tosylarginine-methyl-ester and benzoylarginine-ethyl-ester

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous-organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.

Topics: Acetonitriles; Arginine; Proteins; Solvents; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Tosylarginine Methyl Ester; Trypsin; Water

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Topics: Ammonium Sulfate; Animals; Arginine; Cattle; Heparin; Humans; Kinetics; Mast Cells; Peptide Hydrolases; Potassium Chloride; Skin; Tosylarginine Methyl Ester

Added N alpha-p-tosyl-l-arginine methyl ester or N alpha-benzoyl-l-arginine ethyl ester inhibited the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of the insulin receptor both in a partially purified insulin receptor fraction from rat adipocytes and in a highly purified insulin receptor preparation from human placenta. N-alpha-p-tosyl-l-lysine chloromethyl ketone, N alpha-p-tosyl-l-lysine methyl ester, or N-acetyl-l-phenylalanine ethyl ester were much less potent, while N-benzoyl-1-alanine methyl ester was without effect. Inhibition of the phosphorylation by the arginine analogues did not require preincubation of the insulin receptor with inhibitors in the presence of insulin prior to phosphorylation. Inhibition by N alpha-p-tosyl-l-arginine methyl ester was decreased by preincubation of the receptor fraction with cold ATP and MnCl2. These results suggest that N alpha-p-tosyl-l-arginine methyl ester inhibits an initial ATP and Mn2+ dependent reaction in insulin-stimulated phosphorylation process.

Topics: Adipose Tissue; Animals; Arginine; Insulin; Macromolecular Substances; Male; Molecular Weight; Phosphorylation; Rats; Rats, Inbred Strains; Receptor, Insulin; Tosylarginine Methyl Ester

The presence of three trypsinogens (Try-III, Try-I, and Try-II) in the mouse is demonstrated by DEAE-cellulose column chromatography. Two genetic variants of Try-I are detected, because the activity of Try-I is different between the Mol-A strain and seven other strains. The Prt-3 locus controls the activity of Try-I. The Prt-3a gene exists in CFO, BS, KR, BALB/cJ, C57BL/6J, CBA/J, and 129/Sv-S1-CP strains, whereas the Prt-3b gene is present only in the Mol-A strain. Each Try-I from the CFO or Mol-A strain was purified. The fact that Try-I activity of the Mol-A strain is much higher than those of other strains is because of a difference in the specific activity; the ratio of the Kcat (sec-1) value with Tos-Arg-OMe to that with Bz-Arg-OEt is different between the variants from the CFO and those from the Mol-A strains, being much higher in the Mol-A strain. Also, chicken ovomucoid inhibited Try-I activity of the CFO strain at a molar ratio of one ovomucoid to two trypsins; Try-I activity of the Mol-A strain was only 50% inhibited even with an excess of ovomucoid. There was no difference between genetic variants of Try-I in molecular weight, Km values with Bz-Arg-OEt or Tos-Arg-OMe, pH optimum profile, or inhibition by soybean trypsin inhibitor.

Topics: Animals; Arginine; Genetic Variation; Mice; Mice, Inbred Strains; Species Specificity; Substrate Specificity; Tosylarginine Methyl Ester; Trypsin Inhibitors; Trypsinogen

Topics: Animals; Arginine; Chromatography, DEAE-Cellulose; Pancreas; Rats; Rats, Inbred Strains; Tosylarginine Methyl Ester; Trypsinogen