Modifications of connective tissue matrices by an enzyme extracted from cartilage. A histochemical, autoradiographic, and electronmicroscopic investigation.

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The degradative properties of an enzyme extracted from bovine costal cartilage were studied histochemically, autoradiographically, and electronmicroscopically. Previous work had indicated that this enzyme catalyzes the degradation of proteinpolysaccharide, light fraction (PP-L) from bovine costal cartilage to a proteinpolysaccharide with a lower protein content than the original substrate. Attempts were therefore made to ascertain if this protease was species specific and if proteinpolysaccharides other than those present in cartilage were susceptible to enzyme digestion. To this end, the effect of the enzyme on human fetal cartilage and jaws and the epiphyseal plates of neonatal and postnatal rats was studied. Furthermore, 35S-H2SO4 was injected into pregnant rats, the fetuses were removed and sections of them were digested with the enzyme and then autoradiographed. In the histochemical experiments Alcian Blue with MgCl2 was used for the staining of tissue polyanions and Bromphenol Blue for the detection of free basic groups. Finally, the limbs of 20 day-old rats were utilized in electronmicroscopic studies. Within the limitations of the techniques utilized the results obtained elucidated the following characteristics of the action of the enzyme on the tissues: 1. the products resulting from the action of this protease are more soluble than the proteinpolysaccharides originally present in the tissues, 2. the extent to which the enzyme affects the tissue depends directly on the state of maturation of the tissue and, therefore, on the state of aggregation of the matrix, and 3. the enzyme is not species or tissue specific. Autoradiograms of sections incubated with the enzyme also indicate an enhanced solubility of proteinpolysaccharides. The effects of the enzyme on ultrathin sections were manifested in an increased affinity for phosphotungstic acid staining of the extrafibrillar matrix and of particles in the lacunae of degenerating chondrocytes. The latter showed a distribution similar to that of particles seen in semithin sections stained with Azure A.