PURIFICATION, CHARACTERIZATION, AND BIOSYNTHESIS OF BOVINE CARTILAGE LYSOZYME ISOFORMS

The cationic protein, lysozyme, has an extracellular distribution in c artilage; however, its biological role in this tissue still remains un clear. This study describes a simple and high yielding procedure for t he purification of four novel isoforms of lysozyme from the functional ly different articular (metacarpalphalangeal joint) and nonarticular ( nasal septum) bovine cartilages. Chromatography of the cartilage extra cts on S-Sepharose revealed the presence of four major lysozyme active peaks each of which was further purified to homogeneity by gel filtra tion and reversed-phase chromatography. Each peak yielded a different molecular mass when analyzed by ion spray mass spectrometry, and mater ial isolated from either cartilage source displayed an identical molec ular mass for each lysozyme preparation. N-terminal amino acid sequenc e and amino acid composition analyses confirmed the presence of four n ovel lysozyme isoforms in both bovine articular and nonarticular carti lages. The lytic activity of each lysozyme isoform toward Micrococcus lysodeikticus was dependent on both the ionic strength and pH of the b uffer, where an increase in activity accompanied an increase in ionic strength. The lysozymes were shown to be synthesized by chondrocytes i n vitro, which in addition to the relatively high chemical amounts of lysozyme present in cartilage, would suggest that this small cationic protein has some as yet undetermined biological role within the cartil age extracellular matrix. (C) 1997 Academic Press.