Sulfur-containing Amino Acid Residues Research Articles

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgCl2 and 0.1 microM S-adenosyl-L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in Km for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1 microM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.

Determination of sulfur-containing amino acid residues in proteins by pyrolysis-gas chromatography with flame photometric detection

A new method for the determination of sulfur-containing amino acid residues, cysteine (Cys) and methionine (Met) in proteins has been developed with pyrolysis-gas chromatography. Characteristic sulfur compounds among the complicated pyrolysis products formed from protein samples were separated by using a high resolution fused silica capillary column and selectively detected by flame photometric detector (FPD). In order to improve the reproducibility, an internal standard substance, benzothiophene, was pyrolyzed at 600 °C together with the protein samples. Thus the observed pyrograms were interpreted in terms of the compositional analysis. On the basis of the relative peak intensities of CH3SH and H2S, the simultaneous determinations of Cys and Met residues in various enzyme proteins were successfully carried out with 10–12% and 5–6% of relative standard deviations, respectively.

Purification and molecular characterization of two inhibitors of yeast proteinase B.

A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.

Physicochemical properties of a lipase from pseudomonas fluorescens

The molecular weight of triacylglycerol lipase (EC 3.1.1.3) from Pseudomonas fluorescens is estimated to be approx. 33 000 by sodium dodecyl sulfate electrophoresis and Sephadex G-75 gel filtration. The lipase appears to be a single-chain protein and contains neither sugar nor lipid. The enzyme has a sedimentation coefficient ( s 20, w ) of 3.06, an intrinsic viscosity of 3.0 g/ml and a partial specific volume of 0.730 g/ml, with an isoelectric point of pH 4.46. Amino acid analysis showed that the enzyme contained few sulfur-containing amino acid residues with no disulfide links. The N-terminal residue of the enzyme was found to be alanine and optical rotation dispersion analysis showed that about 20% of the enzyme structure was in a helical configuration.

Fast Intramolecular Radical Processes in Reactions of Aqueous Solutions of Ribonuclease-S-Peptide and Ribonuclease-S-Protein with H Atoms

The reactions of H atoms with ribonuclease-S-peptide and ribonuclease-S-protein at pH 2.2 and 6.6 have been investigated by means of pulse radiolysis-kinetic spectrometry and compared with those of intact ribonuclease. Initial spectra were observed after 100-nsec pulses of 5 MeV electrons under conditions such that only H atoms reacted with substrate. The resulting spectra are due to addition of one H atom to a substrate molecule. Their subsequent transformations were studied over periods ranging from several microseconds to several seconds. The specific rates of reaction of H atoms with S-protein and with intact ribonuclease (1) are the same, while S-peptide reacts about half as rapidly. For both substrates the subsequent transformations are first order and presumably correspond to intramolecular chemical reactions. The data implicate aromatic and sulfur-containing amino acid residues as transient radical sites. The data for S-peptide establish that intramolecular radical transfer is a reaction of polype...

Fast consecutive radical processes within the ribonuclease molecule in aqueous solution II. Reaction with OH radicals and hydrated electrons

Transient absorption spectra generated by the action on aqueous solutions of ribonuclease of OH radicals in the pH range 3.5–7.3 and of hydrated electrons in the pH range 6.0–7.6 have been investigated by the technique of pulse radiolysis-kinetic spectroscopy. Using 100-ns pulses with both reagents a sequence of spectral changes occurs. Using sweep rates of 1 μs/cm or higher a series of spectra were obtained over a time range from about 10 μs after pulsing up to several seconds later. The first spectrum observed is that due to addition of the radical to ribonuclease. The subsequent transformations are first order and presumably correspond to intramolecular reactions in which radical sites migrate within the protein. Spectral data implicate aromatic and sulfur-containing amino acid residues as sites of transient radical intermediates. Attack of OH also appears to involve abstraction of hydrogen from saturated carbon atoms of the peptide link. Both the occurrence of consecutive reactions and the sites of transient radicals resemble behavior observed when H atoms react with ribonuclease 1,2. There are, however, significant differences characteristic of OH and e − aq attack. Values of k[ribonuclease + OH] and the rates of the first order intramolecular changes are reported. The implications of intramolecular free radical migration in protein for enzyme chemistry are discussed.

Tryptophan Oxygenase of Pseudomonas acidovorans: Purification, Composition, and Subunit Structure

Abstract Tryptophan oxygenase (l-tryptophan:oxygen oxidoreductase, EC 1.13.1.12) was purified to a state of homogeneity from substrate-induced Pseudomonas acidovorans. The sedimentation and diffusion coefficients of the homogeneous protein are s20,w, 6.26 x 10-13 sec, and D20,w, 4.78 x 10-7 cm2 sec-1, and from these values a molecular weight, Ms,d, of 121,000 is obtained. High and low speed equilibrium sedimentation experiments give molecular weights, Mequil, of 118,000 and 123,000, respectively. Amino acid analyses reveal no sulfur-containing amino acid residues other than methionine. Quantitative analytical data show 1 mole of ferriprotoporphyrin IX and only trace amounts of copper and nonheme iron per mole of holoenzyme. The major absorption bands in the optical spectrum of the native holoenzyme occur at 280 and 405 mµ, and the specific absorbance values (E1 cm0.1%) are 1.20 and 1.88, respectively. Optical and electron paramagnetic resonance spectral data indicate that the single protoheme iron moiety of both the native (ferric) and the chemically reduced (ferrous) form of the holoenzyme exists predominantly in the high spin configuration. Any of three kinds of treatment (alkaline pH, guanidinium chloride, or sodium dodecyl sulfate) results in dissociation of the native protein into subunits which are devoid of enzymatic activity and which have profoundly altered optical spectra. The subunit species produced in each case appear monodisperse by velocity and equilibrium sedimentation criteria and are characterized by the following sedimentation coefficients (s20,w) and molecular weights (Mequil): alkaline pH, 2.46 S and 31,300; guanidinium chloride, 1.73 S and 33,200; sodium dodecyl sulfate, 2.67 S and 35,800. These data indicate that native tryptophan oxygenase is composed of a single heme moiety affixed to a protein molecule containing four polypeptide chains, of essentially equivalent mass, which are stabilized in a specific tetrameric structure exclusively by noncovalent interactions.