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S Ronchi

Publications and source records attributed to S Ronchi.

At least 55 records · Page 3Linked to original sources
PubMed1991

Identification of Lys116 as the target of N-ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase.

Oxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N-ethylmaleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N-ethyl[2,3-14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110-117 of the enzyme: Lys116 was shown to be the residue alkylated by N-ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5-dimethylaminoaphthalene-1-sulfonyl(dansyl) chloride at the putative NADP(H)-binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295-299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N-ethylmaleimide.

Amino Acids↗
PubMed1991

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.

Amino Acid Sequence↗
PubMed1991

Identification of a conserved epitope in class I alcohol dehydrogenase isoenzymes using monoclonal antibodies.

Two monoclonal antibodies raised against native horse alcohol dehydrogenase (HADH) bind preferentially to the enzyme attached to solid supports and recognize the denatured and carboxymethylated HADH subunits. Both antibodies cross-react with the human class I isoenzymes but do not recognize the class III ADH isoenzyme. Protease digestion, electrophoresis and HPLC have been used to identify the linear epitope which is contained in the sequence Pro344-Glu357 of the HADH subunit.

Alcohol Dehydrogenase↗
PubMed1990

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins.

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.

Amino Acid Sequence↗
PubMed1990

Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence.

A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.

Amino Acid Sequence↗
PubMed1990

Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis.

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.

Amino Acid Sequence↗
PubMed1990

Structural studies on the subunits of glutamate synthase from Azospirillum brasilense.

The amino acid composition and the N-terminal sequences of the two dissimilar subunits of glutamate synthase from Azospirillum brasilense have been determined along with the sequences of selected CNBr peptides. Comparison of our data with those available for Escherichia coli glutamate synthase revealed an overall good homology between the enzymes from the two sources. This is more evident for the heavy subunits where the highly conserved N-terminal sequence containing Cys-1, suggests that this region may be involved in catalysis. However, it appears that the light subunits are different with respect to both their amino acid composition and their N-terminal region, suggesting that the latter may not be part of the enzyme active site. Finally, an extinction coefficient at 444 nm of 62.66 +/- 4.61 mM-1.cm-1 was determined.

Amino Acid Sequence↗
PubMed1990

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules.

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.

Amino Acid Sequence↗
PubMed1989

Properties of D-amino-acid oxidase from Rhodotorula gracilis.

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.

Amino Acids↗
PubMed1989

Purification of Parj I, a major allergen from Parietaria, judaica pollen.

A major allergen, the Parj I, was purified to homogeneity from Parietaria judaica pollen by means of ultrafiltration dialysis, preparative polyacrylamide gel chromatography and affinity chromatography through a column of Sepharose-monoclonal antibody specific for Parj I. The homogeneity of the Parj I was assessed by one single arc of immunoprecipitation both in cross immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis, by one single band of radiostaining after a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose and by one single peak after a size exclusion chromatography on high-performance liquid chromatography (HPLC). The homogeneity was further supported by crossed Laurell immunoelectrophoretic analysis, in that only one arc of precipitation was magnified in CIE after addition of the purified allergen. The purified Parj I allergen was capable of interacting in vitro with 70% of the human IgE specific for a crude P. judaica extract, as determined by radioallergosorbent test inhibition. The purified Parj I was capable of inducing positive reactions in vivo in skin prick tests, and of inducing release of histamine from blood containing basophils as determined by a histamine release assay. The amino acid analysis of the Parj I showed 118 amino acid residues per monomer analyzed and, among other residues, three methionine residues were detected. The molecular weight of the Parj I estimated by HPLC and amino acid composition was 26 kilodaltons.

Allergens↗
PubMed1988

Identification of nucleus-encoded F0I protein of bovine heart mitochondrial H+-ATPase as a functional part of the F0 moiety.

The F0I protein of apparent Mr 27,000, previously characterized [(1988) Eur. J. Biochem. 173, 1-8] as a genuine component of bovine heart F0, has been sequenced and shown to be identical with the nucleus encoded 24,668 Da protein characterized earlier [(1987) J. Mol. Biol. 197, 89-100]. It is directly shown by proteolytic cleavage and reconstitution experiments that this protein, denoted here as PVP from the single-letter codes of the last three residues of the N-terminus, is involved in proton conduction by F0 and in its sensitivity to oligomycin.

Amino Acid Sequence↗
PubMed1988

A biotin-avidin sandwich enzyme-linked immunosorbent assay of growth hormone in bovine plasma.

Purified pituitary bovine growth hormone (bGH) has been used to develop a homologous sandwich enzyme-linked immunosorbent assay in which affinity-purified antibodies are immobilized on microtiter plates. Bovine GH bound to specific antibody is then revealed with a second anti-bGH antibody labeled with biotin and peroxidase-conjugated avidin. The method requires only 48 h, including the coating step, and has a sensitivity as low as 0.25 ng/ml of bGH. Statistical analyses (test of parallelism, cross-reactivity among bGH and GH of various species and bovine prolactin, recovery test, within- and between-assay variation, comparison with radioimmunoassay) confirm the high specificity and reproducibility of the method.

Animals↗
PubMed1987

Phenylglyoxal modification of arginines in mammalian D-amino-acid oxidase.

The presence of arginine in the active center of D-amino-acid oxidase is well documented although its role has been differently interpreted as being part of the substrate-binding site or the positively charged residue near the N1-C2 = O locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in D-amino-acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine-directed reagent. Loss of catalytic activity followed pseudo-first-order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8-mercapto-FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme-binding ability. Analyses of time-dependent activity loss versus arginine modification or [14C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active-site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8-mercapto derivative of D-amino-acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N-terminal portion of the polypeptide chain and point to a role of phenylglyoxal-modifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme.

Aldehydes↗
PubMed1986

C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies.

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.

Amino Acid Sequence↗
PubMed1986

Detection of desamido forms of purified bovine growth hormone.

Tryptic peptide mapping and amino acid sequencing were used for the identification of desamido forms of bovine growth hormone. The major desamido forms of bovine growth hormone resulted from deamidation of asparagine in position 13, 148 and glutamine 140. These forms did not seem to be produced by prolonged treatment of bovine growth hormone in tryptic hydrolysis.

Amino Acid Sequence↗
PubMed1985

The NADP+-binding site of ferredoxin-NADP+ reductase. Sequence of the peptide containing the essential lysine residue.

The flavoprotein ferredoxin-NADP+ reductase is inactivated and loses its ability to bind NADP+ during covalent modification of a lysine by 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, 14-24]. The substrate NADP+ gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl-lysine which was isolated by high-performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP+. The sequence of the dansyl-peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl-peptide. Thus, the octapeptide containing the essential lysine residue has the following sequence: H2N-Ser-Val-Ser-Leu-Cys-Val-Lys-Arg-COOH. A comparison with corresponding sequences of other known NADP+-dependent dehydrogenases is attempted.

Amino Acid Sequence↗