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

Andreas Papapetropoulos, Lynn A. Elmore, John D. Catravas

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume271
Issue number3 15-3
StatePublished - Sep 1 1996
Externally publishedYes

Keywords

  • 5'- nucleotidase
  • angiotensin-converting enzyme
  • bovine pulmonary arterial endothelial cells
  • capillary plasma volume
  • cell culture
  • enzyme activity
  • vascular endothelium

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology
  • Physiology (medical)

Fingerprint Dive into the research topics of 'Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC'. Together they form a unique fingerprint.

  • Cite this