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At least 973 records · Page 54Linked to original sources
PubMed1987

Radiolabeling of a wound-inducible pyridoxal phosphate-utilizing enzyme: evidence for its identification as ACC synthase.

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase, a pyridoxal phosphate-utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate-limiting step in the biosynthesis of the plant hormone ethylene. We report the partial purification (400-fold) of ACC synthase from wounded pink tomato pericarp. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase-enriched fraction was achieved by reduction using tritiated sodium borohydride. Evidence that this radiolabeled protein is ACC synthase is presented.

Autoradiography↗
PubMed1974

Involvement of vitamin B6 in the dethiomethylation of methionine by rumen microorganisms.

The dethiomethylation of methionine by a dialyzed extract obtained from the protozoa-rich fraction of rumen fluid is stimulated 2.5-fold by pyridoxal phosphate and strongly inhibited by deoxypyridoxine, a pyridoxal phosphate antagonist. These effects are not seen with undialyzed extracts or with whole rumen fluid. It is suggested that the anaerobic dethiomethylation of methionine by rumen microorganisms requires pyridoxal phosphate as a cofactor.

Alcohols↗
PubMed1977

Inactivation of avian myeloblastosis virus DNA polymerase by specific binding of pyridoxal 5'-phosphate to deoxynucleoside triphosphate binding site.

Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.

Avian Leukosis Virus↗
PubMed1989

Pyridoxal 5'-phosphate dependent histidine decarboxylase: overproduction, purification, biosynthesis of soluble site-directed mutant proteins, and replacement of conserved residues.

The hdc gene coding for the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii has been expressed in Escherichia coli under control of the lac promoter. The enzyme accumulates to 7-8% of total cell protein and is purified to homogeneity by passage through three columns. Fourteen site-directed mutant enzymes were constructed to explore the roles of residues of interest, especially those in the sequence Ser229-X230-His231-N epsilon-(phosphopyridoxylidene)Lys232, since identical sequences also appear in several other decarboxylases. Most of the overproduced mutant proteins were aggregated into inclusion bodies, but when the late log phase cultures were cooled from 37 to 25 degrees C before induction, the mutant proteins were obtained as soluble products. Ala or Cys in place of Ser-229 yielded mutant enzymes about 7% as active as wild-type, indicating that this serine residue is not essential for catalysis but contributes to activity through conformational or other effects. Of the replacements made for His-231 (Asn, Gln, Phe, and Arg), only Gln and Asn gave partially active enzymes (about 12% and 0.2% of wild-type, respectively). The side-chain amide of Gln may act by mimicking the positionally equivalent tau-nitrogen on the imidazole ring of histidine to provide an interaction (e.g., a hydrogen bond) required for efficient catalysis. The Lys-232 residue that interacts with pyridoxal 5'-phosphate appears central to catalytic efficiency since replacing it with Ala yields a mutant protein that is virtually inactive but retains the ability to bind both pyridoxal 5'-phosphate and histidine efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes.

We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.

Colorimetry↗
PubMed1983

Affinity labeling of erythrocyte band 3 protein with pyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport.

Transport of pyridoxal 5-phosphate (PLP) into erythrocytes was inhibited by inhibitors of anion transport including stilbene disulfonate compounds, indicating that it is mediated by Band 3 protein. When erythrocytes were treated with PLP and large amounts of free lysine and NaBH4, two membrane-spanning fragments of Band 3 (Mr = 17,000 and 35,000) were specifically labeled. When the cells were pretreated with 4,4'-dinitrostilbene 2,2'-disulfonate, the labeling in the 35,000-dalton fragment was inhibited. Erythrocytes labeled by PLP in both the 17,000- and 35,000-dalton fragments transported PLP at a decreased rate, whereas the cells labeled in only the 17,000-dalton fragment had essentially the same transport activity as the control when 4,4'-dinitrostilbene 2,2'-disulfonate was removed. The extent of inhibition of transport of inorganic phosphate in the labeled cells was similar to that of PLP. The results indicate that the 35,000-dalton fragment participates in the anion transport of the cell membrane.

Affinity Labels↗
PubMed1996

Highly sensitive and simple liquid chromatographic determination in plasma of B6 vitamers, especially pyridoxal 5'-phosphate.

A simple and sensitive high-performance liquid chromatographic method for measuring the major vitamers of vitamin B6, i.e. pyridoxal and pyridoxal 5'-phosphate (PLP), and 4-pyridoxic acid (4-PA) in plasma was developed. The vitamers and 4-PA from plasma were extracted with 0.8 mol/l perchloric acid. The separation by HPLC is accomplished using an ODS reversed-phase column and a mobile phase of 0.1 mol/l potassium dihydrogen phosphate containing 0.1 mol/l sodium perchlorate, 0.5 g/l sodium bisulfite adjusted to pH 3, at a flow-rate of 1.0 ml/min. The vitamers and 4-PA were eluted within 13 min and their concentration is determined with a fluorometric detector (excitation, 300 nm; emission, 400 nm). In this method, PLP in plasma can be determined with high sensitivity using derivatization with sodium bisulfite in the mobile phase.

Animals↗
PubMed1982

Characterization of pyridoxal 5'-phosphate disappearance from in vivo perfused segments of rat jejunum.

The disappearance of pyridoxal 5'-phosphate (PLP) from the lumen of in vivo perfused segments of rat jejunum was evaluated utilizing a single-pass technique. Net water flux was monitored by [14C] dextran, a nonabsorbable volume marker. Unlabeled PLP was measured by the tyrosine apodecarboxylase assay. PLP disappearance was linear with respect to PLP concentration at concentrations below 300 micrometers but was saturable at high concentrations (3 micrometers). PLP disappearance was significantly inhibited by 1 micrometer pyridoxamine 5'-phosphate and 5 micrometers and 10 micrometers l-phenylalanine but not by 1 micrometer pyridoxamine. Both in vivo disappearance (during perfusion) and in vitro PLP decay (exiting perfusate used as medium) correlated with the measured alkaline phosphatase activity of exiting perfusate under low-phosphate (1.1 micrometer) conditions. In contrast, in vivo PLP disappearance was not correlated with perfusate alkaline phosphatase activity under high-phosphate (80 micrometer) conditions. When exiting perfusate was ultracentrifuged at 105,000 x g for 1 hour, only 35% of the initial alkaline phosphatase activity remained in the supernatant. Conclusions were: 1) PLP disappearance from the lumen of an in vivo perfused segment of rat jejunum is saturable and inhibited by l-phenylalanine; 2) PLP disappearance appears in part to be a function of intraluminal alkaline phosphatase; and 3) A major portion of the alkaline phosphatase activity measured in the exiting perfusate represents membrane-bound enzyme.

Alkaline Phosphatase↗
PubMed1987

The effect of pyridoxine supplementation on plasma pyridoxal-5'-phosphate levels in children with the nephrotic syndrome.

Nineteen children with low plasma pyridoxal-5'-phosphate (PLP) levels in the nephrotic syndrome due to a variety of histological entities were successfully supplemented with oral pyridoxine hydrochloride. A dose of approximately 2 mg/kg/day of pyridoxine hydrochloride was found to effect adequate plasma PLP levels in most cases. A significant (p less than 0.001) positive correlation (r = 0.83) was found between plasma albumin and PLP levels prior to supplementation and after supplementation both in relapse and remission. Although vitamin B6 repletion led to no demonstrable effect on the clinical course of the syndrome, in view of the possible role which vitamin B6 deficiency may play in defective immune response and thromboembolic phenomena, both being known complications of the nephrotic syndrome, the administration of vitamin B6 supplements is recommended in the active disease state.

Administration, Oral↗
PubMed1978

Validation of the diagnostic value of plasma pyridoxal 5'-phosphate measurements in vitamin B6 nutrition of the rat.

The relationship of plasma pyridoxal 5'-phosphate (PLP) to PLP content of tissues and activities of PLP-dependent enzymes was examined to establish its value in assessing vitamin B6 nutrition. Weanling rats were fed ad libitum for 9 weeks purified diets which supplied 0, 4, 12, 24, and 100 micrograms of pyridoxine daily. Growth increased with increasing pyridoxine intake, reaching a maximum at 24 micrograms/day. Liver and brain PLP also increased, attaining maximal values at 12 micrograms. By contrast, muscle and plasma PLP did not saturate when vitamin B6 intake was increased to 100 micrograms. Erythrocytic holoenzyme activity of aspartate (Asp) aminotransferase became maximal with 24 micrograms but that of alanine (Ala) aminotransferase did not. Hepatic holoenzyme activities of Ala, Asp and tyrosine aminotransferases reached maximal values with only 4 micrograms vitamin B6 but that of serine dehydratase became maximal with 12 micrograms. Measurement of coenzyme saturability suggested that apoenzyme degradation, coenzyme affinity and PLP transfer determine the activities of these enzymes. It is concluded that plasma and muscle PLP behave as mobilizable storage pools and that plasma PLP is a sensitive and reliable indicator of vitamin B6 nutrition.

Animals↗
PubMed1975

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.

Alkaline Phosphatase↗
PubMed1989

Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase.

The amino acid sequences associated with pyridoxal 5'-phosphate binding sites in chicken liver fatty acid synthase have been determined: a site whose modification causes selective inhibition of the enoyl reductase activity and a site (site I) that is not associated with enzymatic activity. The amino acid sequences of peptides obtained by trypsin hydrolysis of the pyridoxamine 5'-phosphate labeled enzyme were determined. For the site associated with enoyl reductase activity, the sequence similarities between chicken and goose are extensive and include the sequence Ser-X-X-Lys, a characteristic structural feature of pyridoxamine enzymes. In addition, the spatial relationships between the pyridoxal 5'-phosphate binding sites and reductase site(s) have been studied with fluorescence resonance energy-transfer techniques. The distances between site I and the enoyl reductase and beta-ketoacyl reductase sites are greater than 50 and 41-44 A, respectively. The distance between the two reductase sites is greater than 49 A.

Amino Acid Sequence↗
PubMed2001

Pyridoxal 5'-phosphate inhibits DNA binding of HNF1.

An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH(2)-terminal. The cDNA encoding HNF1 was ligated to 6 x His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH(2)-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5'-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5'-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5'-phosphate that inhibited 50% of DNA binding was around 100 microM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5'-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.

Amino Acid Sequence↗
PubMed1996

Regulation of pyridoxal-5'-phosphate level by biogenic amines in mouse brain.

We investigated the relationship between the concentration of pyridoxal-5'-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.

Animals↗
PubMed1988

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine.

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.

Algorithms↗
PubMed1983

Tyrosine decarboxylase. Spectrophotometric assay and application in determining pyridoxal-5'-phosphate.

We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.

Enterococcus faecalis↗