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Biomedical subjects

G Irace

Publications and source records attributed to G Irace.

At least 55 records · Page 3Linked to original sources
PubMed1982

Purification and molecular properties of rabbit lung indolamine N-methyltransferase.

Indolamine N-methyltransferase (INMT) has been purified to an apparent homogeneity from rabbit lung, and some of its catalytic and physicochemical properties have been examined. The enzyme is a monomeric protein with a molecular weight of 31,500 +/- 1000, a molecular Stokes radius of 21.5 A, and a diffusion coefficient of 8.7 X 10(-7) cm2/s. The frictional ratio of the native enzyme (1.05) suggests that the shape of the molecule is nearly spherical. Denaturation experiments performed with increasing concentrations of guanidine hydrochloride (Gdn-HCl) at neural pH indicated that the active site of the enzyme was destroyed by a structural rearrangement of the protein molecule without large change in its size and shape. The final state reached in 6.0 M Gdn . HCl seemed to correspond to a disulfide cross-linked randomly coiled polypeptide. Full normalization of the fluorescent parameter was attained only in the presence of 0.1 M beta-mercaptoethanol. A structural rearrangement has been observed upon acidification of INMT from pH 7.0 to pH 2.0. At pH 4.5, most of the peptide backbone appeared to be unorganized, but further acidification to pH 2.0 produced a reorganization of protein structure which became able to bind 8-anilino-1-naphthalenesulfonate. The data support the hypothesis that the enzyme structure results from the close package of organized regions joined by structureless segments.

Animals↗
PubMed1982

Heme and cysteine microenvironments of tuna apomyoglobin. Evidence of two independent unfolding regions.

The heme and cysteine microenvironments of bluefin tuna apomyoglobin have been investigated by examining the fluorescence properties of two extrinsic chromophores, i.e., ANS and 1,5-AEDANS. 1,5-AEDANS was covalently bound to the single cysteine residue found in the primary structure of tuna apomyoglobin. Recombination experiments with hemin showed that tuna apomyoglobin does not bind 1,5-AEDANS in the same binding site of the heme, although the fluorescence properties of the covalently bound 1,5-AEDANS strongly suggest that the dye is embedded in a rather nonpolar microenvironment. ANS was selected because of its ability to bind the apomyoglobin in the same nonpolar moiety of the heme. Acidification of apoMb--AEDANS to pH 3.0 produced an increase of 1,5-AEDANS fluorescence intensity and a shift of its emission maximum from 475 to 470 nm. In the same pH range apomyoglobin lost its ability to bind ANS. Two independent transitions were observed with increasing concentrations of guanidine. Low guanidine concentration (less than 1.0 M) unfolded the heme binding site as indicated by the disappearance of ANS fluorescence, whereas higher denaturant concentration was required to produce full normalization of 1,5-AEDANS emission spectrum.

Animals↗
PubMed1982

Instability of coated vesicles in concentrated sucrose solutions.

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.

Animals↗
PubMed1981

Tryptophanyl fluorescence heterogeneity of apomyoglobins. Correlation with the presence of two distinct structural domains.

The individual fluorescence of the two tryptophan residues (Trp-7 and Trp-14) of mammalian apomyoglobins has been resolved by comparing the fluorescence properties of these proteins to those of bluefin tuna apomyoglobin, which contains only Trp-14. The two tryptophan residues have been found to have different emission maxima, i.e., 321 for Trp-14 and 333 for Trp-7. The fluorescence of Trp-14 depends on the protonation of a sterically related histidyl residue in the pH range between 8.3 and 5.6, where no conformational change was detected. This residue has been identified as His-119. At pH 8.3 the quantum yield of Trp-7 is lower than that of Trp-14. An increase of the fluorescence intensity of Trp-7 occurs when the heme binding site of apomyoglobin is destroyed by acid or a low concentration of guanidine hydrochloride. An independent unfolding of the N-terminal district of the apomyoglobin molecular occurs on increasing the guanidine concentration. The two distinct structural transitions have been discussed in terms of two domains of tertiary structure.

Animals↗
PubMed1979

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.

Antithyroid Agents↗
PubMed1979

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.

Iodides↗
PubMed1978

Thyroxine-induced conformational changes in prealbumin.

The effects of thyroxine binding on the conformation of human prealbumin and bovine serum albumin have been examined. A blue shift in protein absorption was observed with prealbumin, whereas a red shift was observed with bovine serum albumin. In the case of prealbumin, where the two binding sites are identical, the total absorption change was confined to the binding of the first ligand and has been interpreted as resulting from a conformational change. A blue shift observed in the absorption spectrum of thyroxine, however, was the same for the first and second bound molecules. These data have been interpreted in terms of two identical and interacting sites on prealbumin and explain the origin of the difference in binding affinities between the first and second sites. Fluorescence quenching by thyroxine and thyroxine effects on tryptic hydrolysis of prealbumin are in accord with the above interpretation.

Humans↗
PubMed1978

Second-derivative spectroscopy of proteins. A method for the quantitative determination of aromatic amino acids in proteins.

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

Phenylalanine↗
PubMed1978

The effect of evolution on homologous proteins: a comparison between the chromophore microenvironments of Italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin.

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.

Animals↗
PubMed1978

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.

Amino Acids↗
PubMed1975

Covalent structure of fibrinopeptides from buffaloes breeding in Italy.

The primary structure of fibrinopeptides A and B from buffaloes breeding in Italy has been determined with a view to establishing whether this animal is an autochthonous species or is the result of recent radiation mutation. Some differences exist at the morphologic and physiologic levels between the Indian buffalo and that breeding in Italy, But they do not allow a clear evolutionary line to be traced between these species. The amino acid sequences, if compared with homologous sequences of the Indian buffalo, show one difference in the most variable region of fibrino-peptides A, in particular the substitution of a serine residue by a glycine residue in position 8. This difference supports the hypothesis of the autochthonous origin of the Italian buffalo.

Amino Acid Sequence↗