ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE Their Subeellular Distribution in Isolated Chondrocytes

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution . It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences . Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage . tion of various enzymatic activities have been carried out for a number of tissues, both normal (2-6) and pathological (7-9), in many species. Cartilage has thus far defied such an analysis, primarily since it has not been possible to homogenize this tissue under conditions which preserve the integrity of the different subcellular particles because of the large amount of connective tissue surrounding its cells . Lytic enzymes have been used for the dispersion of tissues and the subsequent separation of cells into homogeneous populations (10, 11) . The development of such a method, employing sequential digestion with trypsin and collagenase, for the separation of a large number of viable chondrocytes has been described in the accompanying publication (12) . We here describe the fractionation of chondrocytes isolated by this method and THE JOURNAL OF CELL BIOLOGY • VOLUME 49, 1971 . pages 459-467 459 on O cber 9, 2017 jcb.rress.org D ow nladed fom the distribution of various enzymes characterizing the different cell particulates. The distribution patterns for mitochondrial, lysosomal, microsomal, and soluble enzymes obtained are basically similar to those found in a prototype tissue, mammalian liver, although some qualitative and quantitative differences were observed. While this work was in progress, two reports on the enzymatic composition of cartilage appeared (13, 14) . The present report deals with some enzymes which have not been examined in detail thus far in their intracellular localization . EXPERIMENTAL PROCEDURE