SynopsisPhotochemically induced dynamic nuclear polarization was used to study the accessibility of surface tyrosine and tryptophan residues in proteinases, in their protein inhibitors and in the proteinase-inhibitor complexes. The accessibility probe is the triplet of 10-(carboxyethyl) flavin formed by optical excitation. On complex formation we observe accessibility loss in the surface tyrosines and tryptophans in the proximity of the proteinase-inhibitor contact site, and in the case of bovine pancreatic trypsin inhibitor, in more distant tyrosines as well.
Elastin was fully solubilized by digestion with elastase I or elastase II. Each digest was separated into high‐molecular weight and low‐molecular weight fractions that were characterized by the correspondence to their amino acid content, N‐terminal sequence and C‐terminal amino acids. It was found that although the relative amount of amino acids in the low‐molecular weight fraction obtained by digestion with elastase I was lower than in digestion with elastase II, no major difference in the type of bonds cleaved in the low‐ or high‐molecular weight fractions of each digest could be seen. There is, however, a remarkable difference in the type of bond cleaved by the two enzymes. While elastase I cleaves mostly Ala‐Ala and also Ala‐Gly bonds, elastase II hydrolyzes Leu‐Ala, Leu‐Gly, Phe‐Ala, Phe‐Gly and Tyr‐Ala, Tyr‐Gly bonds. Theoretical calculations led us to suggest both digests are composed of cross‐linked peptides that vary not only in the molecular size but also in the number of cross‐links found in peptides of the same size.
Human neutrophils contain large amounts of a neutral serine protease, human neutrophil elastase (HNE), which has been implicated as a mediator of acute and chronic lung injury. We found that this enzyme is effectively inhibited, at physiological ionic strength, by several synthetic non-base-paired polyribonucleotides. Among the most active of these is polyguanylic acid (poly G). Inhibitory activity is greatest with high-molecular-weight poly G fractions, but poly G fractions even as low as 60K Mr (app) are effective. Both amidolysis of synthetic elastase substrates, such as succinyl-ala-ala-ala-p-nitroanilide, and proteolysis of elastin are blocked. Poly G inhibits elastin proteolysis even when subsequently added to mixtures of elastin and HNE that have first been preincubated together for 10 min. Under these conditions, polyribosylribitol phosphate, a polyanion derived from Haemophilus influenzae capsular polysaccharide, is not inhibitory. Complex formation between HNE and poly G is dependent on ionic rather than covalent interactions, since it is blocked by 0.6 M NaCl but not by inactivation of the enzyme's catalytic-site serine residue with diisopropylfluorophosphate. However, nonspecific ionic interactions alone cannot explain complex formation, since pancreatic elastase and cathepsin G, an even more basic serine protease from human neutrophils, do not form complexes with poly G, even at low ionic strength. Moreover, in the presence of the amphiphiles taurocholic acid and glycocholic acid, HNE is much less effectively blocked by poly G. Peptide chloromethyl ketone-inactivate HNE (which has its extended substrate-binding pocket occupied by the peptidyl inactivator) also fails to form complexes with poly G. These results indicate that HNE may utilize both hydrophobic and ionic binding sites to couple with poly G, and suggest that these sites may be close to or within the extended substrate-binding pocket of the enzyme.
Interaction o f porcine elastase II with native and modified chicken egg-white ovoinhibitor was studied by determining the residual activity of the partially inhibited enzyme and by direct measurement of the stoichiometry of interaction using affinity chromatography, electrophoresis and gel filtration. It was found that the chymotrypsin binding site that is not modified by mild oxidation with N-chlorosuccinimide (Shechter et al., Biochemistry, (1977) 16,[992][993][994][995][996][997] is capable o f binding elastase II as well. The binding o f chymotrypsin and elastase II is mutually exclusive and the affinity for chymotrypsin is stronger. Binding of 2 mol trypsin as 1 rnol elastase I by ovoinhibitor does not interfere with the binding of elastase II. There is also an indication that the second binding site for chymotrypsin is capable o f forming a complex with an additional molecule o f elastase I . but the binding is so weak that it could be detected only by electrophoresis.Key words: chicken ovoinhibitor; inhibition; porcine elastase 11; protein-protein inter- action.Chicken ovoinhibitor, an egg-white glyco-exhibits chymotrypsin-like specificity toward protein with a molecular weight of 48000, hydrolysis of synthetic substrates and its is a multiheaded inhibitor of several mammalian amino terminal sequence strongly resembles serine pancreatic proteinases (1-3). It has been the B chain of bovine chymotrypsin B. Howdemonstrated by kinetic studies (1, 2, 4, 5 ) ever, unlike chymotrypsin, it hydrolyzes and by direct stoichiometric measurements elastin and is not inhibited by turkey ovo-(6) that it can simultaneously inhibit two mucoid (8). molecules of trypsin, two molecules of c h y m e ,We have reported that ovoinhibitor is trypsin and one molecule of elastase I. capable of inhibiting elastase I1 (9). The present Porcine elastase I1 is a recently described study was devoted t o further characterization pancreatic proteinase (7, 8). This enzyme of the interaction between these proteins and identification of the ovoinhibitor binding site(s) Abbrevhti ons: Ac, N-acetyl; Glut, glutaryl; Tos, that reacts with elastase 11. This was achieved N-p-toluenesulfonyl; OMe, methyl ester; OEt, ethyl by using both kinetic studies and measuring ester. the stoichiometry of interaction by affinity 169 0367-8377/81/070169-11 $02.00/0 o 1981 Munkspmd, Copenhagen
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