The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosphate dehydrogenase. (1/1590)

Staphylococcus aureus and Staphylococcus epidermidis possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD+ agarose. Both S. epidermidis and S. aureus Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast, Staphylococcus saprophyticus, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH.  (+info)

Inhibitory sites in enzymes: zinc removal and reactivation by thionein. (2/1590)

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.  (+info)

Effect of mebendazole and praziquantel on glucosephosphate isomerase and glyceraldehydephosphate dehydrogenase in Echinococcus granulosus cyst wall harbored in mice. (3/1590)

AIM: To study effects of antihydatid drugs on glucosephosphate isomerase (GPI) and glyceraldehydephosphate dehydrogenase (GAPDH) in Echinococcus granulosus cyst wall. METHODS: Mice infected with the parasite for 8-10 months were treated i.g. with mebendazole (Meb) or praziquantel (Pra). The activities of GPI and GAPDH in the cysts were measured by the formation of NADH or NADPH. RESULTS: GPI activity in the cyst wall was 197 +/- 103 U, while that of GAPDH was 25 +/- 13 U. When infected mice were treated i.g. with Meb 25-50 mg.kg-1.d-1 for 7-14 d, no apparent effect on the GAPDH activity in the cyst was found. In mice treated i.g. with praziquantel (Pra) 500 mg.kg-1.d-1 for 14 d, the GAPDH activity in the cyst wall was inhibited by 26.5%. As to GPI activity only the group treated i.g. with Meb 25 mg.kg-1.d-1 for 14 d showed 33.2% inhibition of the enzyme in the collapsed cyst wall. CONCLUSION: GPI and GAPDH are not the major targets attacked by the antihydatid drug.  (+info)

Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress. (4/1590)

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeast Saccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.  (+info)

Transcriptional regulation of interleukin-2 gene expression is impaired by copper deficiency in Jurkat human T lymphocytes. (5/1590)

Copper deficiency reduces secretion of the cytokine interleukin-2 (IL-2) by activated rodent splenocytes, human peripheral blood mononuclear cells and Jurkat cells, a human T lymphocyte cell line. Previous studies showed that low Cu status also decreased the level of IL-2 mRNA in activated Jurkat cells by 50%. Synthesis of this cytokine is regulated by alterations in transcription of the IL-2 gene and the stability of IL-2 mRNA. To determine if Cu status influenced promoter activity of the IL-2 gene, Jurkat cells were transfected with a luciferase reporter gene construct containing the entire 300 bp human IL-2 promoter/enhancer sequence. Cu deficiency was induced by incubating stably transfected cells with the Cu chelator 2,3,2-tetraamine for 35 h prior to activating cells with phytohemagglutinin-P and phorbol myristate acetate. Luciferase activity in lysates of Cu-deficient cells was approximately 50% lower in several multiclonal and clonal cell lines of stably transfected cells than in replicate cultures that were not exposed to chelator. The relative levels of endogenous IL-2 bioactivity and luciferase activity were highly correlated in the transfected cell lines. The chelator-mediated reduction in reporter gene activity was dose-dependent at levels of 5-40 micromol 2,3,2-tetraamine/L. The addition of a slight molar excess of Cu, but not Zn or Fe, to medium containing 2,3,2-tetraamine prevented the decline in luciferase activity. IL-2 mRNA stability in parental Jurkat cells was independent of Cu status. These data indicate that decreased cellular Cu attenuates IL-2 synthesis in T lymphocytes by inhibiting transcription of the IL-2 gene.  (+info)

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase. (6/1590)

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.  (+info)

Expression of menin gene mRNA in pituitary tumours. (7/1590)

OBJECTIVE: Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant inherited disorder characterised by the combined occurrence of parathyroid, endocrine pancreas and anterior pituitary tumours. The gene responsible for MEN 1, the menin gene, a putative tumour-suppressor gene located on human chromosome 11q13, has been cloned. To investigate the role of the menin gene in sporadic anterior pituitary tumorigenesis, its mRNA was assessed in a group of pituitary tumours. METHODS: Menin gene expression, along with glyceraldehyde phosphate dehydrogenase (GAPDH) gene expression, has been studied in a group of normal pituitaries and in 23 pituitary tumours not associated with the MEN 1 syndrome. The pituitary tumours included 4 prolactinomas, 11 growth-hormone-secreting tumours and 8 non-functional tumours. Total RNA was extracted from the normal pituitaries and tumours, and cDNA was synthesised with standard reverse transcriptase methods. Duplex polymerase chain reaction (PCR) was standardised in order to quantify the expression of the menin gene using intron-spanning primers across exons 9 and 10 in relation to the 'house-keeping' gene GAPDH. The PCR products were separated on agarose gel and densitometric analysis of the bands allowed semi-quantification. RESULTS: There was no evidence for a change in menin gene expression in any of the pituitary tumours when compared with normal pituitaries. CONCLUSIONS: These studies complement previous work on mutational analysis, and do not suggest a major role for the menin suppressor gene in sporadic pituitary tumorigenesis.  (+info)

Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa. (8/1590)

The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.  (+info)