Alveolar surfactant is known to exist in at least 3 subtypes that are in metabolic relation with each other: a lamellar body form that evolves in sequence into tubular myelin and vesicular form. A unique way of mimicking this metabolic sequence in vitro has been developed in this laboratory, based on repeated cyclic expansion and contraction of the air-fluid interface of surfactant at 38 C (""""""""cycling""""""""). Manipulating nascent surfactant in this way generates products that exactly resemble the subtypes found in alveolar lavage fluid with respect to phospholipid and protein content, buoyant density, ultrastructure, and surface properties. This technique makes it possible for the first time to study the extra-cellular metabolism of surfactant in detail. Using this methodology, the applicant found that any of large number of serine protease inhibitors inhibited the conversion of tubular myelin to the vesicular form on cycling in vitro, suggesting that a serine protease (called """"""""convertase"""""""" for convenience) was required for this conversion. Alpha-1 antitrypsin was found to be the most potent inhibitor, resulting in 50% inhibition at in vivo alveolar concentrations suggesting physiologic relevance. The enzyme, which appears to be concentrated in a lamellar body-rich preparation and secreted with its substrate, has a number of unique biochemical properties including the absolute requirement for a surface that is cyclically expanded and contracted. The present project seeks to purify the convertase, identify its cell source, and explore some of its actions on the physiology and turnover of alveolar surfactant. Using an assay of the convertase developed for this purpose, the enzyme will be purified from a lamellar body-rich preparation of rat lungs using affinity chromatography. Purity will be checked by 2D-SDS-PAGE and Western transfer of the 3H-DFP reacted yield. Its cellular source will be determined using HRP-linked antibody raised in rabbits against the purified enzyme. The applicants propose to investigate the in vivo physiologic role of convertase by examining the effect of a1-antitrypsin inhibition on the surface tension properties of nascent surfactant, and determining the effect of administering to mice a 5-10 fold excess of a1-AT on surfactant subtype proportions, turnover rates, and lung pressure-volume behavior.
