Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

Isolation of transketolase from rabbit liver and comparison of some of its kinetic properties with transketolase from other sources

1. Rabbit liver transketolase activity was purified 56-fold using the following steps: ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-25, concentration through an Amicon ultrafiltration cell and rechromatography on DEAE-Sephadex A-25. 2. The enzyme showed an optimum PH for activity at 7.8-8.0. 3. The optimum temperature was around 40 degrees C and the activation energy calculated from the Arrhenius plot was found to be 11.4 kcal/mole. 4. The molecular weight of the enzyme, as determined by gel filtration, was found to be approximately 162,000, while the content of thiamin diphosphate was between 1.8 and 2 mumole per mole protein. 5. Addition of thiamin diphosphate and magnesium chloride did not influence the activity. 6. From the kinetic studies of the enzyme, the Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 3.8 x 10(-5) M, 9.5 x 10(-5) M and 1.1 x 10(-2) M, respectively.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Purification of skeletal muscle hexokinase by affinity elution chromatography

Type II hexokinase (EC 2.7.1.1) has been purified from rat skeletal muscle by a simple procedure involving chromatography on DEAE-cellulose, affinity elution chromatography from phosphocellulose, and gel filtration on Sephadex G-200. The key to the preparation of homogeneous enzyme is the affinity elution step in which an effector molecule, glucose 6-phosphate, is used as the eluting ligand. A 5300-fold purification is obtained by the procedure and over 400-fold purification is obtained in the affinity elution step alone. Approximately 3.3 mg of homogeneous hexokinase with a specific activity of 120 units/mg is obtained from 800 g of rat limb.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50 degrees C. Km and Vmax for NADP(+) and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.

The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.     

A large-scale purification of recombinant histone H1.5 from Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

Two hexokinases of Homarus americanus (lobster), one having great affinity for mannose and fructose and low affinity for glucose

Two major hexokinases (ATP: d-hexose 6-phosphotransferases, EC 2.7.1.1) have been identified in tissues of Homarus americanus (lobster) and separated from each other by DEAE-cellulose ion-exchange chromatography and by polyacrylamide gel electrophoresis. The molecular weight of each, determined by gel filtration, is about 50 000.Hexokinase II, named for its column elution order, resembles hexokinase isozymes I and II of vertebrates. K_m values jfor glucose, mannose and fructose are 0.08, 0.13 and 6.7 mM, respectively. It is strongly inhibited by the reaction products, ADP and glucose-6-P (K_i = 0.8 mM).Hexokinase I appears to be different from any animal hexokinase previously described. It has a high affinity for mannose and fructose and low affinity for glucose. K_m values are 6, 0.07 and 1.2 mM and relative maximum rates 100, 520 and 1070 for glucose, mannose and fructose, respectively. Hexokinase I is not inhibited by physiological concentrations of ATP nor by glucose-6-P, mannose-6-P or fructose-6-P even at high concentrations. Both enzymes occur in muscle at about 10% of the concentration found in the hepatopancreas.The use of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), with NAD as cofactor, is recommended for measuring hexokinases in crude tissue preparations to avoid the variable further reduction of nucleotide caused by the action of 6-phosphogluconate dehydrogenase when NADP is used with yeast glucose-6-phosphate dehydrogenase.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Heart hexokinase: quaternary structure changes accompanying the binding of regulatory molecules.

Heart hexokinase monomer has a molecular weight of 97000 and so20,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and so20,w 8.1 S. The proportions of monomer and dimer presence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography. In the presence of the regulatory molecule glucose 6-phosphate, the dimer form of the enzyme is favoured. The glucose 6-phosphate mediated dimerisation is abolished in the presence of phosphate or ATP-Mg and less effectively by free ATP. Glucose has no effect on the manomer-dimer equilibrium. On prolonged storage of hexokinase in glucose 6-phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600

A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.