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P D van Poelje

Publications and source records attributed to P D van Poelje.

8 recordsLinked to original sources
PubMed2001

Isolation of a family of organic anion transporters from human liver and kidney.

Five distinct organic anion transporter cDNAs, hOAT1-5, were isolated from human liver and kidney. hOAT1, 2, and 3 are homologous to their respective rat orthologues OAT1-3, whereas hOAT4 and 5 are novel clones that have not been identified in other species. hOAT1- and hOAT3-transfected cells showed uptake of p-aminohippurate and fluorescein. Cells expressing hOAT2 showed uptake of p-aminohippurate, methotrexate, cAMP, and alpha-ketoglutarate. Northern blot analysis indicated differential tissue distribution for the transporter transcripts. These results indicate the existence of a family of organic anion transporting proteins in humans distinct from the oatp-like family of transporters.

Amino Acid Sequence↗
PubMed1994

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Adenosine Monophosphate↗
PubMed1990

Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens.

To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli. The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis. The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure. The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a [Gallagher, T., Snell, E. E., & Hackert, M. L. (1989) J. Biol. Chem. 264, 12737-12743]. Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing, expression, and site-directed mutagenesis of the gene from Clostridium perfringens encoding pyruvoyl-dependent histidine decarboxylase.

The DNA encoding pyruvoyl-dependent histidine decarboxylase (HisDCase) of Clostridium perfringens was cloned, sequenced, and overexpressed in Escherichia coli. The gene encodes a single polypeptide of 320 amino acids, Mr 35,526, demonstrating that clostridial HisDCase, which has an (alpha beta)6 structure, is synthesized as a precursor (proHisDCase, pi 6). No pi subunits of proHisDCase were observed in crude or purified preparations of the cloned HisDCase; they appear to undergo rapid cleavage in vivo to the alpha (Mr 24,887) and beta (Mr 10,526) subunits characteristic of this HisDCase. This cleavage occurs between Ser-96 and Ser-97; Ser-97 gives rise to the catalytically essential pyruvoyl group blocking the N-termini of the alpha subunits of the active enzyme. When Ser-97 was converted to an alanyl residue by site-specific mutagenesis, the expressed, inactive protein (pi' 6) contained a single peptide species (pi', Mr 35,510) that was not cleaved either in vivo or in vitro. These results support previous conclusions that activation of the wild-type clostridial proenzyme occurs via nonhydrolytic serinolysis. Although clostridial HisDCase has only a 47% sequence similarity to HisDCase from Lactobacillus 30a, all of the residues known to be important for substrate binding and catalytic action of the Lactobacillus HisDCase are conserved in the C. perfringens enzyme. While the encoded N-terminal Met of clostridial HisDCase is removed by E. coli, the cloned enzyme retains a 10-residue presequence (NKNLEANRNR) not present in the mature enzyme isolated from C. perfringens.

Alanine↗
PubMed1988

Amine cations promote concurrent conversion of prohistidine decarboxylase from Lactobacillus 30a to active enzyme and a modified proenzyme.

Activation of prohistidine decarboxylase (pi 6) from Lactobacillus 30a proceeds by an intramolecular, pH- and monovalent cation-dependent reaction in which its constituent pi chains are cleaved nonhydrolytically between Ser-81 and Ser-82 with loss of NH3 and conversion of Ser-82 to the pyruvoyl residue of active histidine decarboxylase (alpha beta)6. Amines with pKa values more than 7.0 substitute for K+ or NH4+ in the activation of prohistidine decarboxylase, but they also catalyze its inactivation in a competing reaction, pi 6----pi'6. Sequence analysis of the appropriate tryptic peptide from amine-inactivated prohistidine decarboxylase established that inactivation results from conversion of Ser-82 of the pi chain to an aminoacrylate residue. The inactivated proenzyme (pi'6) does not form histidine decarboxylase; this fact eliminates one of two postulated mechanisms of activation and, thus, favors activation by beta-elimination of the acyl group of an intermediate ester formed between Ser-81 and Ser-82. L-Histidine is bound by the proenzyme (Kd = 1.7 x 10(-4) M) and is an effective activator; one binding site is present per pi subunit. K+, NH4+, and Na+ competitively inhibit (Ki values = 2.8-4.4 x 10(-3) M) activation by histidine. The data suggest the presence of two classes of monovalent cation binding sites on prohistidine decarboxylase: one (near Ser-82) is readily saturable and one is unsaturable even by 2.4 M K+.

Amines↗
PubMed1987

Use of p-aminobenzoic acid and tritiated cyanoborohydride for the detection of pyruvoyl residues in proteins.

A procedure for the detection of covalently bound pyruvic acid in purified proteins or in crude extracts is described. The dialyzed sample is first treated with sodium cyanoborohydride to reduce any Schiff bases present and then incubated with p-aminobenzoic acid and sodium [3H]cyanoborohydride. Derivatized proteins are visualized by fluorography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel slices containing the labeled proteins are hydrolyzed, and, after removal of polyacrylic acid, the hydrolysate is subjected to ion-exchange high-performance liquid chromatography. The presence of pyruvic acid is established by the detection of a tritiated, 280-nm absorbing compound with a retention time corresponding to that of synthetic N-(p-carboxyphenyl)alanine. The procedure is capable of detecting protein-bound pyruvic acid in the picomolar range and is easily modified to screen for other covalently bound keto acids.

4-Aminobenzoic Acid↗