Characteristics of bovine alveolar macrophage elastase.

Lavage of isolated bovine lung lobes was used to retrieve pulmonary alveolar macrophages (PAM). Nominal yields of 72 million viable PAM were routinely obtained using 500 ml of calcium- and magnesium-free phosphate-buffered saline. Bovine PAM readily attached to glass coverslips and within 24 hours, provided cell monolayers consisting exclusively of macrophages. Bovine PAM synthesized and secreted a calcium-dependent elastase in serum-free media. Optimal proteolytic activity using a radiolabeled elastin substrate was observed at pH 7.6. The elastase was insensitive to synthetic peptide chloromethyl ketone elastase inhibitors and to the serine protease inhibitor, phenylmethylsulfonyl fluoride. Enzyme activity, however, was effectively inhibited by the metal chelator, ethylenediaminetetraacetic acid, or suppressed by inhibition of protein synthesis with cycloheximide.