Pyridoxal 5'-phosphate disappearance from perfused rat jejunal segments: correlation with perfusate alkaline phosphatase and water absorption

The relative effects of perfusate alkaline phosphatase activity and net water absorption on 2 microM pyridoxal 5'-phosphate (PLP) luminal disappearance from rat jejunum were studied in a single-pass, in vivo perfused intestinal segment model. Perfusate consisted of unlabeled PLP in buffer (pH = 7.4). Net water flux was monitored by inclusion of [3H]polyethylene glycol. PLP was measured by the [14C]tyrosine apodecarboxylase assay. Single and multiple regression analysis of results during perfusion of 2 microM PLP in Krebs bicarbonate buffer demonstrated no correlation between perfusate alkaline phosphatase activity and net water absorption and significant correlations between PLP luminal disappearance and both perfusate alkaline phosphatase activity and net water absorption. Correlation for the latter was improved when disappearance results were corrected for variations in perfusate alkaline phosphatase activity. When perfusate buffers were selected to yield divergent rates of net water absorption, the one associated with greater net water absorption was also associated with greater PLP disappearance. That this could not be explained by changes in perfusate alkaline phosphatase activity was demonstrated both by assessment of the rate of decay of PLP added in vitro to exited perfusate incubated at 37 degrees C and by measurement of alkaline phosphatase activity under conditions defined by the buffers using a modified spectrophotometric assay. Conclusions were: (1) In vivo PLP luminal disappearance correlates significantly with both perfusate alkaline phosphatase activity and net water absorption; (2) these two factors appear to act as independent variables; and (3) future studies on PLP intestinal absorption will need to take both of these variables into account in the interpretation of results.     

Phosphorylcholine as a unique substrate for human intestinal alkaline phosphatase

• 1.1. The enzymatic nature of human liver, bone, placental and intestinal alkaline phosphatases (ALPs) were investigated with phosphorylcholine (PC), phosphoryl-ethanolamine, pyridoxal-5'-phosphate and p-nitrophenylphosphate at a weakly alkaline pH.
• 2.2. The apparent K_m value of the intestinal ALP with PC was the highest of all ALPs tested. Intestinal ALP hydrolyzes PC the most and has higher affinity for choline as a transphorsphorylating acceptor than the other ALPs. In addition, the intestinal ALP activity with PC was most susceptible to Na₂HPO₄, in the tested ALPs.
• 3.3. The present results suggest that PC is a unique substrate for human intestinal ALP, which may be related to the metabolism of PC or choline as part of phosphatidylcholine.

     

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

Pyridoxal 5'-phosphate dependent enzymes in the nematode Nippostrongylus brasiliensis.

Eight classes of pyridoxal 5'-phosphate dependent enzymes have been investigated in Nippostrongylus brasiliensis in parallel with rat tissues. The range of decarboxylases detected in N. brasiliensis was limited in comparison with rat tissues. N. brasiliensis possessed a highly active L-serine hydroxymethyltransferase, but in contrast with rat liver, 5-aminolevulinic acid synthetase was absent. Similar levels of L-serine and L-threonine dehydratase activities were detected in N. brasiliensis and rat liver, and both organisms lacked L-alanine racemase, L-tryptophan synthetase and L-methionine gamma-lyase. The demonstration of cystathionine beta-synthase and gamma-cystathionase in N. brasiliensis suggests the presence of a functional trans-sulphuration sequence. The substrate specificities of the nematode cystathionine beta-synthase and gamma-cystathionase varied significantly from those of the corresponding mammalian enzymes. Particularly striking was the ability of N. brasiliensis cystathionine beta-synthase to catalyse the non-mammalian 'activated L-serine sulphydrase' reaction (L-cysteine + R-SH----cysteine thioether + H2S). N. brasiliensis and rat liver exhibited comparable abilities to transaminate amino acids via the 2-oxoglutarate: glutamate system.     

Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon

Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.     

A possible new locus of alkaline phosphatase expressed in human testis

Summary Human testes contain trace amounts of heat-stable placental-like alkaline phosphatase. Using a recently described allotype-specific monoclonal antibody (F₁₁) toward placental alkaline phosphatase (PLAP), we show that the frequencies of reactivity of the testis enzymes differ greatly from those of the placental phenotypes. By means of the enzyme inhibitors L-Phe, L-Phe-gly-gly, L-Leu, and L-Leu-gly-gly, the testis enzyme can be clearly distinguished in all cases from the placental enzyme. These results argue that the testis enzyme is not a product of the placental gene and suggest the possible existence of a new locus of alkaline phosphatase.     

Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.     

Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B

This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5"-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5"-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.     

The hydrocortisone 21-phosphate as substrate for alkaline phosphatase

Summary The use of the hydrocortisone 21-phosphate as substrate for alkaline phosphatase is suggested for the localization of phosphatases hydrolyzing steroid-phosphates. The localization of these phosphatases in mouse tissues is described.This work was partially supported by a grant (M 68.0108) of The Population Council.     

Activation of human erythrocyte 2,3-bisphosphoglycerate phosphatase at physiological concentrations of substrate

In human erythrocytes the reactions of the 2,3-bisphosphoglycerate shunt are catalyzed primarily by one protein, 2,3-bisphosphoglycerate synthase-phosphatase. At low concentrations of 2,3-bisphosphoglycerate the phosphatase is activated by several anions including inorganic phosphate and sulfite, and the phosphate activation is inhibited by low concentrations of 3-phosphoglycerate [Z. B. Rose and J. Liebowitz (1970) J. Biol. Chem. 245, 3232-3241]. Phosphate and sulfite also activate at high but physiological concentrations of 2,3-bisphosphoglycerate (5 mM), but the inhibition by 3-phosphoglycerate is much weaker. The basal activity (without added phosphate or sulfite) was also found to be higher and to be 3-phosphoglycerate sensitive; this is attributed to activation either by 2,3-bisphosphoglycerate itself or by a contaminant in it. These results allow previous observations of 2,3-bisphosphoglycerate hydrolysis in intact erythrocytes to be reconciled with the properties of the purified enzyme under near-physiological conditions.