Studies on cartilaginous enzymes, degrading protein-polysaccharide complex:A simple electrophoretic method for estimating the enzyme activities and its application to the purification of the enzymes from puppy articular cartilage

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The light fraction of protein-polysaccharide complex (PPL) was isolated from bovine nasal septum. The enzymic degradation of PPL by protease (trypsin) or polysaccharidase (testicular hyaluronidase (HAse)) was apparently demonstrated by paper electrophoresis (Beckman No. 320046 paper strips, length 7cm, pH, 8.6, 0.05M borate buffer, 60V, 30min), followed by toluidine blue staining. Under these conditions, PPL remaind at the starting line, while the protease-degraded polysaccharide chains migrated towards anode as a broad band. HAse digestion, however, yielded two or three bands which were different in shape and mobility from that of the protease digestion products. Applying the paper electrophoretic method for estimating the activities, the PPL-degrading enzymes were purified from puppy articular cartilage minces by water extraction, protamine treatment, Sephadex G-75 gel filtration, and electrofocusing (pH 3-10), successively. There were separated HAse- and protease-type enzymes with isoelectric points of 5.8 and 7.6, respectively. These enzymes exhibited the optimum pH at about 3-4. The present method was shown to be advantageous with respect to its simplicity, applicability to micro-quantity of the sample, and reliable discrimination between protease- and HAse-type enzyme activities.
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