IDENTIFICATION OF HUMAN PULMONARY ALKALINE-PHOSPHATASE ISOENZYMES

An increase of alkaline phosphatase (ALP) activity has been observed i n the bronchoalveolar lavage fluid (BALF) of patients affected by pulm onary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ ag glutinin (WGA) precipitation, and an immunoassay specific for the bone -isoform of ALP. Only one anodic band representing a high-molecular-we ight isoform of ALP (Mr similar to 2,000 kDa) was observed on electrop horesis of BALF. The inhibition assay results were consistent for a ti ssue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and re sistant to L-phenylalanine and L-leucine. Less than 13% of ALP activit y was heat-stable. After incubation of BALF specimens with glycosyl-ph osphatidyl inositol-phospholipase D plus Nonidet P-40, or with phospha tidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr similar to 220 kDa) appeared near the bone band of a standard seru m. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less th an 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.