Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.

Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe.

Polycyclic aromatic hydrocarbons (PAHs) are a class of widespread environmental carcinogens. Most of our knowledge of their mechanisms of metabolic activation to DNA-binding "ultimate carcinogenic" metabolites has come from analysis of the DNA interaction products formed by these highly reactive intermediates. Studies of their role in forming DNA-binding intermediates identical to those formed in vivo from the PAH itself have also allowed identification of the particular cytochrome P450 enzymes involved in activating various structural classes of carcinogenic PAHs. It has been established that PAHs, after metabolic activation in vivo, are capable of inducing mutations in oncogenes and, by inducing multiple mutations, may result in tumors. PAHs also cause changes in cellular gap-junction communication similar to those caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Thus, PAHs may also act through a promotional mechanism in addition to serving as tumor initiators. Previous studies on these mechanisms are described and summarized.

Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action.

Background—Intravascular red cell hemolysis impairs nitric oxide (NO)–redox homeostasis, producing endothelial dysfunction, platelet activation, and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or microparticles and hemolysis, which we hypothesized could impair vascular function and contribute to the putative storage lesion of banked blood. Methods and Results—We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell-free and microparticle-encapsulated hemoglobin, which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator NO. Using stopped-flow spectroscopy and laser-triggered NO release from a caged NO compound, we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythro...

Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion.

PROBLEM TO BE SOLVED: To perform molecular weight measurement and separation of a fluorine containing organic substance slightly soluble in a general organic solvent by means of size exclusion chromatography by using a solvent containing dichloro pentafluoropropane as a mobile phase and using polymethyl methacrylate with a known molecular weight as a molecular weight measurement standard specimen. SOLUTION: As a mobile phase. a solvent containing HCFC225 such as a solvent containing only HCFC225 and a mixture solvent containing HCFC225 and other solvents are available. HCFC225 is selected from one or more kinds of substances selected from chlorinated fluorinated hydrocarbons with a molecular formula represented by C3 HF3 Cl2 . As the HCFC225, several kinds of isomers with the same molecular formula and different structures are known, however, HCFC225ca (CF3 CF2 CHCl2 ), HCFC225cb (CF2 ClCF2 CFHC1), and a mixture of them are desirable. As a molecular weight measurement standard specimen, polymethyl methacrylate with a known molecular weight is used.

https://www.springer.com/productFlyer_978-3-540-65635-7.pdf?SGWID=0-0-1297-1427978-0

Size Exclusion Chromatography

Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.

Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues.

Test reactions for a stopped-flow apparatus:Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1-5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335-340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]L-phenyl-alanyl-L-alanine amide, and the activation was found to be brought about only by an increase in k(cat'). The effectiveness of monovalent cations on the increase of k(cat) was determined to follow the order of Na(+)>K(+)>Li(+).

/pdf/effect-of-amino-acid-residues-at-the-cleavable-site-of-1g881zf67i.pdf

Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts

Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

Ben'ichiro Tonomura

Papers

Test reactions for a stopped-flow apparatus:Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts

Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.