuwada N Nagano K MacLennan N Havens J Kumar M Dipple KM McCabe ER 《Biochemical and biophysical research communications》2005,335(1):247-255
A glycerol kinase (Gyk) knock-out (KO) mouse model permits improved understanding of glycerol kinase (GK) deficiency (GKD) pathogenesis, however, early death of affected mice limits its utility. The purpose of this work was to delay death of affected males to investigate thoroughly their phenotypes. An adenoviral vector carrying the human (Adeno-XGK) or mouse (Adeno-XGyk) GK gene was injected into KO mice within 24 h of birth. Adeno-XGK did not change KO mouse survival time despite liver GK activity greater than 100% of wild type. However, Adeno-XGyk improved KO mouse survival time greater than two-fold. These investigations demonstrate that gene replacement therapy for Gyk KO mice is more efficacious using murine Gyk than human GK. These studies expand our understanding of GKD pathogenesis in the murine model, and show that while murine GKD is more severe than in humans, GKD mice have similar metabolic disturbances to affected humans with hypoglycemia and acidemia. 相似文献
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We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues. 相似文献
6.
Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase. 相似文献
7.
David M. Koeller Kathleen Axtell DiGiulio Stephen V. Angeloni Lisa L. Dowler Frank E. Frerman Robert A. White Stephen I. Goodman 《Genomics》1995,28(3)
Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped. 相似文献
8.
Novel acyl-CoA synthetase in adrenoleukodystrophy target tissues 总被引:2,自引:0,他引:2
Moriya-Sato A Hida A Inagawa-Ogashiwa M Wada MR Sugiyama K Shimizu J Yabuki T Seyama Y Hashimoto N 《Biochemical and biophysical research communications》2000,279(1):62-68
X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by demyelination of white matter. The X-ALD gene product adrenoleukodystrophy protein (ALDP) is expressed broadly among various tissues. However, deficiency of functional ALDP exclusively impairs brain, adrenal gland, and testis. Thus, loss of ALDP function is assumed to involve inactivation of a putative mediating factor that functions in a tissue-specific manner. Here we cloned a mouse cDNA encoding a novel protein, Lipidosin, that possesses long-chain acyl-CoA synthetase (LCAS) activity. Lipidosin is expressed exclusively in mouse brain, adrenal gland, and testis, which are affected by X-ALD. LCAS activity of Lipidosin was diminished by mutation of conserved amino acids within the AMP-binding domain. Mutation of the Drosophila homologue of Lipidosin has been reported to cause neuronal degeneration. Thus, Lipidosin may mediate the link between ALDP dysfunction and the impairment of fatty acid metabolism in X-ALD. 相似文献
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Ohira RH Dipple KM Zhang YH McCabe ER 《Biochemical and biophysical research communications》2005,331(1):239-246
Glycerol kinase (GK) is a key enzyme in glycerol metabolism with two alternatively spliced forms-one with an 87bp insertion corresponding to exon 18 (GK+EX18), and one lacking exon 18 (GK-EX18). We report the expression of GK+/-EX18 in various tissues and cell lines, as well as their enzymatic characteristics and subcellular localization. RT-PCR revealed differential expression in tissues and cell lines. Northern blot analysis revealed that both forms of the murine ortholog, Gyk, were highly expressed in murine heart and increased during embryonic development. K(m) values for glycerol for GK+/-EX18 were not significantly different, although GK-EX18 had a higher V(max) for glycerol. GK-EX18 had a lower K(m) and V(max) for ATP than GK+EX18. Immunofluorescence experiments showed that GK+EX18 co-localized to the mitochondria and the perinuclear region while GK-EX18 had a diffuse expression pattern. These data suggest specific and divergent roles for GK+EX18 and GK-EX18 in cellular metabolism and development. 相似文献
10.
Myopathy in complex glycerol kinase deficiency patients is due to 3'' deletions of the dystrophin gene. 总被引:6,自引:4,他引:2 下载免费PDF全文
DNA samples from nine previously reported patients with X-linked recessive glycerol kinase deficiency, associated in seven of them with adrenal hypoplasia and in five with developmental delay and myopathy, have been studied for deletions of the Duchenne/Becker muscular dystrophy gene by probing with the entire cDNA for the dystrophin protein. All five patients with myopathy, including two in whom no deletions had been detected before, were found to have variable-sized deletions extending through the 3' end of this gene. The 5' deletion breakpoints are intragenic in four cases and have been mapped precisely on the exon-containing HindIII fragment map. A correlation was found between severity and progression of the muscular dystrophy phenotype and the sizes of the gene deletions. In cases in which there was glycerol kinase deficiency/adrenal hypoplasia microdeletion syndrome without myopathy, no deletions were found with the dystrophin cDNA. 相似文献
11.
Denis R. Joanisse Kenneth B. Storey 《Archives of insect biochemistry and physiology》1995,28(4):339-349
Changes in the activity of over 20 enzymes of intermediary metabolism in 15°C or ?4°C acclimated goldenrod gall moth (Epiblema scudderiana) and gall fly (Eurosta solidaginis) larvae were measured. Increased activities of glyco-genolytic and hexose monophosphate shunt enzymes in cold-acclimated Epiblema scudderiana suggest a role for coarse control in the conversion of glycogen reserves into glycerol cryoprotectant synthesis. In Eurosta solidaginis, high glycogen phosphorylase activity with decreased activities of glycolytic enzymes may account in part for the temperature-dependent switch from glycerol to sorbitol synthesis in these larvae upon cold acclimation. Isoelectric focusing analyses of five enzymes in overwintering Epiblema scudderiana revealed transient mid-winter changes in the isoelectric points of phosphofructokinase and pyruvate kinase, suggesting seasonal changes in the phosphorylation state of these enzymes. A distinct developmental pattern of aldolase isozymes suggests a role for a new isozyme during overwintering or upon spring emergence. Regulation of metabolism by changes in enzyme activities is indicated for both larvae. © 1995 Wiley-Liss, Inc. 相似文献
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D. R. Joanisse K. B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(3):247-255
The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH
6-Phosphogluconate dehydrogenase
- DHAP
dihydroxy acetone phosphate
- F6P
fructose-6-phosphate
- F6Pase
fructose-6-phospha-tase
- FBPase
fructose-bisphosphatase
- G3P
glycerol-3-phosphate
- G3Pase
glycerol-3-phosphate phophatase
- G3PDH
glycerol-3-phosphate dehydrogenase
- G6P
glucose-6-phosphate
- G6Pase
glucose-6-phosphatase
- G6PDH
glucose-6-phosphate dehydrogenase
- GAK
glyceraldehyde kinase
- GAP
glyceraldehyde-3-phosphate
- GAPase
glyceraldehyde-3-phosphatase
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- GDH
glycerol dehydrogenase
- GPase
glycogen phosphorylase
- HMS
hexose monophosphate shunt
- LDH
lactate dehydrogenase
- NADP-IDH
NADP+-dependent isocitrate dehydrogenase
- PDHald
polyol dehydrogenase, glyceraldehyde activity
- PDHgluc
polyol dehydrogenase, glucose activity
- PFK
phosphofructokinase
- PGI
phosphoglucoisomerase
- PGK
phosphoglycerate kinase
- PGM
phosphoglucomutase
- PK
pyruvate kinase
- PMSF
phenylmethylsulfonylfluoride
- SoDH
sorbitol dehydrogenase
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V
max
maximal enzyme activity
- ww
wet weight 相似文献
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The murine Xlr (X-linked, lymphocyte-regulated) gene family was originally identified by subtractive cDNA hybridization and cloning. It was found to encode two 30-kDa nuclear proteins expressed in lymphoid cells and in primary spermatocytes in a developmentally regulated manner. Our data show that, in contrast to most X-linked genes, the Xlr family is not conserved at the DNA level between mouse and human. However, using anti-Xlr antibodies, an Xlr-immunoreactive nuclear protein of Mr 30,000 was characterized in human RAJI B-lymphoblastoid cells by flow cytofluorimetry, by immunoblotting, and by immuno-cytolabeling. An Xlr-like molecule was also found to be expressed in human activated lymphocytes and in human primary spermatocytes, with a stage specificity similar to that known in the mouse. In contrast, no Xlr-immunoreactive protein was detected in a series of human tissues including brain, skeletal muscle, colon, liver, and kidney, revealing a tissue-specific expression pattern similar to that of murine Xlr. These findings most likely identify a human equivalent of Xlr. The Xlr genes belong to a small category of X-linked genes, including STS, MIC2, CSF2RA, and KAL, that diverge at the DNA level in human and in mice. Characterization of the human XLR gene(s) should now be feasible with anti-Xlr antibodies and an expression cloning system. It should provide new insights into the evolution of mammalian X Chromosome (Chr). 相似文献
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Nobuo Kato Hisataka Kobayashi Masayuki Shimao Chikahiro Sakazawa 《Applied microbiology and biotechnology》1986,23(3-4):180-186
Summary A mutant, No. 65, of Hansenula polymorpha CBS 4732 was isolated which was impaired in its ability to grow on methanol and dihydroxyacetone. Mutant No. 65 produced dihydroxyacetone and glycerol from methanol with a 18.8% yield in a resting-cells reaction. The absence of dihydroxyacetone kinase activity in the mutant is believed to be the reason for its inability to grow on methanol and for the accumulation of trioses. This mutant, however, was able to grow on glycerol, and dihydroxyacetone kinase was found in the cells. The growth on glycerol was almost completely inhibited by the addition of methanol (0.1% v/v). As far as tested with partially purified enzymes, no property was found that could be used to distinguish between the kinases from methanol- and glycerol-grown cells. The evidence suggests that the phenotype of No. 65 is a lesion not in the structural gene but in its regulatory gene. 相似文献
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Glycerol-insensitive Arabidopsis mutants: gli1 seedlings lack glycerol kinase, accumulate glycerol and are more resistant to abiotic stress 总被引:1,自引:0,他引:1
Eastmond PJ 《The Plant journal : for cell and molecular biology》2004,37(4):617-625
The aim of this study was to investigate the process of glycerol catabolism in germinating Arabidopsis seed. A genetic screen was performed to isolate glycerol-insensitive (gli) mutant seedlings. Three separate mutant loci were identified (gli1, gli2 and gli3). Of these, only gli1 is unable to utilise glycerol. Following germination, gli1 seedlings transiently accumulate glycerol derived from the breakdown of storage oil and are more resistant to hyperosmotic stress, salt stress, oxidative stress, freezing and desiccation. Enzyme assays revealed that gli1 lacks glycerol kinase activity. GLI1 mapped to chromosome 1 near the putative glycerol kinase gene NHO1. Mutations in this gene were identified in three independent gli1 alleles. A cDNA encoding GLI1 was cloned and its function was proven by complementation of an Escherichia coli glycerol kinase (glpK) deletion strain. Quantitative RT-PCR analysis showed that GLI1 is expressed in all tissues, but is transiently upregulated during early post-germinative growth and leaf senescence. These data show that glycerol kinase is required for glycerol catabolism in Arabidopsis and that the accumulation of glycerol can enhance resistance to a variety of abiotic stresses associated with dehydration. 相似文献
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Kuramochi S Matsuda Y Okamoto M Kitamura F Yonekawa H Karasuyama H 《Immunogenetics》1999,49(5):369-375
LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted
mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced
from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus
and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western
blot analysis with mouse LOK-specific antibody detected 130 000 M
r LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome
10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11.
Received: 28 September 1998 / Revised: 2 November 1998 相似文献
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Yuzuru Sanemitsu Tamon Uematsu Satoru Inoue Katsutoshi Tanaka 《Bioscience, biotechnology, and biochemistry》2013,77(7):1927-1929
Enzyme activities involved in the initial step of glycerol metabolism were determined in cells of methylotrophic yeasts grown on glycerol, methanol or glucose. In Candida boidinii (Kloeckera sp.) No. 2201, the activities of glycerol kinase and dihydroxyacetone kinase were detected in cells grown on glycerol and methanol, respectively. The activity of NAD+-linked glycerol dehydrogenase of Hansenula polymorpha dl-1 was induced by glycerol and methanol, while that of Hansenula ofunaensis was induced by glycerol. The enzymes of both strains were subject to catabolite repression by glucose.The yeasts tested were divided into three groups as to the glycerol dissimilation patterns. Strains of the genera Candida, Saccharomyces, Pichia and Torulopsis had the phosphorylative pathway, in which glycerol is first phosphorylated. H. ofunaensis had the oxidative pathway, in which glycerol is first oxidized. H. polymorpha dl-1 had both the phosphorylative and oxidative pathways. 相似文献
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Subcloning, expression, purification, and characterization of Haemophilus influenzae glycerol kinase
Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia coli is an allosteric regulatory enzyme whose activity is inhibited by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously termed III(Glc)). Comparison of its primary structure with that of the highly similar Haemophilus influenzae glycerol kinase reveals that the amino acid sequence for the binding site for FBP is conserved while the amino acid sequence for the binding site for IIA(Glc) contains differences that are predicted to prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was assembled from DNA library fragments and subcloned into pUC18. The enzyme is expressed at high levels in E. coli. It was purified to greater than 90% homogeneity by taking advantage of its solubility behavior in a procedure that requires no column chromatography. The initial-velocity kinetic parameters of the purified enzyme are similar to those of the E. coli glycerol kinase. The H. influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement with the prediction based on sequence comparison. Sedimentation velocity experiments reveal that inhibition of HiGK by FBP is associated with oligomerization, behavior which is similar to EcGK. The possibility of utilizing mutagenesis studies to exploit the high degree of similarity of these two enzymes to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed. 相似文献
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Heterogeneous mutations in the X-linked gene RPS6KA3, encoding the protein kinase RSK2, are responsible for Coffin-Lowry Syndrome. Here we have further studied a male patient
with a highly suggestive clinical diagnosis of CLS but in whom no mutation was found by exon sequencing. Western blot analysis
revealed a protein much larger than the normal expected size. Sequencing of the RSK2 cDNA, showed the presence of an in-frame
tandem duplication of exons 17–20. The mutated RSK2 protein was found to be inactive in an in-vitro kinase assay. This event,
which was the result of a homologous unequal recombination between Alu sequences, is the first reported large duplication
of the RPS6KA3 gene. Our finding provides further evidence that immunoblot analysis, or a molecular assay capable to detect large genomic
mutational events, is essential for patients with a highly suggestive CLS clinical diagnosis but remaining without mutation
after exon sequencing.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献