The somatic isoform of angiotensin-converting enzyme (ACE) consists of two homologous domains (N-and C-domains), each bearing a catalytic site. We have used the twodomain ACE form and its individual domains to compare characteristics of di¡erent domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz-Phe-His-Leu (N-carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine; from the panel of seven) was hydrolyzed faster by the N-domain, the substrates FA-Phe-Gly-Gly (N-(3-[2-furyl]acryloyl)-L-phenylalanyl-glycyl-glycine) and Hip-His-Leu (N-benzoyl-glycyl-L-histidyl-L-leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C-domain. The inhibitor captopril ((2S)-1-(3-mercapto-2-methylpropionyl)-L-proline) bound to the N-domain more e¡ectively than to the C-domain, whereas lisinopril ((S)-N K -(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA-PheGly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two-domain or single-domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k cat values obtained for somatic enzyme was the sum of k cat values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two-domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single-domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity. ß

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Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [(3)H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real-time beta-imager, [(3)H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5-aminocaproyl-isatin-Sepharose, we have investigated the distribution of [(3)H]isatin specific binding sites in the rat brain, characterized their K(d) and B(max), and identified some individual brain isatin binding proteins. The binding of [(3)H]isatin to rat brain sections was saturable and characterized by K(d) values (of 0.2-0.3 microM) consistent with physiological concentrations. The highest B(max) was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [(3)H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X-100 lysates of whole-brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde-3-phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton-related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. sigma=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold iinmunocytochemical method. Duodenase exhibits trypsin-like and chyinotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be a-N-tosylglycylprolyllysine 4-nitroanilide (kJK,,, of 35 000 M-' s-'), a-N-succinylthreonylprolyllysine 4-nitroanilide (kc4,/K1,! of 18 000 M-' s-') and a-N-serylprolyllysine 4-nitroanilide (k,JK,, of 2600 M-' s-I), all of which contain the P I -P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9 -PS'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (K,,, of 87 pM; k,;,, of 1.4 s-I; k,,,/K,,, of 16000 M-' s-I). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron Xrays to 0.2-nm resolution.