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.     

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate

Homogeneous D-ribulose 1,5-bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, $\text{Chlamydomonas reinhardtii}$, and $\text{Hydrogenomonas eutropha}$ are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from $\text{Rhodospirillum rubrum}$, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from $\text{Rhodospirillum rubrum}$.     

Conditions for the measurement of nuclear estrogen receptor at low temperature

This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time (${}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 35}\text{h}$). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract.     

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.     

Inhibition by pyridoxal-5'-phosphate of gamma-aminobutyric acid receptor binding to synaptic membranes of cat cerebellum.

The binding of $\text{[}{}^3\text{H]γ-aminobutyric}$ acid to cat cerebellar membranes is $\text{reversibly}$ inhibited in a competitive manner by pyridoxal-5′-phosphate present during the binding assay. Structural analogues of the inhibitor have no such effect. If, on the other hand, the membranes are preincubated with pyridoxal-5′-phosphate followed by the addition of sodium borohydride, a rapid, $\text{irreversible}$ inhibition of subsequent γ-aminobutyric acid binding is observed. Since pyridoxal-5′-phosphate is known to inactivate certain enzymes by reacting with essential lysine residues, the present results suggest that such a lysine residue may be present within the γ-aminobutyric acid receptor.     

Acylcoenzyme A:cholesterol acyltransferase activity: solubilization and reconstitution in liposomes

P¹,P²-bis(5′-pyridoxal)diphosphate inactivates apophosphorylase $\text{b}$ from rabbit muscle, but not holophosphorylase. Inactivation is stoichiometric with the incorporation of 1 mol of the pyridoxal 5′-phosphate analog per mol of enzyme monomer. One of the two pyridoxal groups of the analog is kinked to the cofactor site forming a Schiff base, and is not reduced with NaBH₄. The other also forms a Schiff base, but is easily reduced by the same treatment. The residue involving in the latter binding has been identified as Lys-573. Its ε-amino group may interact with the phosphate group of the cofactor or of the substrate in the native enzyme.     

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.     

Stringent control of glycolysis in Escherichia coli.

When $\text{Escherichia}\text{coli}\text{CP78}\text{(}\text{rel}{}^{\mathrm{+}}$) growing on glucose was starved for isoleucine by the addition of valine, the intracellular levels of fructose 6-phosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were abruptly decreased to one-half, but those of glucose 6-phosphate and ATP remained constant. In contrast, this was not the case with CP79($\text{rel}{}^{\mathrm{?}}$). Chloramphenicol released the response observed in CP78. These results suggest that the glycolytic activity is also under the stringent control. Since only glucosephosphate isomerase[EC 5.3.1.9] was significantly inhibited by guanosine 5′-diphosphate 3′-diphosphate among several glycolytic enzymes tested, the enzyme might be responsible for the decrease observed in CP78.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain

The relationship between the susceptibility to convulsions, the content of pyridoxal 5′-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-σ-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5′-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5′-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5′-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5′-phosphate levels. The correlation between pyridoxal 5′-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

5′-Deoxypyridoxal inhibition of glucocorticoid receptor binding in HeLa S3 cells and rat thymocytes

Recent evidence suggests a possible role for pyridoxal 5′-phosphate, the active physiological form of vitamin B₆, in steroid hormone action (1–3). We now report that 5′-deoxypyridoxal, a synthetic vitamin B₆ antagonist, causes a rapid and complete loss of dexamethasone receptor binding in cytosol preparations in whole HeLa S₃ cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5′-deoxypyridoxal. In whole cell incubations of either HeLa S₃ cells or rat thymocytes at 37°C, a 50% reduction in [³H]dexamethasone binding was observed in the presence of 0.25 mM 5′-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S₃ cells incubated with 3.0mM or 1.0mM 5′-deoxypyridoxal respectively. Pyridoxal 5′-phosphate, 5′-deoxypyridoxine and 5′-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5′-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Irreversible inactivation of rat liver tyrosine aminotransferase

Homogenates prepared from rat livers irreversibly inactivate tyrosine aminotransferase, both endogenous and purified exogenous enzyme, in the presence of certain compounds which bind to pyridoxal 5′-P. The rate of inactivation ranged from a half-life of 0.72 to greater than 15 hr. The pyridoxal 5′-P binding compounds may be considered to be structural analogs for α-ketoglutarate or l-tyrosine, both of which are substrates for the enzyme. l-Cysteine and l-DOPA are the most effective compounds tested of each of the two structural analog classes, respectively. Absence of the carboxyl group from l-cysteine or l-DOPA has little effect on the half-life of the enzyme, whereas absence or substitution of the amino group results in an increased enzyme half-life. Absence of the —SH group from l-cysteine or of the 3′-OH group from l-DOPA results in little or no inactivation of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increased to greater than 15 hr). Semicarbazide and hydroxylamine have little effect on the stability of the enzyme. Addition of pyridoxal 5′-P to homogenates incubated with l-cysteine or l-DOPA inhibits the inactivation of the enzyme. However, the addition of cofactor to inactivated enzyme does not restore lost activity.There is a disappearance of antigenic cross-reacting material during inactivation of the enzyme. This loss of specific cross-reacting material occurs at a slower rate than the loss of enzyme activity, indicating that enzymatic activity is lost prior to loss of antigenic recognition. A three-step proposal is presented to explain the data observed in which the first step is a reversible loss of pyridoxal 5′-P from the enzyme, followed by a specific irreversible inactivation of the enzyme, and ending with nonspecific proteolysis or degradation of the inactivated enzyme molecules.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Cyclic AMP inhibition of reactions of glyceraldehyde 3-phosphate dehydrogenase

Adenosine 3′,5′-monophosphate (cyclic AMP) is an inhibitor of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate and benzaldehyde. Inhibition appears to be competitive toward glyceraldehyde 3-phosphate and of a mixed type toward NAD⁺. In the absence of arsenate a plot of $\text{1}\text{V}$ vs (I) is sigmoidal at constant concentrations of glyceraldehyde 3-phosphate and NAD⁺ and linear at constant concentrations of benzaldehyde and NAD⁺. Thus, sigmoidal inhibition plots are dependent on the nature of the aldehyde substrate as was found previously to be the case with inhibition of these reactions by highly branched acyl phosphates. In the presence of 0.013 m arsenate the plots of $\text{1}\text{V}$ vs [I] are linear.     

Cyclic 3',5'-adenosine monophosphate stimulates trehalose degradation in baker's yeast

The levels of cyclic 3′,5′-AMP and trehalose, as well as the specific activity of the trehalase have been investigated in cells of baker's yeast ($\text{Saccharomyces cerevisiae}$) during the lag phase preceding growth. During the first few minutes a substantial increase in the intracellular concentration of cyclic 3′,5′-AMP was observed, followed by a 6–8 fold increase in trehalase activity concomitant with the rapid degradation of trehalose. Cell free extracts prepared from resting yeast were shown to contain a cryptic trehalase, which under physiological conditions could be activated by cyclic 3′,5′-AMP to the same degree as $\text{in vivo}$. These observations suggest that in the lag phase of growth, the level of trehalose in baker's yeast is under control of a system, regulated by the level of cyclic 3′,5′-AMP.     

S1 nuclease of Aspergillus oryzae: characterization of the associated phosphomonoesterase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae was found to catalyze the hydrolysis of 2′- or 3′-phosphomonoester groups from several mono- and oligonucleotides. The specificity of the enzyme for mononucleotide substrates was determined by steady-state kinetic measurements at pH 4.5. The values of V were similar for all ribonucleoside 3′-phosphates tested, and they were 50–400 times greater than those for the corresponding deoxyribonucleotides or ribonucleoside 2′-phosphates. Purine nucleotides had lower apparent K_m values than pyrimidine nucleotides. Apparent K_m values of mononucleotides were also strongly dependent on the type of sugar and the positions of phosphoryl groups. Substrate specificity, as expressed by $\text{V}\text{K}{}_{\mathrm{m}}$, occurred in the following order: ribonucleoside 3′,5′-bisphosphate > ribonucleoside 3′-phosphate > deoxyribonucleoside 3′,5'-bisphosphate > deoxyribonucleoside 3′-phosphate ≈ ribonucleoside 2′-phosphate. S₁ nuclease also catalyzed the dephosphorylation of the dinucleotide ApAp at a high rate and the release of PP_i from adenosine 3′-diphosphate 5′-phosphate at a low rate. The phosphomonoesterase activity of the enzyme was competitively inhibited by single-stranded DNA and 5′-nucleotides. Apparent K_i values for adenosine compounds occurred in the order ATP < ADP < AMP ? adenosine. Tests of S₁ nuclease for phosphotransferase activity at pH 4.5 and 7.0 were negative.