Purification, characterization, and biosynthesis of bovine cartilage lysozyme isoforms.
The cationic protein, lysozyme, has an extracellular distribution in cartilage; however, its biological role in this tissue still remains unclear. This study describes a simple and high yielding procedure for the purification of four novel isoforms of lysozyme from the functionally different articular (metacarpalphalangeal joint) and nonarticular (nasal septum) bovine cartilages. Chromatography of the cartilage extracts on S-Sepharose revealed the presence of four major lysozyme active peaks each of which was further purified to homogeneity by gel filtration and reversed-phase chromatography. Each peak yielded a different molecular mass when analyzed by ion spray mass spectrometry, and material isolated from either cartilage source displayed an identical molecular mass for each lysozyme preparation. N-terminal amino acid sequence and amino acid composition analyses confirmed the presence of four novel lysozyme isoforms in both bovine articular and nonarticular cartilages. The lytic activity of each lysozyme isoform toward Micrococcus lysodeikticus was dependent on both the ionic strength and pH of the buffer, where an increase in activity accompanied an increase in ionic strength. The lysozymes were shown to be synthesized by chondrocytes in 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 cartilage extracellular matrix.
Moss
J M
JM
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Van Damme
M P
MP
Murphy
W H
WH
Stanton
P G
PG
Thomas
P
P
Preston
B N
BN
D000595:Amino Acid Sequence; D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002358:Cartilage, Articular / Q000201:enzymology*; D002417:Cattle; D002853:Chromatography, Liquid; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D009994:Osmolar Concentration; D016415:Sequence Alignment
D007527:Isoenzymes; D009113:Muramidase