Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC

It is unclear whether all or a fraction of the capillary plasma volume (V(cp)) serves as the reaction volume (V(r)) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-V(r) relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (V(max)) E x k(cat)/V(r), where E is enzyme mass and k(cat) is the constant of product formation] is inversely proportional to V(r), we hypothesized that increasing the volume of medium (V(m)) bathing EC monolayers would proportionally reduce the calculated V(max) (or V(max)/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, V(m) = V(r). To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [³H]benzoyl-Phe-Ala-Pro ([³H]BPAP) and 5'-[¹⁴C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP]= 1 μM) V(max)/K(m) ratios of ACE and NCT declined to 20% of their original values, as V(m) increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (V(max)/K(m)) declined proportionally to increasing V(m). Under zero-order reaction conditions ([BPAP] = 250 μM), ACE activity (V(max)) was similarly related to V(m). Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml V(m), a volume vastly exceeding that of V(cp) in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that V(cp) could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.