The effect of cathepsins from varied sources on proteinpolysaccharide (PP-L) of bovine costal cartilage and on proteoglycan subunit (PGS) of bovine nasal cartilage.

Abstract

The death of the hypertrophic cartilage cells of epiphyseM plates may be causally linked to the provisional calcification of extraterritorial matrix in the vicinity of such cells. An increasing number of cytoplasmic organelles, limited by a single membrane and morphologically identified as lysosomes are found in maturing chondrocytes (8). Moreover, when the hypertrophic cells degenerate, the marker enzyme for lysosomes, acid phosphatase, is seen in the adjacent cartilage matrix (1, 4). Among the complement of enzymes in lysosomes there are acid proteases. These have been implicated as playing a role in the modification of eartilagenous matrices (3, 7, 9). On the other hand, hydrolases released from elements of invading capillary sprouts may also have a lyric effect on the matrix (7). In view of the two possibilities mentioned above, an acid protease isolated from bovine costal cartilage has been compared with an acid protease isolated from alveolar macrophages of rabbits as regards their effects on proteinpolysacch aride light component (PP-L)isolated from costal cartilages of calves (5) and on the proteoglycan subunit isolated from bovine nasal cartilage (6). Thin slices of fresh costal cartilage from calves were extracted two times at 4oC for 24 hours each time with 10 volumes of deionized water which was saturated with chloroform. After filtration through a sintered-glass filter, the filtrate was made 0.05 M in KC1 and 0.005 M in ~a~ttPO4. The pH of the solution was adjusted to 7.0 by the addition of concentrated HC1. To this solution at 20~ a 10 per cent solution of eetylpyridinium chloride in a 0.005 M sodium phosphate buffer, pit 7.0, which was also 0.05 M in KC], was slowly added with stirring until a floeeulent end point was reached. The resultant precipitate was removed by filtration through a sinteredglass filter of medium porosity. Thereafter the clear filtrate was set aside at 4oC overnight. If excess cetylpyridinium chloride precipitated during this time, it was removed by filtration at 4oC. The solution was dialyzed against a 0.005 M sodium phosphate buffer, pit 7.0, which was also 0.05 M in KC1, for 6 hours at room temperature and then against water saturated with chloroform for 16 hours. It was next equilibrated by dialysis against a 0.005 M potassium acetate buffer, pit 4.0, which was also 0.05 M in KCI. Of the resultant retent~te, 1000 ml were passed through a 2 x30 em column of SE-Sephadex C-25, which too had been previously equilibrated