Purification and characterization of pyridoxal kinase from human erythrocytes

Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.     

Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine.

Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.     

Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.

Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.     

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.     

Cloning and characterization of Arabidopsis thaliana pyridoxal kinase

Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum

A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized. The enzyme was a dimeric protein with a subunit molecular weight of 38,000. It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1. The optimum pH (8.0-8.5) was 0.8-1.3 lower than that of the Pseudomonas enzyme. The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140 +/- 0.008 mM) and pyridoxal (0.473 +/- 0.109 mM), respectively, than those of the Pseudomonas enzyme. The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+. The enzyme was much more tolerant to metal-chelating agents. Irreversibility of the enzymatic reaction was shared by the two enzymes. No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.     

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

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.     

Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Purification and characterization of rhodopsin kinase

Rhodopsin kinase was purified to near homogeneity by affinity binding to light-exposed rod cell outer segment membranes, followed by DEAE-cellulose and hydroxyapatite chromatography. This resulted in a 1055-fold purification of highly active rhodopsin kinase with an overall recovery of 19%. Rhodopsin kinase is a single polypeptide chain with Mr = 67,000-70,000 as determined by gel filtration and SDS-PAGE. The kinetic parameters of the enzyme for freshly bleached rhodopsin are Km = 4 microM and Vmax = 700 nmol/min/mg whereas for ATP Km = 2 microM (which is a low value for kinases generally, and about 20 times lower than comparable measurements for a kinase of a similar type, the beta-adrenergic-receptor kinase (Benovic, J.L., Mayor, F. Jr., Staniszewski, C., Lefkowitz, R.J., and Caron, M.G. (1987) J. Biol. Chem. 262, 9026-9032). GTP, on the other hand, is a very poor substrate (Km = 1 mM, Vmax = 10 nmol/min/mg). Rhodopsin kinase is competitively inhibited by adenosine and its mono- and diphosphate derivatives, but not by most other adenosine derivatives. Based upon measurements with 28 nucleotide derivatives, the ATP-binding site of rhodopsin kinase appears to have more specific requirements than that for other kinases. Compounds such as cGMP, inositol trisphosphate, and others that change concentration during exposure of rod cells to light have only minor inhibitory effects on the kinase activity, with the exception of inositol monophosphate, which can activate the kinase about 20% at 50-100 microM. Rhodopsin kinase has been difficult to store with retention of activity, but can be successfully stored frozen at -20 degrees C in 20% adonitol.     

Purification of mitochondrial creatine kinase. Biochemical and immunological characterization

Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.     

Purification and characterization of Novikoff ascites tumor protein kinase.

A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.     

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn²⁺ and Mg²⁺ and inactivation by Co²⁺ and Zn²⁺ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. K_m values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Purification and characterization of arginine kinase from locust

L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.