ESTIMATION OF RATE CONSTANTS FOR REACTIONS OF PULMONARY MICROVASCULARANGIOTENSIN-CONVERTING ENZYME WITH AN INHIBITOR AND A SUBSTRATE IN-VIVO

We developed a method of measuring the mole quantity of pulmonary angi otensin-converting enzyme (ACE) bound by a partially saturating dose o f an ACE inhibitor injected i.v. For each test animal (11 guinea pigs) , tracer (nonsaturating) doses of the ACE substrate [C-14]benzoyl-Ala- Gly-Pro (C-14-BAGP) and the ACE inhibitor H-3-RAC-X-65 were coinjected at timed intervals for a total of four studies per animal. The inject ate used for the second study contained, in addition, a partially satu rating dose of unlabeled RAC-X-65. With indicator-dilution techniques supplemented with measurements of fractional hydrolysis of C-14-BAGP a nd uptake of 3H-RAC-X-65 during a single transit through the pulmonary vascular bed, the following parameters were computed: plasma flow (Q( p)), (k(cat)/K-m)[E]t(c1) k(1)[E]t(c) and E(b), where [E] is the conce ntration of active ACE, Eb is the mole quantity of ACE bound by inhibi tor, k(cat)/K-m is the second-order rate constant for substrate hydrol ysis, k(1) is the inhibitor-ACE association rate constant and E is cap illary mean transit time. As shown elsewhere (Catravas et al., 1990; C atravas and White, 1984), the product of Q(p) (in liters per second) m ultiplied by (k(cat)/K-m)[E]t(c) is (k(cat)/K-m)E(1) and the product o f Q(p) multiplied by k(1)[E]t(c) is k(1)E, where E is the mole quantit y of ACE. Values of (k(cat)/K-m)E(b) and k(1)E(b) were computed and di vided by E(b) to obtain k(cat)/K-m and k(1). The fractional degree of inhibition conferred by a partially saturating dose of an ACE inhibito r can be understood to be the ratio E(b)/E(T1) where E(T) is total ACE . With E(B) in moles and the ratio E(b)/E(T), we computed the mole qua ntity of E(T). By measuring the rate of recovery of ACE activity follo wing partial inhibition of ACE, an apparent dissociation rate constant , k(dissoc), was computed. With k(dissoc) and k(1), an apparent K-I wa s computed. The following computations were obtained: E(T) of 0.90 +/- 0.20 (S.E.M.) nmol; k(cat)/K-m, 5.16 +/- 0.89E + 06 M(-1).sec(-1); k( 1), 1.26 +/- 0.21E + 06 M(-1) sec(-1); k(dissoc), 6.47 +/- 0.63E - 04 Sec(-1) and K-I, 5.13E - 10 M. Although we focused on the characteriza tion of ACE, the methods developed are general and may be applicable t o studies of other vascular surface proteins, including other enzymes and hormone receptors.