Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5'-phosphate

3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH₄. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl₂ was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.     

Chemoenzymatic synthesis of 1-deaza-pyridoxal 5′-phosphate

The first synthesis of 1-deaza-pyridoxal 5′-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate) is described. The chemoenzymatic approach described here is a reliable route to this important isosteric pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyridoxal 5′-phosphate dependent enzymes.     

Preparation and characterization of several new immobilized derivatives of pyridoxal 5′-phosphate

Two types of new Sepharose-bound pyridoxal 5′-phosphate, N-immobilized and 3-0-immobilized pyridoxal 5′-phosphate analogues, were prepared by reacting pyridoxal 5′-phosphate with a bromoacetyl derivative of Sepharose 4B in dimethylformamide (50% v/v) and in potassium phosphate buffer (pH 6.0) for approx. 70 h at room temperature in the dark, respectively. The properties of these immobilized pyridoxal 5′-phosphate derivatives including their catalytic activities in the non-enzymatic cleavage reaction of tryptophan were studied in comparison with those of the 6-immobilized pyridoxal 5′-phosphate analogue reported previously by the present authors. The usefulness of these pyridoxal 5′-phosphate analogues in the preparation of immobilized tryptophanase was demonstrated.     

Partial purification of androgen receptor from hypertrophic human prostate

For purification of androgen receptor from hypertrophic human prostate, solutions used for elution of androgen receptor from DNA Sepharose, affinity labeling of the receptor and ability of affinity gel to retain the receptor were examined. Elution with 20 mM pyridoxal 5'-phosphate of the receptor from DNA Sepharose was more efficient than that with diluted pyridoxal 5'-phosphate, high ionic solution or various concentrations of Mg++, 3H-dihydrotestosterone bromoacetate was applicable to covalent binding with partially purified androgen receptor regardless of the low specificity of the ligand. Affinity gel of thiopropyl-Sepharose 6B coupled to 17 alpha-(2', 3'-epoxy-propyl)-5 alpha-dihydrotestosterone was better than Affigel 102 coupled to N-[3-(3-oxo-5 alpha-androstane-17 beta-yloxycarbonyl) propionyloxy] succimide or aminoethyl-Sepharose 4B coupled to 17 alpha-carboxyethynyl testosterone with respect to the rate of retention of androgen receptor. In view of these observations, the following purification procedures were constructed: Removal of DNA Sepharose-binders from the cytosol, 40% ammonium sulfate precipitation, affinity chromatography using thiopropyl-Sepharose 6B coupled to 17 alpha-(2',3'-epoxypropyl)-5 alpha-dihydrotestosterone, and DNA Sepharose chromatography. After affinity labeling of the receptor thus obtained, the molecular weight was estimated. Some 1300-fold purification with a yield of 0.25% of the androgen receptor was achieved. The molecular weight of the receptor was mainly 45 K with 90 K in a lesser amount. The Stokes radius was calculated as 30 A.     

Extraction of nuclear glucocorticoid-receptor complexes with pyridoxal phosphate

Rat thymic lymphocytes have saturable, specific receptors for glucocorticoids, which are localized predominantly in the nucleus following exposure of thymocytes to dexamethasone at 37°C. The present results demonstrate the dose-dependent extraction by pyridoxal phosphate of dexamethasone-receptor complexes from isolated thymocyte nuclei. On an equal molar basis, pyridoxal phosphate is considerably more effective than pyridoxal; pyridoxine, pyridoxamine phosphate and 5-deoxypyridoxal are ineffective. The release of the nuclear dexamethasone receptor complex is dependent on the integrity of the C4′ carboxaldehyde group of pyridoxal phosphate as evidenced by the inhibition of extraction of dexamethasone-receptor complexes by either hydroxylamine or semicarbazide. The dexamethasone which pyridoxal phosphate liberates from thymus nuclei is bound to a macromolecule which is of smaller size than unactivated cytoplasmic dexamethasone receptor.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Regulation of pyridoxal 5'-phosphate metabolism in liver

The pyridoxal 5′-phosphate content of liver $\text{in vivo}$ and of hepatocytes $\text{in vitro}$ remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INHIBITION STUDIES WITH N-(5′-PHOSPHOPYRIDOXYL) AMINO ACIDS1

Abstract— Seven N-(5′-phosphopyridoxyl) amino acids, reduced analogs of the glutamate-pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul-finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non-linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.     

Lysyl oxidase: evidence that pyridoxal phosphate is a cofactor

Both crude and partially purified preparations of embryonic chick aortic lysyl oxidase tend to gradually lose enzymic activity when illuminated, or when urea is removed by dialysis. Full activity is restored to such preparations by dialysis versus low concentrations of pyridoxal 5′-phosphate prior to assay. Upon treatment with potassium cyanide or semicarbazide, purified embryonic chick aortic lysyl oxidase gives rise to fluorescent derivatives. The fluorescence spectrum of the semicarbazide adduct closely resembles that of pyridoxal phosphate semicarbazone. A preliminary ultraviolet/visible spectrum of bovine aortic lysyl oxidase is also presented; this shows features which add to the existing evidence that lysyl oxidase contains an essential pyridoxal phosphate cofactor.     

Preparation of pyridoxal 5'-phosphate-bound sepharose and its use for immobilization of tryptophanase

Pyridoxal 5′-phosphate-bound Sepharose (SP) was prepared by coupling pyridoxal 5′-phosphate (PLP) to diazotized p-aminobenzamidohexyl-Sepharose. A derivative of pyridoxine having an absorption maximum at $\text{ca.}$ 316 nm (possibly, 6-amino-pyridoxine 5′-phosphate) was liberated from SP by treatment with 0.1 M sodium dithionite at pH 9.0. SP catalyzed the cleavage of tryptophan in the presence of Cu²⁺, a typical non-enzymatic model of tryptophanase reaction. From the spectrophotometric data and catalytic activity, it was estimated that SP contained about 1.5 μmoles of bound PLP per gram of Sepharose. Tetrameric apotryptophanase was immobilized by incubation with SP, followed by reduction with NaBH₄. The resulting immobilized tryptophanase retained $\text{ca.}$ 60 % of the catalytic activity of free tryptophanase used. This method was much superior to other methods used commonly for preparation of immobilized enzymes.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Rapid large-scale purification of pig heart nucleoside diphosphate kinase by affinity chromatography on Cibacron Blue 3G-A Sepharose

A rapid procedure for the large-scale purification of pig heart nucleoside diphosphate kinase is described. The purification procedure involves extraction of the enzyme, absorption on cibacron Blue 3G-A Sepharose, elution with ATP, ammonium sulfate precipitation, heat treatment, and rechromatography on Cibacron Blue 3G-A Sepharose. Typically, 10–12 mg of pure nucleoside diphosphate kinase is obtained from 1 kg of heart muscle (50% yield), with a purification factor of 1200 over the extract. The specific activity is 1500 units/mg at 25°C with 8-bromoinosine 5′-diphosphate as acceptor nucleotide. This method may be easily scaled up.     

Inactivation of avian progesterone receptor binding to ATP-Sepharose by pyridoxal 5'-phosphate

The affinity of progesterone receptor from hen oviduct for ATP-Sepharose was diminished by preincubation with pyridoxal 5′-phosphate. This effect was specific for pyridoxal 5′-phosphate since the related compounds, pyridoxal, pyridoxine, pyridoxamine and pyridoxamine 5′-phosphate, were not effectors. The inactivation was easily reversed by the addition of the primary amine, Tris. However, in the presence of the reducing agent NaBH₄, the inhibitory effect of pyridoxal 5′-phosphate was irreversible. The results suggest that pyridoxal 5′-phosphate forms a Schiff base with a critical amino group, presumably at the nucleotide binding site of the progesterone receptor.     

Pyridoxylation of essential lysine residues of mitochondrial adenosine triphosphatase

1. Soluble beef-heart mitochondrial ATPase (F₁) was incubated with [³H]pyridoxal 5′-phosphate and the Schiffbase complex formed was reduced with sodium borohydride. Spectral measurements indicate that lysine residues are modified and gel electrophoresis in the presence of detergent shows the tritium label to be associated with the two largest subunits, α and β. 2. In the absence of protecting ligands, the loss of ATP hydrolysis activity is linearly dependent on the level of pyridoxylation with complete inactivation correlating to 10 mol pyridoxamine phosphate incorporated per mol enzyme. Partial inactivation of F₁ with pyridoxal phosphate has no effect on either the K_m for ATP or the ability of bicarbonate to stimulate residual hydrolysis activity, suggesting a mixed population of fully active and fully inactive enzyme. 3. In the presence of excess magnesium, the addition of ADP or ATP, but not AMP, decreases the rate and extent of modification of F₁ by pyridoxal phosphate. The non-hydrolyzable ATP analog, 5′-adenylyl-β, γ-imidodiphosphate, is particularly effective in protecting F₁ against both modification and inactivation. Efrapeptin and P_i have no effect on the modification reaction. 4. Prior modification of F₁ with pyridoxal phosphate decreases the number of exchangeable nucleotide binding sites by one. However, pyridoxylation of F₁ is ineffective in displacing endogenous nucleotides bound at non-catalytic sites and does not affect the stoichiometry of P_i binding. 5. The ability of nucleotides to protect against modification and inactivation by pyridoxal phosphate and the loss of one exchangeable nucleotide site with the pyridoxylation of F₁ suggest the presence of a positively charged lysine residue at the catalytic site of an enzyme that binds two negatively charged substrates.     

Properties of taurine: alpha-ketoglutarate aminotransferase of Achromobacter superficialis. Inactivation and reactivation of enzyme

The activity of taurine: alpha-ketoglutarate aminotransferase (taurine: 2-oxoglutarate aminotransferase, EC 2.6.1.55) from Achromobacter superficialis is significantly diminished by treatment of the enzyme with (NH4)2SO4 in the course of purification, and recovered by incubation with pyridoxal phosphate at high temperatures such as 60 degrees C. The inactive form of enzyme absorbing at 280 and 345 nm contains 3 mol of pyridoxal phosphate per mol. The activated enzyme contains additional 1 mol of pyridoxal phosphate with a maximum at 430 nm. This peak is shifted to about 400 nm as a shoulder by dialysis of the enzyme, but the activity is not influenced. The inactive form is regarded as a partially resolved form, i.e. a semiapoenzyme. The enzyme catalyzes transamination of various omega-amino aicds with alpha-ketoglutarate, which is the exclusive amino acceptor. Hypotaurine, DL-beta-aminoisobutyrate, beta-alanine and taurine are the preferred amino donors. The apparent Michaelis constants are as follows; taurine 12 mM, hypotaurine 16 mM, DL-beta-aminoisobutyrate 11 mM, beta-alanine 17 mM, alpha ketoglutarate 11 mM and pyridoxal phosphate 5 micron.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.     

Direct phosphate-phosphate interaction between coenzyme and substrate in the catalytic reaction of glycogen phosphorylase

Glycogen phosphorylase contains firmly bound pyridoxal 5′-phosphate (PLP), and catalyzes the reversible transfer of a glucosyl moiety between glucose-1-phosphate (G-1-P) and α-1,4-glucan. X-ray crystallographic studies revealed that PLP is located in a pocket where the phosphate group of PLP is pointed toward the G-1-P binding site. We have synthesized pyridoxal(5′)diphospho(1)-α-d-glucose, as a model compound for the phosphate-phosphate interaction between PLP and G-1-P, and reconstituted the enzyme with this compound. The resulting enzyme is catalytically inactive in itself, but, in the presence of glucan, the glycosyl moiety of this compound is transferred to the glucan forming a new α-1,4-glucosidic linkage along with the production of pyridoxal 5′-diphosphate. This glucosyltransfer is similar to the normal catalytic reaction in various aspects, although the rate is smaller in the order of three. AMP accelerates the transfer about 24 times compared with the reaction in its absence. We have more recently used pyridoxal(5′)triphospho(1)-α-D-glucose to reconstitute the enzyme. In the presence of glucan, the compound bound to enzyme is gradually degraded to pyridoxal 5′-triphosphate. This reaction is essentially dependent on AMP, and proceeds several times more slowly than the glucosyltransfer from the diphospho compound. These results provide evidence for the direct phosphate-phosphate interaction between the coenzyme and the substrate in the normal enzyme reaction, and seem to reflect a rather wide allowance in regard to this interaction.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.