Inhibitory effect of αS1- and αS2-casein hydrolysates on angiotensin I-converting enzyme in human endothelial cells in vitro, rat aortic tissue ex vivo, and renovascular hypertensive rats in vivo.

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from alpha(S1)- and alpha(S2)-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood.

Regulation of trypsin activity by Cu2+ chelation of the substrate binding site.

Lysine 188 of trypsin was replaced with histidine in order to create a metal chelation site in the substrate binding pocket of this protease, built in a metal binding 'switch,' and to be able to modulate its activity at lower pH. The catalytic properties of wild-type and mutant trypsin were measured with tetrapeptide substrates containing a nitroanilide leaving group and whole native protein substrate: beta-casein. The results obtained reveal that K188H mutation does not affect catalytic efficiency, raising only slightly (from 6 to 8) the arginine/lysine preference of the mutant and increasing 1.8- and 1.2-fold the second-order rate constant k(cat)/Km for arginine- and lysine-containing substrates, respectively. Compared with wild-type trypsin, K188H mutant shows, in the absence of Cu2+, a different catalytic activity pattern as a function of pH. The addition of Cu2+ to trypsin K188H induces a 30-100-fold increase in Km, while k(cat) is scarcely decreased. The hydrolytic activity of this mutant can be fully restored by addition of EDTA. In contrast to a chelating active site, a novel mode of metal-dependent inhibition activity of trypsin with copper is presented. As suggested by molecular modelling studies, the substrate binding pocket of the protease is considerably perturbed by vicinal chelation. More generally, this type of transition metal chelate may present wider possibilities of trypsin activity and specificity modulation than the previously accomplished chelation of a histidine moiety from a catalytic triad.