Pig liver phosphomevalone kinase. 1. Purification and properties

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.     

Steady-state kinetic properties of phosphoglycerate kinase of mung beans

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. I. Purification and physical properties

Deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria has been partially purified and compared with partially purified AdR kinase from the cytosol of the same biological material. Some physical properties of both enzymes, including molecular weight, gel electrophoresis and gel isoelectric focusing were investigated and considerable differences between these data for mitochondrial and cytosol AdR kinase were found.     

Purification and kinetic properties of rabbit liver paraoxonase 1

Paraoxonase 1 (PON1) is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDL. In this study, rabbit liver PON1 enzyme was purified to homogeneity using a new purification approach, and the kinetic properties of the enzyme were investigated using phenyl acetate and homocysteine thiolactone as substrates. Rabbit liver PON1 was purified through the preparation of liver microsomal fraction, Sephacryl S300 HR gel filtration chromatography, DEAE Trisacryl M ion-exchange chromatography and hydroxyapatite chromatography steps. Using this method, rabbit liver PON1 was purified 576 times with a specific activity of 2726 U/mg protein. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the obtained enzyme as a single protein band close to 40 kDa. The Km of the this enzyme was found as 0.55 ± 0.024 mM for phenyl acetate and 17.31 ± 1.2 mM for homocysteine thiolactone. In this study, a new approach was used to purify PON1 enzyme from rabbit liver and for the first time in the literature, its kinetics was studied with homocysteine thiolactone as substrate.     

Purification, assay, and kinetic properties of sheep liver pyridoxal kinase

Pyridoxal kinase from sheep liver has been purified 1100-fold by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephadex G-150, and ADP-Sepharose. Polyacrylamide gel electrophoresis indicated the enzyme to be nearly homogeneous. Initial velocity studies were consistent with a sequential mechanism. The Michaelis constants for pyridoxal and ZnATP²⁻ complex are 160 and 31 μ , respectively. A new assay was developed in which [³H]pyridoxine was used as substrate. The product, [³H]pyridoxine 5′-P, was separated from the substrate with DEAE-cellulose disks. Determination of the Michaelis constants for pyridoxine and the ZnATP²⁻ complex by this new method gave values of 110 and 32 μ , respectively.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Rat liver alcohol dehydrogenase. Purification and properties

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

Purification and kinetic properties of pyruvate kinase isoenzymes of Salmonella typhimurium.

Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.