DEGRADATION OF SURFACTANT-ASSOCIATED PROTEIN-B (SP-B) DURING IN-VITROCONVERSION OF LARGE TO SMALL SURFACTANT AGGREGATES

Pulmonary surfactant obtained from lung lavages can be separated by di fferential centrifugation into two distinct subfractions known as larg e surfactant aggregates and small surfactant aggregates. The large-agg regate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfacta nt aggregates increases after birth and in several types of lung injur y. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates gener ated by surface area cycling were separated from large aggregates by c entrifugation at 40000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by m orphological studies. Surface-tension-reducing activity of total surfa ctant extracts. as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to t he generation of small aggregates. Immunoblot analysis of large and sm all aggregates separated by sucrose gradient centrifugation revealed t he presence of detectable amounts of surfactant-associated protein B ( SP-B) in large aggregates but not in small aggregates. SP-A was detect able in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregate s in vitro and that a change in surface area appears to be necessary f or exposing SP-B to protease activity.