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D B McCormick

Publications and source records attributed to D B McCormick.

At least 37 records · Page 2Linked to original sources
PubMed1992

Modification of arginyl and lysyl residues of flavokinase from rat small intestine.

The amino acid composition of flavokinase has been determined to contain five arginyl and four lysyl residues per mole. Flavokinase is inactivated by arginine-specific reagents. The substrates riboflavin and especially ATP impede inactivation, whereas neither of the products, ADP or FMN, protect. Among lysine-modifying reagents, only 2,4,6-trinitrobenzene sulfonic acid caused inactivation of the enzyme, especially under conditions for denaturation. In this case it was noted that the activity was enhanced at the early stage of the reaction but this enhancement was repressed in the presence of ATP along with the significant protection of activity. Results with modification of arginyl residues and incorporation of 14C-phenylglyoxal suggest that one such residue is involved in the substrate-binding site. The involvement of lysyl residues in catalytic function remains unclear, but seems less critical.

Adenosine Diphosphate↗
PubMed1991

Uptake of N-(4'-pyridoxyl)amines and release of amines by renal cells: a model for transporter-enhanced delivery of bioactive compounds.

The importing of vitamin B6 by renal proximal tubular cells from the rat is facilitated and Na(+)-dependent and reflects specificity for the meta-phenolate pyridinium structure with a 5-hydroxymethyl function. This transporter can, however, accept competitively each of the natural nonphosphorylated vitamers (pyridoxine, pyridoxamine, and pyridoxal) and other B6 analogues differing only in the groups at position 4. A series of N-(4'-pyridoxyl)amines was synthesized by sodium borohydride or boro[3H]hydride reduction of aldimines formed by condensing the amines with pyridoxal. The unlabeled B6-secondary amine compounds were found to competitively inhibit the uptake of [4'-3H]pyridoxine by the renal cells. Moreover, the 3H-labeled N-(4'-pyridoxyl)amines were shown to enter the cells by the process facilitated by the B6 transporter. Upon entry the labeled compounds were converted to N-(5'-phospho-4'-pyridoxyl)amines in a reaction catalyzed by pyridoxal kinase, an enzyme that tolerates considerable functional variation in position 4 of the B6 structure. The 5'-phosphates were subsequently converted within the cell to pyridoxal 5'-phosphate with liberation of the original amine in a reaction catalyzed by pyridoxamine (pyridoxine) 5'-phosphate oxidase, an enzyme with broad specificity for 4'-substituted amines on the 5'-phospho-B6 structure. This system illustrates how knowledge of transporter specificity can permit design of a compound with potential biologic activity. A drug or other intracellular effector may be piggybacked onto a transported solute (e.g., vitamin or other nutrient) that gains facilitated entry to a cell and is, thereafter, metabolized to release the active compound.

Amines↗
PubMed1991

Stereospecificity of the metal.ATP complex in flavokinase from rat small intestine.

Transfer of the gamma-phosphoryl group of ATP to riboflavin is catalyzed by flavokinase, which prefers Zn(II), and is essential in the biosynthesis of the flavocoenzyme, FMN. To study the mechanism and steric disposition of ATP binding, adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) were tested in comparison to ATP with representative divalent metal ions. Activities with 0.1 mM Zn(II) for 1 mM ATP thio analogues compared to ATP with flavokinase were 60% for the S-isomer of ATP beta S, 312% for the R-isomer of ATP beta S, and 14% for ATP gamma S. With Mg(II), flavokinase showed stereospecificity for the R-isomer of ATP beta S with a Vmax ratio, S/R = 0.125. With Cd(II), the enzyme showed preference for the S-isomer of ATP beta S with a Vmax ratio, S/R = 1.261. The Vmax ratio progressively increases from Mg(II) to Cd(II) in the order of affinity for sulfur. The ratios, (Vmax/Km)Mg/(Vmax/Km)Cd, for the diastereomers of ATP beta S were expectedly greater than 1 for one diastereomer (R = 6.597) and less than 1 for the other (S = 0.292). Activities with ATP gamma S were much lower than with ATP or ATP beta S. With Mg(II), the gamma analogue was a good substrate; however, with Cd(II), it was hardly active. Altogether these results indicate that flavokinase uses the lambda, beta, gamma-bidentate Zn.ATP as a substrate.

Adenosine Triphosphate↗
PubMed1991

Characterization of ascorbic acid uptake by isolated rat kidney cells.

Isolated kidney cells accumulated L[1-14C]ascorbic acid in a time-dependent manner and reached a steady state after 15 min at 37 degrees C. Initial velocity for uptake was over 300 pmol/mg protein per min when cells were separated from the bathing solution using a density gradient established during centrifugation. The uptake process was saturable with an apparent concentration at half maximal uptake of 36 mumols/L. Ascorbate uptake was reduced by metabolic inhibitors and was temperature dependent. Although ascorbic acid is an acid anion at pH 7.4, uptake did not appear to be inhibited by other acid anions such as p-aminohippurate and probenecid; however, involvement of the ion gradient established by Na+, H(+)-adenosine triphosphatase could not be confirmed. Replacing the sodium ion with other monovalent ions reduced the accumulation of ascorbate significantly. Isoascorbic and dehydroascorbic acids inhibited ascorbate uptake (34 and 13 mmol/L, respectively), whereas high concentrations of glucose showed some stimulation. These findings indicated that ascorbic acid is reabsorbed by the kidney in a sodium-dependent active transport process that is not common to other acid anions and has some specificity for the ascorbic acid structure.

Adenosine Triphosphate↗
PubMed1991

Urinary riboflavin and its metabolites: effects of riboflavin supplementation in healthy residents of rural Georgia (USA).

The following study was designed to provide a comprehensive assessment of urinary riboflavin and flavin metabolites before and after ingestion of a riboflavin load in normal human volunteers. With respect to riboflavin, the total flavin excretion, the flavin-to-creatinine ratio, and the response of the flavin catabolites to the supplement indicate a well-nourished population. Differences in the effects of supplementation on the specific flavin catabolites across sex and age groups are included in detail for the first time. The feasibility of the use of flavin catabolites for determination of riboflavin status should be tested further in individuals with clinical signs of ariboflavinosis.

Adolescent↗
PubMed1990

Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver.

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.

Adenosine Triphosphate↗
PubMed1990

Flavin composition of human milk.

The identity and quantity of greater than 95% of the flavins present in human milk were assessed by acid-phenol extractions followed by high-performance liquid chromatography. Flavin adenine dinucleotide (FAD) and riboflavin were the predominant flavins, followed by 10-(2'-hydroxyethyl)-flavin. In addition, traces of 7 alpha- and 8 alpha-hydroxyriboflavins (7-hydroxymethylriboflavin and 8-hydroxymethylriboflavin, respectively) were detected. The flavin content of human milk samples in this study was higher than contents reported in earlier studies where no correction for the internal fluorescence quenching of FAD was made. This finding may have implications for dietary recommendations concerning both lactating women and infants. In practical terms, the types and amounts of flavins in human milk are very similar to those recently reported for cow milk.

Animals↗
PubMed1990

Qualitative and quantitative assessment of flavins in cow's milk.

Although milk is one of the primary sources of riboflavin in the North American diet, the amounts of riboflavin and other flavin derivatives in milk have not been accurately quantified. In this study, a comprehensive assessment of milk was performed to identify and quantify greater than 95% of the flavins in this food. Riboflavin and flavin adenine dinucleotide (FAD) were the predominant flavins in all milk samples; 10-(2'-hydroxyethyl)flavin was next most common. The latter is potentially an antivitamin that may exert its effect at either the absorption or utilization level. Therefore, the presence of this analog should be considered when evaluating the riboflavin efficiency of milk. The large quantity of FAD indicates that precautions against underestimating FAD are warranted when determining total flavin content by fluorescence. This is particularly important because the fluorescence of FAD at pH 7.0 is only 10-20% of that for riboflavin. The presence of 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) in milk was ascertained by extensive testing. Traces of 8 alpha-hydroxyriboflavin (8-hydroxymethylriboflavin) were also detected. These metabolites may serve as indicators of cellular biochemical activity.

Animals↗
PubMed1990

Biochemical aspects.

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Biochemical Phenomena↗
PubMed1989

Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase.

Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Pyridoxine uptake by rat renal proximal tubular cells.

Uptake of [3H]pyridoxine by freshly isolated rat renal proximal tubular cells was temperature dependent and exhibited saturation kinetics, with estimated Kt and Vmax of 1.3 microM and 14 pmol/(10(6) cells.0.5 min), respectively. Pyridoxamine, 4'-deoxypyridoxine and 5'-deoxypyridoxal were as effective as unlabeled pyridoxine in inhibiting tracer uptake, whereas pyridoxamine-5'-phosphate, pyridoxal and 4'-pyridoxic acid had no effect, and other vitameric forms and structural analogs caused intermediate inhibition of cellular vitamin uptake. After 30 min, 88% of accumulated radioactive vitamin B-6 in cell extracts had been metabolized to phosphorylated forms and pyridoxal, which supports metabolic trapping as a mechanism responsible for the cellular accumulation observed. Initial uptake was significantly decreased in the presence of ethionine and ouabain; however, carbonylcyanide-p-trifluoromethoxyphenylhydrazone had no effect. Partial to complete substitution of NH4Cl and LiCl for NaCl had no effect, while partial substitution with RbCl caused significant dose-related inhibition of initial pyridoxine uptake. Amiloride also significantly decreased initial uptake. These data are consistent with a facilitated uptake process for pyridoxine that has substrate specificity and may be modulated by sodium-hydrogen exchange and/or pH gradient effects. Cellular uptake is followed by intracellular metabolic trapping catalyzed by pyridoxal kinase.

Animals↗