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B Curti

Publications and source records attributed to B Curti.

At least 55 records · Page 3Linked to original sources
PubMed1984

A cross-linked complex between ferredoxin and ferredoxin-NADP+ reductase.

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to effectively cross-link ferredoxin to ferredoxin-NADP+ reductase. The covalent complex has a stoichiometry of 1 mol of ferredoxin per mol of the reductase. The flavoprotein moiety of the cross-linked complex maintains most of its diaphorase activity and more interestingly has gained the capacity to catalyze the NADPH-cytochrome c reaction without addition of free ferredoxin in the assay mixture. Furthermore, the cross-linked complex binds NADP+ with a Kd = 88 microM at an ionic strength of 0.02 M. These results show that a ternary complex among the reductase and its substrates can be formed, suggesting that the binding sites for ferredoxin and the pyridine nucleotides are distinct. The bound ferredoxin can interact with cytochrome c; the iron-sulfur cluster of the cross-linked complex is shown to be reduced under anaerobic conditions by NADPH and to be required for the catalysis of the NADPH-cytochrome c reductase reaction. The cross-linked complex, added to thylakoids inhibited by the antibody against the reductase, catalyzes the H2O-cytochrome c photoreduction, which suggests that the ferredoxin moiety of the complex can interact with its electron donor in the photosynthetic chain. Restoration of NADP+ photoreduction requires the addition of free ferredoxin.

Carbodiimides↗
PubMed1983

Renaturation studies of free and immobilized D-amino-acid oxidase.

The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.

Animals↗
PubMed1983

FAD analogues as mechanistic and 'binding-domain' probes of spinach ferredoxin-NADP+ reductase.

The native flavin, FAD, of spinach ferredoxin--NADP+ reductase, has been replaced by a number of FAD analogues with modifications of the isoalloxazine ring system. The apoenzyme binds 8-mercapto-FAD in its thiolate anion form and 6-hydroxy-FAD in its neutral form. These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with other properties of this class, e.g. stabilization of the neutral flavin semiquinone. The chemical reactivity toward methylmethanethiolsulfonate of the 8-mercapto-FAD . enzyme clearly shows that the flavin 8-position is exposed to solvent. On the other hand, the lack of reactivity with the 2-thio-FAD . enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The seven modified flavins examined all support NADPH--ferricyanide reductase activity, the catalytic velocity being directly proportional to the redox potential of the flavin. No such linear free energy relationship was found between redox potential and activity with ferredoxin or iodonitrotetrazolium as acceptor.

Catalysis↗
PubMed1983

[Computerized methods in the diagnosis and prognosis of surgically treated bilio-pancreatic diseases].

Multivaried and trend analyses were applied to a set of 27 periodic haematochemical checks on 37 patients given surgery for biliopancreatic pathology. Preoperative and postoperative data were separately analysed. The software employed was specially designed for the purpose. The discriminating function was used in the preoperative period to establish the best moment for diagnosis, which turns out to be 5 days after admission to hospital. Variance and regression analyses were used for short term prognosis by calculating survival chances. In one case the prognosis turned out to be distinctly inaccurate.

Biliary Tract Diseases↗
PubMed1982

Preparation of apoprotein from spinach ferredoxin-NADP+ reductase. Studies on the resolution process and characterization of the FAD reconstituted holoenzyme.

1. Ferredoxin-NADP+ reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl2 at pH 7.5 and 2 degrees C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCl. The apoprotein is stable for at least a week at -20 degrees C. 2. The release of the prosthetic group from the protein by the Ca2+ ions is a multi-step process. Three different effects of these ions are identifiable: (a) a rapid 20-25% inhibition of catalytic activity, probably caused by an increase in the ionic strength of the medium; other cations can produce it as well; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the FAD is finally released from the protein; (c) complete conversion of FAD to FMN, which blocks reconstitution to holoenzyme, caused by the well-known hydrolytic action of Ca2+ ions on the pyrophosphate bridge of FAD. 3. Binding of FAD by the apoferredoxin-NADP+ reductase is very rapid and it is complete in a few minutes even at 0 degrees C. A Kd of 3.4 X 10(-9) M has been determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.

Apoenzymes↗
PubMed1982

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides.

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.

Amino Acid Sequence↗
PubMed1981

Reactivity of D-amino acid oxidase with 1,2-cyclohexanedione: evidence for one arginine in the substrate-binding site.

D-Amino acid oxidase is inactivated by reaction with 1,2-cyclohexanedione in borate buffer at pH 8.8. The reaction follows pseudo-first-order kinetics. The present of benzoate, a substrate-competitive inhibitor of the enzyme, protects substantially against inactivation. Partial reactivation could be obtained by removal of borate and its substitution with phosphate buffer. The reaction of 1,2-cyclohexanedione with the enzyme at different inhibitor concentrations appears to follow a saturation kinetics, indicating the formation of an intermediate complex between enzyme and inhibitor prior to the inactivation process. The partially inactivated enzyme shows the same apparent Km but a decreased V as compared to the native D-amino acid oxidase. Similarly, the inhibited enzyme fails to bind benzoate. Amino acid analysis of the 1,2-cyclohexanedione-treated enzyme at various times of inactivation shows no loss of amino acid residues except for arginines. Analysis of the reaction data by statistical methods indicates that three arginine residues react with the inhibitor at slightly different rates, and that one of them is essential for catalytic activity. The presence of benzoate, while it prevents the loss of activity, reduces by one the number of arginine residues hit by the reagent in the reaction of 1,2-cyclohexanedione with D-amino acid oxidase.

Animals↗
PubMed1980

An active site-tyrosine-containing heptapeptide from D-amino acid oxidase.

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.

Amino Acid Sequence↗
PubMed1979

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.

Aldehydes↗
PubMed1978

Crystallographic studies on D-amino acid oxidase.

D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.

D-Amino-Acid Oxidase↗
PubMed1977

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.

Amino Acids↗
PubMed1976

Isolation, characterization and partial sequence of cyanogen bromide fragments and thiol peptides from pig kidney D-amino-acid oxidase.

A partial characterization of the primary structure of D-amino-acid oxidase (D-Amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3.) from hog kidney has been achieved by a CNBr cleavage of the 14C-carboxymethylated protein. Four fragments have been isolated and purified and their alignment made possible by overlapping with methionine-containing peptides derived from tryptic digestion of the 14C-carboxymethylated protein. A partial sequencing of the CNBr fragments has been carried out by the automated Edman procedure and by manual sequence analysis. Chymotryptic peptides containing the 5 alkylated thiols of the monomer enzyme (Curti, B., Ronchi, S., branzoli, U., Ferri, G. and Williams, Jr., C. H. (1973) Biochim. Biophys. Acta 327, 266-273) have been isolated and their sequence determined. The present results do not show any significant homologies with the known sequences of other flavoproteins.

Amino Acid Sequence↗