Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 micrograms/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT form the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.
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PMID:Targeting of hexokinase 1 to liver and hepatoma mitochondria. 130 5

Regulation of lactate dehydrogenase (LDH) (EC 1.1.1.27) isozymes occurs through a multitude of physiological signals. Here, we show that modulation of LDH A subunit occurs via the protein kinase C pathway. Activators of protein kinase C, such as tetradecanoylphorbol acetate (TPA) and dioctanoylglycerol (DG), caused a 3-4-fold accumulation of LDH A subunit mRNA in rat C6 glioma cells. The specific protein kinase C inhibitor bisindolylmaleimide GF 109203X prevented the TPA-induced increase of LDH A subunit mRNA. To analyze the molecular basis of these effects in more detail, the transcription-modulatory effects of TPA and DG were evaluated in transient transfection assays using plasmids which contain LDH A subunit promoter fragments fused to a chloramphenicol acetyltransferase reporter gene. Both effector agents caused a marked increase of the transcriptional activity of an LDH -830/+25 bp promoter/CAT construct. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, had no effect on the LDH promoter activity. Transient transfection analysis of LDH promoter deletion/CAT constructs, DNA/protein binding assays, including footprint and gel shift analyses, identified a TRE/AP-1 enhancer module at position -294 bp which was the target for the protein kinase C-mediated signal transduction pathway. Thus, our data demonstrate an active role of the protein kinase C signal pathway in regulating LDH A subunit gene expression which may be significant in regulating LDH isozyme patterns under various physiologic conditions.
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PMID:Transcriptional regulation of the lactate dehydrogenase A subunit gene by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. 775 43

Two Toxoplasma gondii genes were characterized that are differentially expressed during the parasite's life cycle. The genes named LDH1 and LDH2, respectively, encode polypeptides similar to the enzyme lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from a variety of organisms. They show 64.0% nucleotide identity in the coding region and both have an intron at the same relative position. The deduced amino acid sequences of LDH1 and LDH2 share 71.1% identity. LDH1 and LDH2 are most similar to an LDH of Plasmodium falciparum (46.5% and 48.5% amino acid identities, respectively). The mRNA of LDH2 was only detected in the bradyzoite stage, while the mRNA of LDH1 was detected in both the bradyzoite and tachyzoite stages. However, by isoelectric focusing and immunoblot analysis, only one LDH isoform was found to be expressed in each stage. Furthermore, the expression of a reporter gene carrying chloramphenicol acetyltransferase (CAT) coding sequence and the putative LDH2 promoter sequence was significantly up-regulated by growing parasites in tissue culture in media with alkaline pH (pH 8.2, a condition known to induce the expression of bradyzoite-specific antigens), while the expression of a CAT reporter construct carrying the putative LDH1 promoter sequence was down-regulated by similar treatment. These results indicate that LDH expression is developmentally regulated in T. gondii and suggest a possible correlation between stage conversion and alteration in carbohydrate or energy metabolism in this parasite.
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PMID:Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. 901 46

The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.
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PMID:Cytotoxic potency of cardiotoxin from Naja sputatrix: development of a new cytolytic assay. 1202 4