Clearance of the alpha2 macroglobulin-tripsin complex and uptake of trypsin by perfused liver

The uptake and degradation of the alpha2macroglobulin-trypsin (alpha2m-trypsin) complex have been studied using isolated liver cells but not in the liver as a whole. We report the clearance of the complex by the isolated and exsanguinated liver of Wistar male rats, weighing 150-280 g, and compare it with that of the free enzyme. The hepatic clearance of the alpha2m-trypsin complex follows a pattern with a distribution phase followed by an elimination phase, which contrasts with that of trypsin where only the distribution phase is observed. The extraction of trypsin from the perfusate is Ca2+ -independent (156 + 14 pmol/g liver in the presence of 2.5 mM Ca2+, N = 9, versus 140 + 8 pmol/g liver in its absence, N = 7) and is not affected by 100 mM NH4Cl (152 + 7 pmol/g liver, N = 6), 100 U/ml heparin (164 + 14 p/mol/g liver, N = 5), 30 mul/ml carbon particle suspension (150 + 13 pmol/g liver, N = 7) or an acute-phase situation induced by turpentine (125 + 10 pmol/g liver, N = 6) (P>0.05, ANOVA). The hepatic clearance of the alpha2m-trypsin complex is Ca2+ -dependent (1.8 + 0.2 ml/min in the presence of Ca2+, N = 8, versus 0.6 + 0.03 ml/min in its absence, N = 4), affected by NH4Cl (<0.1 ml/min, N = 7), heparin (1.1 + 0.2 ml/min, N = 6) and the acute-phase (0.6 + 0.1 ml/min, N = 6) but bot by the carbon particle suspension (1.8 + 0.2 ml/min, N = 7). These results show that trypsin is not internalized by hepatocytes (no NH4Cl effect) or Kupffer cells (no carbon particle effect) and that the alpha2m-trypsin complex is internalized in a Ca2+ -dependent process by hepatocytes, but not by Kupffer cells, and is affected by an acute-phase reaction.

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.