Estimation of rate constants for reactions of pulmonary microvascular angiotensin converting enzyme with an inhibitor and a substrate in vivo.
We developed a method of measuring the mole quantity of pulmonary angiotensin-converting enzyme (ACE) bound by a partially saturating dose of an ACE inhibitor injected i.v. For each test animal (11 guinea pigs), tracer (nonsaturating) doses of the ACE substrate [14C]benzoyl-Ala-Gly-Pro (14C-BAGP) and the ACE inhibitor 3H-RAC-X-65 were coinjected at timed intervals for a total of four studies per animal. The injectate used for the second study contained, in addition, a partially saturating dose of unlabeled RAC-X-65. With indicator-dilution techniques supplemented with measurements of fractional hydrolysis of 14CBAGP and uptake of 3H-RAC-X-65 during a single transit through the pulmonary vascular bed, the following parameters were computed: plasma flow (Qp), (kcat/Km)[E] vector c, k1[E] vector c and Eb, where [E] is the concentration of active ACE, Eb is the mole quantity of ACE bound by inhibitor, kcat/Km is the second-order rate constant for substrate hydrolysis, k1 is the inhibitor-ACE association rate constant and vector c is capillary mean transit time. As shown elsewhere (Catravas et al., 1990; Catravas and White, 1984), the product of Qp (in liters per second) multiplied by (kcat/Km)[E] vector c is (kcat/Km)E, and the product of Qp multiplied by k1[E] vector c is k1E, where E is the mole quantity of ACE. Values of (kcat/Km)Eb and k1Eb were computed and divided by Eb to obtain kcat/Km and k1. The fractional degree of inhibition conferred by a partially saturating dose of an ACE inhibitor can be understood to be the ratio Eb/ET, where ET is total ACE. With Eb in moles and the ratio Eb/ET, we computed the mole quantity of ET. By measuring the rate of recovery of ACE activity following partial inhibition of ACE, an apparent dissociation rate constant, k(dissoc), was computed. With k(dissoc) and K1, an apparent Ki was computed. The following computations were obtaine: ET of 0.90 +/- 0.20 (S.E.M.) nmol; kcat/Km, 5.16 +/- 0.89E + 06 M-1.sec-1; k1, 1.26 +/- 0.21E + 06 M-1.sec-1; k(dissoc), 6.47 +/- 0.63E - 04 sec-1 and Ki, 5.13E - 10 M. Although we focused on the characterization of ACE, the methods developed are general and may be applicable to studies of other vascular surface proteins, including other enzymes and hormone receptors.
Ryan
J W
JW
Department of Medicine, University of Miami School of Medicine, Florida.
Valido
F A
FA
Sequeira
M J
MJ
Chung
A Y
AY
Berryer
P
P
Chen
X L
XL
Catravas
J D
JD
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D002250:Carbon Radioisotopes; D004656:Enalapril / Q000031:analogs & derivatives / Q000494:pharmacology; D004795:Enzyme Stability; D005260:Female; D006168:Guinea Pigs; D006868:Hydrolysis; D007700:Kinetics; D008168:Lung / Q000098:blood supply* / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D013379:Substrate Specificity; D014316:Tritium
D000806:Angiotensin-Converting Enzyme Inhibitors; D002250:Carbon Radioisotopes; D009842:Oligopeptides; D014316:Tritium; C051316:N-(1-carboxy-3-carboxanilidopropyl)alanylproline; C085359:benzoylalanyl-glycyl-proline; D004656:Enalapril; D007703:Peptidyl-Dipeptidase A