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Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90-95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyrylcholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. The pH optimum of the peptidase was in the range of 7.5-9.5 and the divalent cations Co2+, Mn2+, and Zn2+ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co2+, Mn2+, and Zn2+ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited alpha-chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.

Several bradykinin potentiating peptides (BPPs) were isolated from the venom of the Brazilian arboricole snake Bothrops insularis by gel filtration on Sephadex G-150-120, followed by sequencial high-voltage paper electrophoreses at pH 3.5, 6.5, and 2.1. The BPPs were assayed by their ability to potentiate the contractile activity, on the isolated guinea pig ileum, and the hypotensive activity, on anesthetized rats, of bradykinin. Eight BPPs, containing 3-13 amino acid residues, were sequenced and their primary structures were shown to have a marked degree of homology with those of several BPPs from other venoms.

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (alpha-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi, J. Protein Chem. 11, 579-581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.

Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.

In the present paper, we demonstrate that alpha1-antichymotrypsin, a serpin with high inhibitory specificity toward cathepsin G, and kallistatin, a human serpin with high specificity toward tissue kallikrein, are digested by cathepsin D. Alpha1-Antichymotrypsin was hydrolyzed essentially in the reactive center loop at L-S, A-L, or L-V bonds; kallistatin was split into small fragments, but we detected the cleavage at F-F and F-S bonds in its reactive center loop in the first 15 min of digestion. In contrast to alpha1-antichymotrypsin, kallistatin is irreversibly inactivated at pH 4.0. Synthetic internally quenched fluorescent peptides containing sequences similar to the reactive center loops of these serpins were hydrolyzed by cathepsin D. The peptides derived from kallistatin were hydrolyzed more efficiently, and particularly relevant was the high susceptibility of the substrates Abz-AIKFFSAQTNRHILRFNRQ-EDDnp (Km = 0.08 microM, kcat = 2.4 s(-1)) and Abz-AIKFFSAQTNRQ-EDDnp (Km = 0.8 microM, kcat = 17.8 s(-1)), which were hydrolyzed at the F-F bond. Therefore, besides the description of a new class of very efficient internally quenched substrates for cathepsin D, we give evidence for the downregulation role of this proteinase on alpha1-antichymotrypsin and kallistatin. The acidification of extracellular milieu by tumor cells can result in activation of cathepsin D; as a consequence, kinins can be released, improving blood supply and leaving more cathepsin G available for the degradation of extracellular matrix.

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.

It has been shown that probable portions which form contacts in a protein can be predicted by means of an average distance map (ADM) as well as regular structures (alpha-helices and beta-turns) defined as short-range compact regions (Kikuchi et al., 1988a,c). In this paper, we analyze the occurrence of those portions and short-range compact regions on ADMs for various proteins regarding their folding types. We have found out that each folding type of proteins shows characteristic distribution of such parts on ADMs. We also discuss the possibility of the prediction of folding types of proteins by ADMs.

Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypolyethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the alpha-NH2 of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N-terminal of an otherwise unaltered peptide molecule.