Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies.
Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H(+)-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (all inhibitors of vacuolar-type H(+)-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-gamma-32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H(+)-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H(+)-ATPase.
Chander
A
A
University of Pennsylvania School of Medicine, Institute for Environmental Medicine, Philadelphia.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects*; D000818:Animals; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D008168:Lung / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D014638:Vanadates / Q000494:pharmacology*
D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases