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.

N-glycosylation of forms of angiotensin converting enzyme from four mammalian species.

To help clarify bases for the molecular weight and surface charge heterogeneities of forms of somatic angiotensin converting enzyme (ACE), we examined for differences in N-glycosylation. ACE preparations purified from human, guinea pig, rat and rabbit tissues were found to be heterogeneous in terms of numbers of N-glycosylated sites (7-8 sites per molecule of ACE) and in types of structures of oligosaccharides used for glycosylation (complex versus high mannose oligosaccharide contents). Our findings, taken with reports of potential N-glycosylation sites and amino acid sequencing data, indicate that ACE forms can differ in terms of degrees of glycosylation, sites of glycosylation and structures of attached oligosaccharide units.

A comparison of guinea pig serum angiotensin converting enzyme with forms of angiotensin converting enzyme from human, rat and rabbit tissues.

Guinea pig serum angiotensin converting enzyme (ACE) activities exceed ACE activities of other mammalian sera by as much as two magnitudes. To examine the possibility that guinea pig ACE has a superior catalytic efficiency, we purified it to apparent homogeneity and compared it to highly purified forms of ACE from human seminal plasma, rat lungs and rabbit lungs. The first 24 amino acid residues of guinea pig and rat ACE forms were 96% identical with the sequence of human ACE. Second order rate constants (kcat/Km) for guinea pig, human and rabbit forms of ACE on reaction with benzoyl-Phe-Ala-Pro were identical (1.6E-09 M-1 min-1). Their dissociation constants on reaction with the ACE inhibitor RAC-X-65 were within a narrow range (10-16 pM). Thus, the high ACE activity of guinea pig serum is owing to high enzyme concentration and not to superior catalytic efficiency.

A radiochemical assay for aminopeptidase N.

We developed an assay for aminopeptidase N (AmN) in which substrate, Arg-Phe-[3H]anilide (24.9 Ci/mmol), can be used at concentrations (1-200 nM) well below Km (12 microM) and at or below enzyme concentration ([E]). Such reaction conditions simulate those in vivo where peptide hormones in picomolar concentrations (<< Km) are degraded by nano- or micromolar concentrations of enzyme. The Arg-Phe-[3H]anilide:AmN reaction obeyed first-order enzyme kinetics when human serum, human seminal plasma, guinea pig serum, or homogeneous porcine kidney AmN was used as enzyme source and substrate was within the concentration range of 1-200 nM. For porcine AmN, kcat/Km was 1.47 x 10(9) M-1 min-1, kcat 17,640 min-1. Human serum AmN was in a concentration (about 4.6 nM) in great excess over those reported for substrates such as angiotensin III. Several advantages accrue under conditions of first-order enzyme kinetics: (1) Vmax/Km is measured directly. (2) When kcat/Km is known, [E] can be computed in mol/liter. (3) IC50 values for alternative substrates can be taken as Km values. (4) IC50 values for inhibitors are Ki values when Ki >> [E]. Arg-Phe-[3H]anilide can be used to measure AmN activity in the presence of chromophores and fluorophores that interfere with photometric and fluorometric assays. We have confirmed that alleged substrates such as angiotensin III and Met-Lys- and Lys-bradykinin are bound by AmN with high affinities (Km values, 5.7, 9.1, and 14.3 microM). Bovine pulmonary artery endothelial cell cultures were found to possess AmN-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)