. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SEA STAR TRYPSIN INHIBITOR 359 O H CD X IOO- 80- 60- 4O- 20- o O CD o CT> > CM I I. —r— 25 15 20 FRACTION NO T" 30 35 FIGURE 2. Gel permeation of sea star cell-free coelomic fluid. Coelomic fluid (200 n\) was gel filtered on Sephadex G-50 ( cm X 115 cm) equilibrated in MNaCl in 50 mMTris-HCl, pH Fractions of ml were collected at a column flow rate of 9 ml/h. Inhibition of bovine trypsin by eluate fractions was determined by measuring residual enzymatic activity with S2222 as outlined above. Molecu
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SEA STAR TRYPSIN INHIBITOR 359 O H CD X IOO- 80- 60- 4O- 20- o O CD o CT> > CM I I. —r— 25 15 20 FRACTION NO T" 30 35 FIGURE 2. Gel permeation of sea star cell-free coelomic fluid. Coelomic fluid (200 n\) was gel filtered on Sephadex G-50 ( cm X 115 cm) equilibrated in MNaCl in 50 mMTris-HCl, pH Fractions of ml were collected at a column flow rate of 9 ml/h. Inhibition of bovine trypsin by eluate fractions was determined by measuring residual enzymatic activity with S2222 as outlined above. Molecular weight standards included carbonic anhydrase (29,000), lyzozyme (14,300), and aprotinin (6500). the inhibitor is only mildly sensitive to heat and acid treatments. A molecular weight of about 6500 was estimated by gel permeation chromatography using Sephadex G- 50 (Fig. 2). The above molecular characteristics of the coelomic fluid inhibitor are similar to those described for other small molecular weight inhibitors (Fritz and Krejci, 1976; Tschesche, 1976; Tschesche and Dietl, 1976). Crude preparations of proteolytic enzymes were obtained from the gastric tissues of A. forbesi and the sea urchin Strongylocentrotus droebachiensis. Briefly, the gut was excised surgically and disrupted with a glass pestle and homogenizer (Radnoti Glass Technology, Inc., Monrovia, California 91016). Cellular debris was removed by centrifugation at 1000 X g for 20 min at 4°C, and the supernate containing the gut enzyme(s) was decanted. These gut proteases represented trypsin-like enzymes based upon the following criteria: (1) Enzymatic activity was inhibited completely by bo- vine pancreatic trypsin inhibitor and soybean trypsin inhibitor, and (2) the proteases hydrolyzed synthetic peptide substrates containing carboxy-terminal arginyl residues (data not shown). Admixture of protease from either sea star or sea urchin with sea star coelomic fluid resulted in rapid inhibition of the respectiv
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