Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface areacycling

Pulmonary surfactant isolated by lavage can be separated into large aggrega tes (LA) and small aggregates (SA). Pulse labeling experiments have shown t hat the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling . The precise mechanisms of surfactant subtype conversion remain unknown. W e have previously reported a decline in surfactant-associated protein B (SP -B) during in vitro subtype conversion of canine surfactant. This led to th e hypothesis that SP-B may be degraded by a serine protease 'convertase' du ring cycling. The current studies used a quantitative slot-blot assay to in vestigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Convers ion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform :methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recover ed SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels. (C) 1999 Elsevier Science B.V. All righ ts reserved.