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PubMed1992

Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus.

Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

Amino Acid Sequence↗
PubMed1995

Molecular associations of flavins with betacarbolines and related indoles.

The interactions of a set of structurally selected betacarbolines (BC), (9H-pyrido[3,4-b]indoles) and indoles (IND) with two representative flavins (FN): riboflavin (RFN) and flavin mononucleotide (FMN) have been investigated by absorption and fluorescence spectroscopies. Spectral results provided evidence on the formation of 1:1 non-fluorescent molecular complexes, whose stability constants and other related thermodynamic parameters have been estimated from Stern-Volmer quenching analysis. The FMN complexes are somewhat more stable than the RFN complexes. The stabilities of the IND and BC complexes for a given FN follow approximately the order IND approximately tetrahydro BC < dehydro BC < fully aromatic BC. Protonation of the pyridinic nitrogen atom of BCs has a destabilizing effect, which is more pronounced for fully aromatic than for tetrahydro derivatives. The influence of structural factors on the stability of the complexes has been discussed and, aided by theoretical AM1 calculations, a qualitative model for the structure (stacking) and binding forces (cooperative localized charge transfer and dispersion forces) of the complexes has been proposed.

Carbolines↗
PubMed1977

[The influence of biophysical factors on biological oxidation and redox processes. 16. The possibility of influencing flavine coenzymes in liver mitochondria by means of ultrasonic treatment (author's transl)].

The quantity of FAD and FMN in mitochondria isolated from the liver of albino rats was determined before and after application of ultrasonic energy with the intensities 0,2,0,6 and 1,0 w/cm2 in a short-term test, and the intensity 0,2 w/cm2 applied at ten successive days in a long-term test. The total amount of the two coenzymes as well as their percentages were ascertained. The following results derive from our experiments: 1. A quantitative increase of FAD in liver mitochondria. This suggests that the capacity of the shortened respiratory chain has increased, since redox systems represent a factor determining its stage. 2. The flavine-mononucleotide responds to ultrasonics as a relatively instable biological macromolecule. 3. The total amount of flavine coenzymes is influenced especially by the quantity of FAD, and, correspondingly, by changes of the latter.

Animals↗
PubMed1990

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia.

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.

Journal Article↗
PubMed1986

Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453.

The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns. It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms. The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component. Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity. FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions. Binding between the two proteins that made up the active complex was fairly weak and freely reversible. It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site. Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein.

Apoenzymes↗
PubMed2004

Role of neighboring FMN side chains in the modulation of flavin reduction potentials and in the energetics of the FMN:apoprotein interaction in Anabaena flavodoxin.

Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.

Amino Acid Sequence↗
PubMed1998

Apoflavodoxin: structure, stability, and FMN binding.

Flavodoxins are one domain alpha/beta electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that confers redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apoflavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form that is competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a two-state mechanism. The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments. The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak.

Anabaena↗
PubMed1993

Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein.

Amino Acid Sequence↗
PubMed2003

Subsecond separation of cellular flavin coenzymes by microchip capillary electrophoresis with laser-induced fluorescence detection.

In this article, it was demonstrated that a subsecond separation of cellular metabolites such as riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) was achieved using microchip capillary electrophoresis with laser-induced fluorescence detection. The influences of crucial parameters that governed analysis time (e.g., channel length and electric field for separation) and separation resolution (e.g., sample size) were investigated, both in theoretical aspects and experimental practice. Quantitative analyses were performed that exhibited linear dynamic range of two orders of magnitude, with calculated detection limits of 34, 201, and 127 nM for RF, FAD, and FMN, respectively. To test the validity of the method, it was successfully applied to characterize several recombinant flavin-binding domains in a human neuronal nitric oxide synthase.

Coenzymes↗
PubMed2004

Native fluorescence detection of flavin derivatives by microchip capillary electrophoresis with laser-induced fluorescence intensified charge-coupled device detection.

To widen the scope of laser-induced fluorescence (LIF) for detection in microchip capillary electrophoresis (CE), a microchip CE LIF-ICCD (intensified charge-coupled device) system based on a tunable wavelength dye laser pumped by a pico-second pulse nitrogen laser for excitation and a spectrograph with ICCD for detection had developed to demonstrate the enhancement in detection sensitivity by the following three approaches: direct detection of native fluorescence, improvement of signal-to-noise ratio by pulse laser excitation and time delay detection, and selective spectral acquisition by multi-channel detection. Riboflavin, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) have been selected as they are dietetically important and microchip CE provides a promising onsite detection method. The results indicate a strong effect of wavelength on detection sensitivity and the need to tune wavelength for direct detection. Under optimized conditions (excitation 450 nm, emission 520 nm, gate delay time 45 ns, 20 mM phosphate buffer at pH 7.1), the following results were obtained under static condition: Working ranges (0.6-350 microg/l, r > 0.99), detection limits (0.15-1.0 microg/l) and peak height repeatability (1.8-2.2% R.S.D.), all within the applicability range for body fluids or beverages such as human urine and cow milk. Baseline separation of three flavins was obtained under dynamic condition and the fluorescence spectra acquired assist the identification of alkaline-degraded products of riboflavin. Thus, the capability to check peak purity and identify unknown peaks has been demonstrated.

Electrophoresis, Capillary↗
PubMed1975

Bacterial luciferase requires one reduced flavin for light emission.

Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A two-flavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. Quantum yield results demonstrate that bioluminescence requires only one reduced flavin per luciferase, ruling out mechanisms involving either two reduced flavins or one reduced flavin plus one oxidized flavin per catalytic cycle.

Flavin Mononucleotide↗
PubMed1997

Crystallization and preliminary X-ray diffraction analysis of the heterotetrameric dihydroorotate dehydrogenase B of Lactococcus lactis, a flavoprotein enzyme system consisting of two PyrDB subunits and two iron-sulfur cluster containing PyrK subunits.

Dihydroorotate dehydrogenases are flavin-containing enzymes which catalyze the conversion of (S)-dihydroorotate to orotate. Dihydroorotate dehydrogenase B (DHODB) from Lactococcus lactis is a heterotetramer containing two subunits of the protein encoded by the pyrDb gene (PyrDB) and two subunits of the protein encoded by the pyrK gene (PyrK). In addition, DHODB contains two molecules of flavin mononucleotide, two molecules of flavin adenine dinucleotide and two [2Fe-2S] iron-sulfur clusters as tightly bound cofactors. Yellow crystals of this enzyme have been grown using the hanging-drop vapour-diffusion technique from solutions of 2.5 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6. The crystals have been shown to contain both the PyrDB and the PyrK subunits and fluorescence measurements indicate that the two different subunits interact very closely with each other in the active-site region. Native data sets have been collected to 2.6 A with a conventional X-ray source and to 2.2 A using synchrotron radiation. The crystals are rhombohedral, space group R32, with correspondin8 hexagonal unit-cell dimensions a = b = 202.3 and c = 81.0 A. The asymmetric unit in the crystal contains one PyrDB subunit and one PyrK subunit, which suggests that the two halves of the heterotetramer are related by a crystallographic twofold axis.

Journal Article↗
PubMedDate not supplied

Water-soluble vitamins.

Simultaneous Determination of Vitamins.--Klejdus et al. described a simultaneous determination of 10 water- and 10 fat-soluble vitamins in pharmaceutical preparations by liquid chromatography-diode-array detection (LC-DAD). A combined isocratic and linear gradient allowed separation of vitamins in 3 distinct groups: polar, low-polar, and nonpolar. The method was applied to pharmaceutical preparations, fortified powdered drinks, and food samples, for which results were in good agreement with values claimed. Heudi et al. described a separation of 9 water-soluble vitamins by LC-UV. The method was applied for the quantification of vitamins in polyvitaminated premixes used for the fortification of infant nutrition products. The repeatability of the method was evaluated at different concentration levels and coefficients of variation were <6.5%. The concentrations of vitamins found in premixes with the method were comparable to the values declared. A disadvantage of the methods mentioned above is that sample composition has to be known in advance. According to European legislation, for example, foods might be fortified with riboflavin phosphate or thiamin phosphate, vitamers which are not included in the simultaneous separations described. Vitamin B2.--Viñas et al. elaborated an LC analysis of riboflavin vitamers in foods. Vitamin B2 can be found in nature as the free riboflavin, but in most biological materials it occurs predominantly in the form of 2 coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD). Several methods usually involve the conversion of these coenzymes into free riboflavin before quantification of total riboflavin. According to the authors, there is growing interest to know flavin composition of foods. The described method separates the individual vitamers isocratically. Accuracy of the method is tested with 2 certified reference materials (CRMs). Vitamin B5.-Methods for the determination of vitamin B5 in foods are limited because of their low sensitivity and poor selectivity. Pakin et al. proposed a post-column derivatization of pantothenic acid as a fluorescent compound and used this principle in a specific and sensitive method for the determination of free and bound pantothenic acid in a large variety of foods. A French laboratory invited European laboratories to participate in a series of collaborative studies for this method, which will be carried out in 2005/2006. A more sophisticated method was described by Mittermayer et al. They developed an LC-mass spectrometry (LC/MS) method for the determination of vitamin B5 in a wide range of fortified food products. Application of the method to various samples showed consistent results with those obtained by microbiology. Vitamin B6.-Method 2004.07, an LC method for the analysis of vitamin B6 in reconstituted infant formula, was published by Mann et al. In contrast with this method, which quantifies vitamin B6 after converting the phosphorylated and free vitamers into pyridoxine, Viñas et al. published an LC method which determines 6 vitamin B6 related compounds, the 3 B6 vitamers, their corresponding phosphorylated esters, and a metabolite. Accuracy was determined using 2 CRMs. Results were within the certified ranges. Vitamin C.-Franke et al. described an extensive study to vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Vitamin C was determined by measuring ascorbic acid in its reduced state by LC and coulometric detection along with UV absorbance detection at 245 nm. No attempts were made to assess levels of dehydroascorbic acid. Most recent research revealed that cell uptake of dehydroascorbic acid is unlikely to play a major role, which may explain the very low vitamin C activity of orally administered L-dehydroascorbic acid in rats. The food levels found by Franke et al. are variably lower, higher, or equal in comparison to other studies. Iwase described a method for the determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization. Electrochemical detection was used for the quantification. Traditionally, metaphosphoric acid was proven to be a useful dissolving agent for the determination of ascorbic acid. However, it dissolves in water very slowly, it is hygroscopic, and accurate weighing is not easy. Adjustment at pH 2-3 takes a long time. It appeared to be possible to replace metaphosphoric acid by 0.2% phosphoric acid. Methionine played an important role on the stability of ascorbic acid. The method seemed to be applicable to the routine analysis of ascorbic acid in foods. Folic Acid.-Microbiological analysis of total folate in foods is often considered as the golden standard compared to other methods based on, for example, LC. Koontz et al. showed results of total folate concentrations measured by microbiological assay in a variety of foods. Samples were submitted in a routine manner to experienced laboratories that regularly perform folate analysis fee-for-service basis in the United States. Each laboratory reported the use of a microbiological method similar to the AOAC Official Method for the determination of folic acid. Striking was, the use of 3 different pH extraction conditions by 4 laboratories. Only one laboratory reported using a tri-enzyme extraction. Results were evaluated. Results for folic acid fortified foods had considerably lower between-laboratory variation, 9-11%, versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. One should realize a large variation in results, which might be caused by slight modifications in the microbiological analysis of total folate in foods or the analysis in various (unfortified) food matrixes. Furthermore, optimal combination of enzymes and reaction conditions may vary depending on the composition of the food. Padrangi and Laborde showed recently that treatment with alpha-amylase had no significant effect on measured folate in spinach, although addition of protease significantly increased the release of folate. LC/MS applications gain increasing attention because of their specificity. Rychlik used stable isotope dilution assays for the determination of the folate content of broccoli and bread. Compared to data in the literature and food data bases, amounts were significantly lower. Pawlosky et al., however, found comparable values for 5-methyltetrahydrofolic acid and folic acid by HPLC analysis with fluorescent detection and HPLC/MS. Among samples analyzed were CRMs and broccoli. Besides folic acid, other water-soluble vitamins were also determined by LC/MS/MS by Leporati et al. The method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the U.S. market. Biotin.-A paper from Staggs et al. included the assertion that existing biotin data in food composition tables are inaccurate because the majority are based on bioassays with all relevant disadvantages. Data in most cases are overestimated with consequences for recommendations for dietary biotin intake. An HPLC/avidin-binding assay was used to analyze 87 foods to support the hypothesis mentioned.

Chemistry Techniques, Analytical↗
PubMed1975

Nitrate reductase of Dunaliella parva: electron donor specificity and heat activation.

Nitrate reductase of the salt tolerant alga Dunaliella parva, in contrast to that of most green algae, can use NADPH as well as NADH as electron donor. Extracts of cells contained various amounts of latent nitrate reductase. The latent enzyme could be activated at 45 degrees C but only in the presence of flavine adenine dinucleotide. The heat activated enzyme did not require flavine adenine dinucleotide for activity and was fully active with NADH, NADPH or reduced flavine mononucleotide as electron donors.

Chlorophyta↗
PubMed2003

Electrochromatographic retention studies on a flavin-binding RNA aptamer sorbent.

Aptamers are oligonucleotides that are isolated and amplified on the basis of their recognition of a target molecule. In this study, an RNA aptamer isolated and amplified on the basis of its affinity for flavin mononucleotide (FMN) was covalently bound to the inner walls of fused-silica capillaries. This aptamer recognizes the flavin moiety of both FMN and flavin adenine dinucleotide (FAD). When an attempt was made to evaluate these capillaries according to existing theory, the theory proved to be insufficient. We describe a new method to evaluate capillaries for use in open-tubular capillary electrochromatography (OTCEC) of charged analytes, which combines OTCEC and flow-counterbalanced capillary electrophoresis. This method enabled us to extract k' and evaluate k(CEC) values for these capillaries, and the dependence of these values on Mg(2+) concentration was explored. The k' values for these capillaries ranged from 0.0951 to 0.2530 and from 0.0255 to 0.1118 for FMN and FAD, respectively.

Algorithms↗
PubMed1995

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.

Amino Acid Sequence↗
PubMed1982

ACP1GUA-1--a low-activity variant of human erythrocyte acid phosphatase: association with increased glutathione reductase activity.

ACP1GUA-1, a variant of human erythrocyte acid phosphatase, exists as a polymorphism (allele frequency of .132) in the Guaymi Indians of Central America. This variant has an electrophoretic mobility similar to the common B- and C-type variants, but individuals of the ACP1GUA-1 phenotype have a level of enzyme activity only 27% of the activity expected for the ACP1C variant. The GUA-1 variant is more thermostable than is the B variant, and the order of responsiveness to the modulation of activity by purine analogs and folate is always (B)-(A)-(GUA-1). Thus, the GUA-1 variant is a low-activity variant with C-like regulatory properties. Erythrocytes from individuals of the ACP1GUA-1 phenotype have increased basal levels of glutathione reductase, and a larger fraction of the glutathione reductase protein is present as the holoenzyme, indicating increased levels of flavin adenine dinucleotide in the erythrocytes of these individuals. This is consistent with the suggestion that ACP1 has a physiological function as a flavin mononucleotide phosphatase.

Acid Phosphatase↗