Donald B. McCormick

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.

The interaction of flavins with egg white riboflavin-binding protein

Abstract The properties of the riboflavin-binding site in the riboflavin-binding protein from egg white have been elucidated by determining constants for binding of flavin analogs to the protein and by changes in absorption spectra of free and bound flavins. The spectral changes and unfavorable interaction of the protein with charged species indicate that the overall flavin environment in the holoprotein is hydrophobic. Modification of either ring or side-chain portions of flavin usually results in a decrease of binding energy. Although no one portion of the structure is absolutely essential, both 7- and 8-methyl groups and 2′-hydroxyl group contribute most significantly to binding. The binding site at the region of C-2 and N-3 of the isoalloxazine is rather insensitive to the relative site of a substituent and thus relatively open, whereas considerable steric limitation is imposed at C-8, N-10, especially C-1′, and 4carbonyl positions. The hydroxyl groups of the N-10 side chain contribute in a stereoselective manner by formation of hydrogen bonds. Studies with model compounds that represent only a part of flavin suggest that the dimethylbenzenoid portion of the ring is involved in primary interactions of binding, and relatively buried in the protein. The quenching of protein fluorescence upon binding is mainly due to ground-state stacking interaction between a trytophanyl residue at the binding site and the quinoxaline portion, and not to Forster energy transfer.

Thyroid hormone regulation of flavocoenzyme biosynthesis

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.

Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible.

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.

High-performance liquid chromatography of biotin and analogues

Biotin, analogues, and chemical intermediates were separated by high-performance liquid chromatography (HPLC) using reversed-phase and anion-exchange chromatographic conditions. Reversed-phase separations provided a wide range of retention times and resolution of nearly all the biotin compounds from mixtures of the analogues. Anion-exchange separations gave generally shorter retention times as compared to reversed-phase separations and greater resolution between biotin l- and d-sulfoxide. However, fewer analogues were resolved from mixtures of the compounds with anion-exchange HPLC.

Isolation and identification of alternative riboflavin-binding proteins from human plasma

Abstract A number of riboflavin-binding proteins have been purified from human blood using faavinyl affinity chromatography materials with biospecific elution by riboflavin. Repeated extraction of plasma with flavinyl agarose beads yields a maximum of 6.5 mg of protein from 100 ml of plasma. These proteins have been identified as IgGs by: (i) electrophoretic mobilities corresponding to plasma γ-globulins on cellulose acetate; (ii) electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gels which indicate molecular weights of 155,000 for the parent molecule and 55,000 and 25,000 for the subunits (obtained by reduction with β-mercaptoethanol); (iii) quantitative binding to Protein A-Sepharose CL-4B (Pharmacia), which is specific for IgG and some subclasses of IgA; and (iv) reaction with antisera to human IgG (but not antisera to human IgA) upon Ouchterlony double diffusion analysis. A fraction of the IgG molecules (approximately 20%) bind [2-14C]riboflavin with KD ≤ 4 μ m , as estimated by equilibrium dialysis. Similar proteins have been obtained from the plasma of male, female, and pregnant female donors and fetal (cord) blood. Fetal blood, however, also yielded small quantities (approximately 25 μg 15 ml ) of protein which binds riboflavin even more tightly. These findings help establish that human blood, like that of other animals, contains proteins which may serve an accessory or auxiliary role to albumin in the utilization of this vitamin, especially during pregnancy.

Riboflavin 5′-Hydroxymethyl Oxidation MOLECULAR CLONING, EXPRESSION, AND GLYCOPROTEIN NATURE OF THE 5′-ALDEHYDE-FORMING ENZYME FROM SCHIZOPHYLLUM COMMUNE

Vitamin B2-aldehyde-forming enzyme catalyzes oxidation of the 5′-hydroxymethyl of riboflavin to the formyl group. We have purified the enzyme from the culture media ofSchizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981)J. Biol. Chem. 256, 6682–6685) for cell-free extract. By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa. The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini. We have isolated a full-length cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences. From the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp. The enzyme was determined to be a glycoprotein; however,O-deglucosylation only slightly affects activity. Computer searches showed that the B2-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins. The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B2-aldehyde-forming enzyme activity.

Correlation of H2O2 production and liver catalase during riboflavin deficiency and repletion in mammals

A substantial decrease in liver peroxisomal catalase was found during riboflavin deficiency in rats. This decrease is greater than that found among other hemoproteins and seems to follow decrease in flavin-dependent peroxisomal oxidases. This is not due to a general depression of peroxisomal enzymes, since Cu-dependent urate oxidase activity was not changed. Furthermore, the level of catalase activity as well as flavin-dependent oxidases was restored by riboflavin repletion. These results suggest that hydrogen peroxide, the substrate for catalase produced by several flavoprotein oxidases, induces catalase in mammals as has been indicated for certain bacteria.

Vitamin/mineral supplements: of questionable benefit for the general population.

In the United States today, there is a multibillion dollar industry in dietary supplements with at least a third sold as vitamin/mineral pills and drinks. Though everyone requires small amounts of these essential micronutrients, and supplements are of benefit to some within the population, the considerable majority of people can fulfill their needs with the intake of healthy diets of mixed foods. In addition, the fortification of some processed foods adds extra amounts of several micronutrients, especially those for which there is deemed to be a need in special segments of our population. In spite of this safe and adequate level of intake, there are many who have been led to believe that the frequent ingestion of supplements will be helpful in their efforts to maintain optimal health, live longer, and even prevent or cure non-deficiency diseases. It is the intent of this article to unravel the causes and misconceptions behind this practice and to emphasize that most of the money spent on unnecessary supplementation could be better used for other purposes.

Lipoic acid (thioctic acid) analogs, tryptophan analogs, and urea do not interfere with the assay of biotin and biotin metabolites by high-performance liquid chromatography/avidin-binding assay

Abstract Lipoic acid, urea, and tryptophan show structural similarities to the vitamin biotin. If these compounds can successfully compete with biotin or a biotinylated protein for binding to avidin, their presence in serum and urine would cause artifacts in avidin-binding assays for biotin. We assessed the ability of lipoic acid and certain analogs, urea, and l -tryptophan and tryptophan derivatives to interfere with the measurement of 16 biotin analogs by a high-performance liquid chromatography (HPLC)/avidin-binding assay. In this assay, compounds are separated by reversed-phase HPLC, followed by assay of each fraction based on binding to avidin-horseradish peroxidase. At physiologic concentrations, neither lipoic acid analogs (d-lipoate, l-lipoate, d,l-lipoamide, bisnorlipoate, β-hydroxybisnorlipoate, and tetranorlipoate) nor tryptophan derivatives exhibited detectable avidin-binding. Minor avidin-binding was seen for urea and l -tryptophan; binding ratios relative to biotin were 1 × 10−9 and 3 × 10−6, respectively. Urea did not co-elute on HPLC with any of the 16 biotin analogs, but l -tryptophan did co-elute with bisnorbiotin methyl ketone. However, because the relative avidin affinity of l -tryptophan is five orders of magnitude smaller than that of bisnorbiotin methyl ketone, we conclude that none of the tested compounds are likely to interfere with the measurement of biotin or its metabolites by an HPLC/avidin-binding assay.