Isolation,Purification,Properties And Modification Of Groups Of The Glutamate Dehydrogenase From Bovine Liver

Glutamate dehydrogenase is a key enzyme which is widely existed in the cell mitochondria matrix of the organism, which is connected with the carbohydrate metabolism and the amino acid metabolism. It plays an important role in many cellular metabolic processes, such as citric acid cycle, nitrogen metabolism, and mainly catalyze the reversibie transforation between the α-ketoglutarate and glutamic acid, and thus participate in the synthesis and decomposition of glutamic acid. In vivo, the GDH exists in the form of tetramers or hexamers. According to the type of coenzyme GDH dependent on the catalytic reaction, it can be divided into three categories: NAD-dependent（NAD-GDH）, NADP-dependent（NADP-GDH） and NAD（P） dependent（NAD（P）-GDH）.GDH is widely used in the field of chemical analysis and biological medicine, So far, there are many sources of material about GDH have been studied deeply, but there is no domestic reports from bovine liver GDH purification method and its physicochemical properties, and the required GDH mainly depends on foreign production. In this study, a wide range of sources and low cost of cattle liver were selected as test materials, and GDH was isolated and purified from the liver. At the same time, using the data from NCBI carried out the bioinformatics analysis of GDH, the results were as follows: 1. Bioinformatics analysis of Bovine GDHThe full-length cDNA of bovine GLUD1 was 2446 bp, contains 13 exons and 12 introns, ORF full length is 1686 bp, encoding 561 amino acids; amino acid sequence consisting essentially of two conserved domains, ELFV_dehydrog_N domain and NAD_bind_1Glu_DH domain; and several other mammalian GLUD1 amino acid sequence of the full-length similar was 93%-99%, similar to chicken was 71%, similar to Xenopus was 85%; In evolutionary relationships, cattle and sheep was on an evolutionary branch, they had the closest genetic relationship. 2. Separation and Purification of GDH from Bovine LiverElectrophoresis-purity GDH from bovine liver was obtained through the procedures of homogenization, buffer solution extraction, butanol degreasing, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography. 23.12% of the GDH activity was obtained, The specific activity was 306.06 U/mg, The purification fold of the GDH was 93.6. 3. Characteristics of GDH from Bovine Liverthe molecular weight of GDH was approximately 380.20 kD, in which the subunit molecular mass was roughly 61.7 kD, which indicated that the enzyme is composed of six identical subunits. Characterization results show that: the optimum temperature of GDH was 50 ℃, relatively stable in the range of 20 ℃-40 ℃; optimum pH was 8.2, stable in the range of pH 5-10, with a strong acid alkali tolerance; its Km was 0.696mmol/L towards NADH. 4. Effects of different metal ions, compounds and organic solvents on the activity of GDH.Effect of Ba2+, Li+, K+, Mg2+ on the activity of GDH has a dual nature, namely low concentration activation, inhibition of high concentration, low concentration of Zn2+, Cu2+, Pb2+, Ag+ of the enzyme showed strong inhibition, Co2+, Ca2+, Cd2+ with the increase of the concentration of inhibition strengthened; oxalic acid, urea, ascorbic acid and SDS on the enzyme showed inhibitory effect, EDTA has some activation effect on this enzyme; methanol, ethanol and isopropanol has strong inhibitory effect on the enzyme. 5. Chemical Modification of GDH from Bovine LiverModified by groups of different chemical modification agent on the function of this enzyme results showed that the sulfur ether methionine, cysteine sulfhydryl and lysine residues constitute the essential groups of bovine liver glutamate dehydrogenase activity center, serine residues, tryptophan residues, two disulfide bonds, arginine residues a histidine and tyrosine phenolic hydroxyl and bovine liver glutamate dehydrogenase activity center is not directly related, is not necessary for the enzyme activity center group.