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Biomedical subjects

P Stover

Publications and source records attributed to P Stover.

17 recordsLinked to original sources
PubMed2001

Relationship between dopamine-stimulated phospholipid methylation and the single-carbon folate pathway.

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single-carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R-mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA-stimulated incorporation of [14C]-labeled formate into phospholipids in cultured SH-SY5Y neuroblastoma cells. These interventions included: (i) Overexpression of methenyltetrahydrofolate synthetase (MTHFS). (ii) Treatment with 5-formylTHF. (iii) Treatment with the MTHFS inhibitor 5-formyltetrahydrohomofolic acid (5-formylTHHF). (iv) Growth in nucleoside-free media. 31P-NMR was also used to follow DA-induced changes in cell phospholipid composition. MTHFS overexpression and 5-formylTHHF treatment, both of which lower 5-methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5-formylTHF, which increases 5-methylTHF, caused a dose-dependent increase in both basal and DA-stimulated PLM. Growth in nucleoside-free media caused time-dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA-stimulated PLM was initially increased followed by a later decrease. Together, these findings indicate a close functional relationship between single-carbon folate metabolism and DA-stimulated PLM, consistent with a role for 5-methylTHF as the methyl donor for the D4R-mediated process.

Carbon Radioisotopes↗
PubMed1996

Tracking skin integrity: a template for hospital and vendor collaboration.

Collaborative endeavors are particularly attractive in a time when resources are under extensive demand, but for organizations to be successful at collaboration, many shared values and qualities are necessary. In 1992, a hospital and a vendor of therapeutic bed surfaces entered into a collaborative partnership to test clinical indicator ability to track skin care outcomes. The vendor contributed major funding for the project, a national database from which clinical indicators on pressure ulcers were determined, and data processing and analysis. The hospital provided project coordination, clinical knowledge, data collectors, and access to patients. The article describes the process, problems, and benefits of collaboration and underscores the need for a trusting psychological climate, strong mutual interest in the project, clearly stated goals with resources to meet them, collaborator consideration of each other as peers, and skill in tension management if collaboration is to be successful.

Humans↗
PubMed1995

Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.

Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed.

Abnormalities, Multiple↗
PubMed1994

Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax.

Amino Acid Sequence↗
PubMed1993

Diffraction grade crystals of Escherichia coli serine hydroxymethyltransferase.

Diffraction grade crystals of serine hydroxymethyltransferase were obtained after extensive screening of source of enzyme, purification procedures, ligand binding and crystallization conditions. Serine hydroxymethyltransferase purified from Escherichia coli crystallizes in two forms that are suitable for crystal structure determination.

Animals↗
PubMed1993

The metabolic role of leucovorin.

Interest in determining if leucovorin, known chemically as 5-formyltetra-hydrofolate, plays a role in one-carbon metabolism is reemerging. While investigations in the 1940s suggested it was an important donor of one-carbon units in folate-mediated biosynthetic reactions, studies between the 1950s and 1980s disproved this hypothesis and dismissed its presence in biological systems as artifactual. Recently, new data has focused attention on the possible biological function of this compound that is widely used in cancer chemotherapy.

Animals↗
PubMed1992

Escherichia coli serine hydroxymethyltransferase. The role of histidine 228 in determining reaction specificity.

Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate. This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase. Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified. The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics. The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site. The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme. As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate. The released formaldehyde inactivates these mutant enzymes. The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.

Base Sequence↗
PubMed1992

Evidence for the accumulation of a stable intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate.

Solutions of 5,10-methenyltetrahydropteroylglutamate can be converted to a stable hydrated adduct by heating solutions at 50 degrees C at pH values of 3-5 for several hours. The adduct is stable at pH values from 4 to 9 for hours, but at pH values below 2 it is converted to 5,10-methenyltetrahydropteroylglutamate and at pH values above 8 it is converted to 5-formyltetrahydropteroylglutamate. Arguments are presented that the adduct is (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate formed from (11S)-5,10-hydroxymethylenetetrahydropteroylglutamate by formation of an ylide at C-11 which undergoes inversion of the electron pair to form the (11R) isomer. The (11R) hydrated adducted is believed to be the isomer of 5,10-methenyltetrahydropteroylglutamate referred to as anhydroleucovorin B by Cosulich et al. [Cosulich, D. C., Roth, B., Smith, J. M., Hultquist, M. E., & Parker, R. P. (1952) J. Am. Chem. Soc. 74, 3252-3263]. In addition, a new mechanism for the formation of 5-formyltetrahydropteroylglutamate from either 5,10-methenyltetrahydropteroylglutamate or 10-formyltetrahydropteroylglutamate via (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate is proposed. A requirement for this pathway is that the formyl proton of 10-formyltetrahydropteroylglutamate exchange with solvent protons. The exchange of this formyl proton was observed at all pH values from 5.5 to 11.5 at a rate which exceeded by more than an order of magnitude the rate of formation of 5-formyltetrahydropteroylglutamate.

Hydrogen-Ion Concentration↗
PubMed1992

Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.

Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6R)-5,10-methenyltetrahydropteroylpolyglutamate believed to be (6R,11R)-5,10-hydroxymethylenetetrahydropteroylpolyglutamate , a putative intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V. (1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates and the role of glycine in the reaction was addressed. Evidence suggests that (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is a catalytically competent intermediate in the enzyme-catalyzed hydrolysis of (6R)-5,10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 microM for (6R)-5,10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R,11R)-5,10-hydroxymethylenetetrahydropteroyltetraglutamate++ + is below 0.5 microM. The kcat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbing at 502 nm which is formed from glycine and (6S)-5-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5,10-methenyltetrahydropteroyltetraglutamate, suggesting that the enzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H]glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity in mechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6R)-5,10-methenyltetrahydropteroylpolyglutamates.

Calorimetry, Differential Scanning↗
PubMed1992

Synthesis of (6S)-5-formyltetrahydropteroyl-polyglutamates and interconversion to other reduced pteroylpolyglutamate derivatives.

5-Formyltetrahydropteroylpolyglutamates can be synthesized and purified directly from dihydropteroylpolyglutamates in a single-step procedure without purification of intermediates and with yields greater than 90%. The procedure involves a coupled enzymatic synthesis of 10-formyltetrahydropteroylpolyglutamates using the enzymes dihydrofolate reductase, serine hydroxymethyltransferase, and C1-tetrahydrofolate synthase with catalytic amounts of NADPH. The 10-formyltetrahydropteroylpolyglutamates are subsequently converted to 5-formyltetrahydropteroylpolyglutamates at 90 degrees C with near quantitative yields. 5-Formyltetrahydropteroylpolyglutamates are the only stable reduced derivatives of tetrahydropteroylpolyglutamates and can be purified and stored indefinitely without decomposition. Additionally, 5-formyltetrahydropteroylpolyglutamates can be readily converted to other derivatives of tetrahydropteroylpolyglutamates with yields greater than 95%. Also described is the synthesis of tetrahydropteroylglutamates labeled at C-11 with either 14C or 13C. Rapid purification procedures for serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from frozen rabbit livers are presented.

Aminohydrolases↗
PubMed1991

5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase.

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.

Animals↗
PubMed1991

Treatment of pyoderma gangrenosum with cyclosporine: results in seven patients.

The mainstay of therapy for pyoderma gangrenosum has been corticosteroids, but many patients respond poorly. During the past 2 years we have treated seven patients who had pyoderma gangrenosum with cyclosporine after their condition proved resistant to conventional therapy. No evidence of permanent toxicity from cyclosporine was detected and treatment with other immunosuppressive agents was discontinued in five of seven cases. Tuberculosis was reactivated in one patient. Three patients had a remission, three had an intermediate response, and one did not respond. These results indicate that cyclosporine is useful in the treatment of patients with refractory pyoderma gangrenosum and suggest an immune mechanism in the pathogenesis of this disorder.

Adult↗
PubMed1990

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate.

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.

Animals↗