Separation and Purification of GST-glycerol-3-phosphate Dehydrogenase

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity

Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycelytic enzyme, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer （RaGAPDH） has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genornic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligandbinding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coil （EHEC）, belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestiferdisplayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism＇s virulence.

Critical protein GAPDH and its regulatory mechanisms in cancer cells

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and pnsttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 （HIF-1）, p53, nitric oxide （NO）, and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications （PTMs） occurring in GAPDH in cancer cells result in new activities unrelated to the original glycnlytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.

The role of AtGPDHc2 in regulating cellular redox homeostasis of Arabidopsis under salt stress

Plants glycerol-3-phosphate dehydrogenase(GPDH)catalyzes the formation of glycerol-3-phosphate,and plays an essential role in glycerolipid metabolism and stress responses.In the present study,the knock-out mutants of cytosolic GPDH(AtGPDHc2)and wild-type Arabidopsis plants were treated with 0,50,100,and 150 mmol L–1 NaCl to reveal the effects of AtGPDHc2 deficiency on salinity stress responses.The fluctuation in redox status,reactive oxygen species(ROS)and antioxidant enzymes as well as the transcripts of genes involved in the relevant processes were measured.In the presence of 100 and 150 mmol L–1 NaCl treatments,AtGPDHc2-deficient plants exhibited a pronounced reduction in germination rate,fresh weight,root length,and overall biomass.Furthermore,loss of AtGPDHc2 resulted in a significant perturbation in cellular redox state(NADH/NAD+and AsA/DHA)and consequent elevation of ROS and thiobarbituric acid-reactive substances(TBARS)content.The elevated ROS level triggered substantial increases in ROS-scavenging enzymes activities,and the up-regulated transcripts of the genes(CSD1,sAPX and PER33)encoding the antioxidant enzymes were also observed.In addition,the transcript levels of COX15,AOX1A and GLDH in gpdhc2 mutants decreased in comparison to wild-type plants,which demonstrated that the deficiency of AtGPDHc2 might also has impact on mitochondrial respiration under salt stress.Together,this work provides some new evidences on illustrating the roles of AtGPDHc2 playing in response to salinity stress by regulating cellular redox homeostasis,ROS metabolism and mitochondrial respiration.

The Effect of Ultra Low Concentrations of Some Biologically Active Substances on the Aerobic Respiration

For today it is known, that primary and secondary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of clinical manifestations and diseases. Therefore, the question about effective correction of various types of energy exchange disorders remains topical. Thus, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (EC 1.3.99.1) (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) (GPD2). The human lymphocytes assays were tested in vitro (22 donors). In negative control lymphocytes, cell culture normal saline solution was added. Normal saline solution with NaN3 was added in positive control lymphocytes cell culture. Experimental cell culture contained NaN3 and BAS. Our investigations had been revealed increase SQR activity in the experimental cell culture as compared with positive control culture throughout the time of experiment (measurements were carried out at 4, 24, 48, 72 h of incubation). The SQR activity of experimental cell culture and negative control lymphocytes cell culture was equal up to 24 h of experiment. It showed neutralization of NaN3 inhibitory effect (during 24 h) due to BAS influence. Activity of base glycerophosphate shunt ferment GPD2 of experimental lymphocyte cell culture was not different from GPD2 index in the negative control, but was lower than GPD2 activity in the positive control. It also indicated neutralization NaN3 inhibitory effect due to BAS influence. So we had found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro could effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link.