Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.2 (alanine aminotransferase)
26,722 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Asparagine-linked glycosylation is initiated by the synthesis of N-acetylglucosaminylpyrophosphoryl dolichol (GlcNAc-P-P-dolichol), which is extended by a series of glycosyltransferases to yield Glc3Man9GlcNAc2-P-P-dolichol (where Glc is glucose and Man is mannose). The oligosaccharide unit is then transferred en bloc to asparagine residues of nascent polypeptides in the lumen of the rough endoplasmic reticulum. The question here is whether GlcNAc-P-P-dolichol biosynthesis is a fixed process unaffected by cellular events, or a regulated reaction responsive to cellular requirements for glycoprotein biosynthesis. Several lines of evidence indicate that the latter is the case and that GlcNAc-P-P-dolichol biosynthesis may be subject to multiple forms of regulation. Recent information about the N-acetylglucosamine-1-P transferase (GPT) responsible for this reaction and the cloning of cDNA candidates for this enzyme have provided further insight into these mechanisms. This review will examine current hypotheses dealing with GPT and its role in the committed step of asparagine-linked glycosylation.
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PMID:Biosynthesis of N-acetylglucosamine-P-P-dolichol, the committed step of asparagine-linked oligosaccharide assembly. 166 6

Dictyostelium discoideum cells appear to be able to recognize particular carbohydrate prosthetic groups at different stages in their life cycle. We therefore used our previously developed model system (consisting of polyacrylamide gels containing putative ligands covalently linked to the polymer) to determine the receptors on these cells capable of recognizing carbohydrates. D. discoideum cells, at different developmental stages from growth phase to late aggregation, were incubated with the derivatized gels, and the number of adherent cells was determined by measuring alanine transaminase after cell lysis. From 70 to 100% of the cells firmly adhered to gels derivatized with glucose, maltose, or cellobiose. The cells were also capable of binding to N-acetylglucosamine and mannose, but both the rate and the extent of binding to these sugars were less than those observed with the glucose derivatives. Furthermore, binding to N-acetylglucosamine decreased to negligible levels during the aggregation stage of development. The cells did not bind to the glucose-derivatized gels in the presence of glucose and a variety of carbohydrates containing glucose at the nonreducing termini, whereas binding was not inhibited by N-acetylglucosamine, mannose, and derivatives of these sugars. Adhesion to all sugars was blocked by 2,4-dinitrophenol. This inhibitor also reversed the binding to gels containing N-acetylglucosamine and mannose, but not to glucose. Differential binding to the three monosaccharides was also observed under conditions affecting the normal amoeboid shape of the cells. In addition, adhesion to N-acetylglucosamine and mannose was trypsin-sensitive, whereas adhesion to glucose was only slightly affected by treating the cells with trypsin (and cycloheximide). These and other results suggest that D. discoideum cell adhesion to derivatized gels is mediated by three different receptors, one highly specific for glucose and two (probably less specific) for N-acetylglucosamine and mannose.
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PMID:Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. I. Evidence for three sugar-specific cell surface receptors. 668 32

Hydropathic profile for several dolichol-coupled enzymes (mammal N-acetylglucosamine-1-phosphate transferases (GPT), yeast products of genes ALG7, ALG1, DMP1 and SEC59) taking part in the biosynthesis of complex oligosaccharide used for N-glycosylation of proteins in endoplasmic reticulum (ER) of eukaryotic cells has been calculated and constructed. On the basis of analysis of present research and available data sites of amino acid sequence (AAS) potentially capable to penetrate ER membrane were identified. A tendency of AAS segments of dolichol-coupled enzymes to form alpha-helices, beta-folded structures and beta-bends was calculated using correlational methods. For COOH-terminal part of ALG1 the prediction about the capability to form helix-helix structures was made. It was concluded that at least three types of topological models in ER membrane were present for the mentioned dolichol-coupled enzymes.
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PMID:[A theoretical analysis of the secondary structure and topology of dolichol-coupled enzymes]. 748 10

The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library. The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified by complementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter. This construct allows to specifically suppress the endogenous enzyme activity. The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced protein sequence shows a homology of over 90% when compared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species. This homology however decreases to 40-50% when compared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the recombinant plasmid and grown on glucose thus suppressing transcription of the endogenous gene. Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin. These results show that we have cloned the human GlcNAc-1-P transferase by heterologous complementation in S. cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.
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PMID:Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae. 945 Oct 16

The cytotoxic drug tunicamycin kills cells because it is a specific inhibitor of UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial step of the biosynthesis of dolichol-linked oligosaccharides. In the presence of tunicamycin, asparagine-linked glycoproteins made in the endoplasmic reticulum are not glycosylated with N-linked glycans, and therefore may not fold correctly. Such proteins may be targeted for breakdown. Cells that are treated with tunicamycin normally experience an unfolded protein response and induce genes that encode endoplasmic reticulum chaperones such as the binding protein (BiP). We isolated a cDNA clone for Arabidopsis GPT and overexpressed it in Arabidopsis. The transgenic plants have a 10-fold higher level of GPT activity and are resistant to 1 microg/mL tunicamycin, a concentration that kills control plants. Transgenic plants grown in the presence of tunicamycin have N-glycosylated proteins and the drug does not induce BiP mRNA levels as it does in control plants. BiP mRNA levels are highly induced in both control and GPT-expressing plants by azetidine-2-carboxylate. These observations suggest that excess GPT activity obviates the normal unfolded protein response that cells experience when exposed to tunicamycin.
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PMID:Overexpression of a gene that encodes the first enzyme in the biosynthesis of asparagine-linked glycans makes plants resistant to tunicamycin and obviates the tunicamycin-induced unfolded protein response. 1051 26

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.
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PMID:Characterisation of the gptA gene, encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase, from the filamentous fungus, Aspergillus niger. 1249 19

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.
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PMID:Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases. 1584 95

The protective effects of chitosan oligosaccharide (COS), d-glucosamine (GlcNH(2)) and N-acetyl-d-glucosamine (GlcNAc) on carbon tetrachloride (CCl(4))-induced hepatotoxicity and the possible mechanisms that involved were investigated in male ICR mice. CCl(4) (20mg/kg body weight, i.p.) administration induced marked increase in serum AST and ALT activities, primed liver lipid peroxidation, depleted sulfhydryl content, impaired total antioxidant capabilities and induced genotoxicity 24h after administration. Pretreatment with COS, GlcNH(2), and GlcNAc (1.5g/kg body weight, i.g.) for 12 consecutive days prior to CCl(4) challenge significantly induced metallothionein (MT) expression. Thus, the antioxidant defensive system in the body was strengthened to counteract the oxidative damage induced by the succedent CCl(4) administration. Serum AST and ALT activities were effectively decreased. Hepatic malondialdehyde formation was inhibited and sulfhydryl contents, total antioxidant capabilities were markedly restored. Genotoxicity as reflected by DNA fragmentation, however, was not mitigated by pretreatment with COS, GlcNH(2), and GlcNAc. Histophathologic results of liver also confirmed their hepato-protective effects. Pretreatment with COS, GlcNH(2), and GlcNAc also could significantly decrease serum creatinine and uric acid levels and inhibit lipid peroxidation in kidney homogenate. Our results suggest that pretreatment with COS, GlcNH(2), and GlcNAc can efficiently protect mice against CCl(4)-induced toxicity.
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PMID:Protective effects of chitosan oligosaccharide and its derivatives against carbon tetrachloride-induced liver damage in mice. 1673 Feb 24

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% alpha-isoform and 45% beta-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.
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PMID:Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation. 1684 3

A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.
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PMID:Construction of a binary vector for transformation of Arabidopsis thaliana with a new selection marker. 1899 21


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