19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Human plasma high density apolipoprotein A-I: Effect of protein-protein interactions on the spontaneous formation of a lipid-protein recombinant

The effect of the self-association of apolipoprotein A-I on the dynamics of lipid-protein complex formation was studied. Treatment of self-associated apolipoprotein A-I with guanidine hydrochloride initially resulted in dissociation of the oligomers into monomers and subsequent denaturation of the monomers. The association of monomeric and oligomeric apolipoprotein A-I with dimyristoylphosphatidylcholine resulted in identical lipid-protein recombinants as determined by chemical analysis and gel-filtration column elution profiles. Denaturation of a recombinant with guanidine hydrochloride indicated that the protein is more stable in a lipid-protein recombinant than as an oligomer; however, self-association does decrease the rate of lipidprotein recombinant formation. Because apolipoprotein A-I is more stable when it is associated with lipid, we conclude that the association of this protein with a variety of lipids is subject to kinetic control.     

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.     

Chemical modifications of D-amino acid oxidase. Evidence for active site histidine, tyrosine, and arginine residues

1. D-amino acid oxidase is inactivated by reaction with a low molar excess of dansyl chloride at pH 6.6, with complete inactivation accompanied by incorporation of 1.7 dansyl residues per mol of enzyme-bound flavin. The presence of benzoate, a potent competitive inhibitor, protects substantially against inactivation. Evidence is presented that the inactivation is due to dansylation of an active site histidine residue. Reactivation may be obtained by incubation with hydroxylamine. Diethylpyrocarbonate also inactivates the enzyme and modifies the labeling pattern with dansyl chloride. 2. Butanedione in the presence of borate reacts rapidly to inactivate D-amino acid oxidase. Reactivation is obtained spontaneously on removal of borate, implicating reaction of butanedione with an active site arginine residue. 3. Fluorodinitrobenzene appears to behave as an active site-directed reagent when mixed with D-amino acid oxidase at pH 7.4. Complete inactivation is obtained with incorporation of 2.0 dinitrophenyl residues per mol of enzyme-bound flavin. Again benzoate protects against inactivation; only one dinitrophenyl residue is incorporated in the presence of benzoate. The active site residue attacked by fluorodinitrobenzene has been identified as tyrosine.     

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.     

Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.     

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins

Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.