Positive Effects of Isopropanol as a Co-Precipitant in Glycerol-3-Phosphate Acyltransferase Crystallization

Glycerol-3-phosphate acyltransferase(GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid(LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It's suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol(PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 ? and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 ?, and α = 62.85, β = 73.04, γ = 80.53?. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.

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.

Overexpression of Sweet Pepper Glycerol-3-Phosphate Acyltransferase Gene Enhanced Thermotolerance of Photosynthetic Apparatus in Transgenic Tobacco

In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase （GPAT） gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol （MGDG）, sulfoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T1 lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.6% in T1-2 and T1-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem Ⅱ （PSⅡ） photochemistry （Fv/Fm）, actual photochemical efficiency of PSll in the light （ФPSⅡ） and net photosynthetic rate （Pn） of the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSⅡ in transgenic tobacco compared with wild-type plants, the entire capability of PSⅡ recovered faster in transgenic tobacco, which was shown in the parameters of PI, Fv/Fm and ФPSⅡ, as a result, the recovery of Pn was accelerated. Conclusively, we proposed that the increase in saturated extent of thylakoid membrane lipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.

Substrate Selectivity of Glycerol-3-phosphate Acyl Transferase in Rice

Substrate selectivity of glycerol-3-phosphate acyltransferase （EC 2. 3. 1. 15） of rice （Oryza sativa L.） was explored in a comparative study of acyltransferases from seven plant species. In vitro labeling of acyl carrier protein （ACP） with ^14C or 3H showed that acyltransferase from chill-sensitive plants, such as rice that uses either oleic （18：1） or palmitic acid （16：0） as acyl donor at comparable rates, displays lower selectivity than the enzyme from chill-resistant plants, such as spinach, which preferentially uses oleic acid （18：1） rather than palmitic acid （16：0） as an acyl donor. This may be a result of the size and character of the substrate-binding pocket of acyltransferase. Homology modeling and protein structure-based sequence alignment of acyltransferases revealed that proteins from either chill-sensitive or chill-tolerant plants shared a highly conserved domain containing the proposed substrate-binding pocket. However, the aligned residues surrounding the substrate-binding pocket are highly heterogeneous and may have an influence mainly on the size of the substrate binding pockets of acyltransferases. The substrate selectivity of acyltransferase of rice can be improved by enlarging the substrate-binding pocket using molecular biological methods.

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.