His-Pro hydrochloride

Hydroxyl or peroxyl radicals and hypochlorous acid (HOCl) are known to cause the oxidation of lipoproteins. Here, we examined Cu(2+)-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu(2+), peroxyl radicals or HOCl. When Cu(2+)-binding property of PON1 was examined spectrophotometrically, the maximal Cu(2+) binding was achieved at 1:1 molar ratio of PON1: Cu(2+). Additionally, Cu(2+)-catalyzed oxidative inactivation of PON1 was prevented by Ca(2+)-depleted PON1 at 1:1 ratio, but not diethylpyrocarbonate (DEPC)-modified PON1, suggesting the participation of His residue in Cu(2+)-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu(2+)-mediated low density lipoprotein (LDL) oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu(2+)-induced LDL oxidation (24 h), indicating a tight binding of Cu(2+) by peptides. In support of this, the peptide/Cu(2+) complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2,2'-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyrosine (Tyr) or Cysteine (Cys) residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu(2+), 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) or HOCl.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.

The unmasking of the low concentration effect of angiotensin II (AII) was identified within the concentration ranges of 10(-13) to 10(-11) M of AII by PD 121981 (5-diphenylacetyl-1-(4-methoxy-3-methylbenzyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid) and 10(-12) to 3 x 10(-10) M of AII by CGP 42112 (nicotinic acid-Tyr-(N alpha-benzyl-oxycarbonyl-Arg)Lys-His-Pro-IIe-OH), AT2 antagonists, in association with the ordinary contraction curve, i.e., high concentration effect (at 3 x 10(-10)-10(-6) M of AII), in the rabbit abdominal aorta. Thus, they showed clear biphasic features of AII-induced contraction curves. However, this was not the case for angiotensin I and angiotensin III. This PD 121981-evoked low concentration effect of AII was selectively inhibited by DuP 753 (0.01-1 nM), dithiothreitol (10 and 100 microM), pertussis toxin (50 and 300 ng/ml, for 2 hr), nifedipine (1 and 10 microM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (1 and 3 microM), which suggests the receptors were the AT1 subtype. However, the high concentration effect of AII was not affected by these drugs within the concentration ranges used in the present studies. These myographic results were almost consistent with the features of the intracellular Ca++ changes. Thus, it was concluded that the receptors that mediate the low concentration effect of AII belong to the AT1 subtype. However, the current study did not determine the mechanism by which PD 121981 and CGP 42112 evoked the up-regulation of the AT1 receptors.