Michaelis-Menten kinetics of pulmonary endothelial angiotensin converting enzyme in the conscious rabbit

Estimations of Michaelis-Menten constants Km and Amax (product of Vmax and pulmonary microvascular plasma volume) of pulmonary, endothelial-bound angiotensin converting enzyme (ACE) for the synthetic substrate 3H-Benzoyl-Phe-Ala-Pro (BPAP) were performed in rabbits, in vivo, utilizing indicator dilution techniques. The animals were conscious and equipped with permanent right atrial and left carotid catheters. For each determination of Km and Amax, two consecutive bolus injections of BPAP were given into the right atrial catheter, the first containing 0.1 and the second 1622 nmol of substrate, producing first order and mixed order substrate concentrations, respectively, in the pulmonary circulation. Arterial blood was withdrawn at 0.7 sec intervals for 15 sec after each injection and from the family of substrate concentrations thus created (usually 14-20) and resulting range of substrate utilization (10-90%), Km and Amax values were calculated utilizing the Lineweaver-Burk, Eadie-Scatchard, Woolf-Augustinsson-Hofstee or Hanes-Woolf transformations of the integrated Henri-Michaelis-Menten equation. All four methods produced similar values of Km (10-12 microM), Amax (6-7 mumol/min) and Amax/Km (600-720 ml/min); however, linear regression analysis of data from the Hanes-Woolf transformation resulted in a higher correlation coefficient (0.96 vs 0.86-0.88 for the other three methods). Values for the kinetic constants reported here were similar to those previously reported in anesthetized rabbits utilizing the Woolf-Augustinsson-Hofstee transformation, but higher than those reported for purified rabbit lung ACE, in vitro.