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Human triosephosphate isomerase cDNA and protein structure. Studies of triosephosphate isomerase deficiency in man 总被引:18,自引:0,他引:18
Nine cDNA clones of human adult liver triosephosphate (TP) isomerase have been isolated and characterized. All nine appear to be derived from a single mRNA species. DNA sequencing of one clone, designated pHTPI-5a, defined the last two nucleotides of the methionine initiation codon, the entire 744-nucleotide coding region of the mature polypeptide, and the entire 448-nucleotide 3' untranslated region. The frequency of TP isomerase clones in the cDNA library suggests that TP isomerase mRNA is present in adult liver at approximately 25 copies/cell. A single, low abundance TP isomerase mRNA species was detected in RNA isolated from normal human fibroblast cell lines. Analysis of TP isomerase mRNA levels in cultured fibroblasts of individuals that are homozygous for TP isomerase deficiency revealed normal levels in one and approximately 40% of normal levels in another. From this small patient sampling, it can be concluded that the genetic basis for TP isomerase deficiency is heterogeneous. 相似文献
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J C Kaplan L Teeple N Shore E Beutler 《Biochemical and biophysical research communications》1968,31(5):768-773
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Cytoplasmic mRNA for human triosephosphate isomerase is immune to nonsense-mediated decay despite forming polysomes 总被引:2,自引:0,他引:2
Nonsense codons between position 14 within the first exon and position 193 within the penultimate exon of the human gene for triosephosphate isomerase reduce mRNA abundance to 25% of normal. The reduction in abundance is due to the decay of newly synthesized mRNA that copurifies with nuclei. TPI mRNA that copurifies with cytoplasm is immune to decay. We show here that immunity is not due to the failure of nonsense-containing mRNA to form polysomes. This finding indicates that cytoplasmic mRNA, in contrast to nucleus-associated mRNA, may have lost one or more factors that are required for nonsense-mediated decay or gained one or more factors that confer immunity to nonsense-mediated decay. 相似文献
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Primary structure of human triosephosphate isomerase 总被引:10,自引:0,他引:10
Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes. 相似文献
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A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice. 相似文献
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In vitro deamidation of human triosephosphate isomerase 总被引:4,自引:0,他引:4
The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo. 相似文献
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G Garza-Ramos M Tuena de Gómez-Puyou A Gómez-Puyou R W Gracy 《European journal of biochemistry》1992,208(2):389-395
The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes. 相似文献
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Molecular dynamics simulations (30-60 ns runs) are performed on free/apo triosephosphate isomerase (TIM) to determine any correlation between collective motions and loop 6 dynamics. Native TIM is reported to be active only as a homodimer even though cooperativity has not been observed between the two identical subunits. Both dimeric and monomeric (isolated from dimer) forms of TIM are simulated in explicit water at 300 K and 1 bar to inspect any differences between the structures in terms of fluctuation dynamics and functionally important loop 6 dynamics/closure. Significant cross-correlations between residue fluctuations are observed in the dimer, which result from the global counter-rotations of the two identical subunits in the essential modes of the dimer. Specifically, the first essential mode contributing to 34% of overall motion of the dimer is strongly coupled to the loop 6's closure over the active site. In contrast, such significant correlations cannot be observed in the monomeric structure, which maintains relatively localized motions of the loops in the essential modes. Thus, the onset of collective motions at ns time scale due to dimerization has functional implications as to the coordination of loop 6 closure. 相似文献
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Structural determinants for ligand binding and catalysis of triosephosphate isomerase. 总被引:1,自引:0,他引:1
I Kursula S Partanen A M Lambeir D M Antonov K Augustyns R K Wierenga 《European journal of biochemistry》2001,268(19):5189-5196
The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding. 相似文献
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I D Campbell R B Jones P A Kiener E Richards S C Waley R Wolfenden 《Biochemical and biophysical research communications》1978,83(1):347-352
In NMR experiments designed to distinguish between possible enzyme-bound forms of the inhibitor 2-phosphoglycollic acid, it is found that neither of the di-anionic species, that would be consistent with the observed pH-dependence of Ki, is in fact correct. Instead, the enzyme appears to bind the tri-anionic species of this inhibitor, taking up a proton at a separate site. 相似文献
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Human triosephosphate isomerase deficiency resulting from mutation of Phe-240. 总被引:5,自引:1,他引:4
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M L Chang P J Artymiuk X Wu S Holln A Lammi L E Maquat 《American journal of human genetics》1993,52(6):1260-1269
Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jó., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe]-->CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by disrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10-20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu]-->GAC [Asp]), which also results in thermolabile protein. 相似文献