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For understanding the characteristics of lysosomal enzymes, cathepsin D was first purified by heat treatment, concanavalin A-Sepharose and Sephadex G-150 chromatographies, and finally preparative electrophoresis. A purification of 244-fold against the crude extract was achieved. The recovery was 2 96. The purified enzyme appeared t o be electrophoretically homogeneous and had a molecular weight of 55 000. Inhibitor study and molecular weight determination indicated this enzyme t o be an aspartic proteinase, cathepsin D. The optimal pH and temperature were 3.

Calpain II (EC 3.4.22.17), calcium-activated neutral proteinase, from tilapia muscle (Tilapia nilotica X Tilapia aurea) was purified by using hydrophobic chromatography with phenyl-Sepharose CL-4B and substrate affinity chromatography with a-casein agarose. Electrophoretic analysis and inhibitor specificity studies indicated that the calpain II was a monomer and purified to homogeneity. The molecular weight was electrophoretically determined to be 80 000. The requirement of Ca2+ concentration was 0.1 mM for half-maximal and 1 mM for maximum activation. This enzyme was fully activated by 10 mM 2-mercaptoethanol. The inhibitory ability of calpain inhibitor I was the highest, followed by leupeptin, calpain inhibitor II, antipain, iodoacetate, PCMB, and PMSF in decreasing order. Accordingly, the active site of this proteinase might contain cysteine and histidine for catalysis. The caseinolytic activity of this purified calpain was 480 units/mg at pH 7.5. The recovery was 35%.

soybean 11s globulin. Agric. Biol. Chem. 1977,41, 647-653. Pleitz, P.; Damaschun, G. The structure of the 11s seed globulins from various plant species: Comparative investigations by physical methods. Stud. Biophys. 1986,116, 153-173. Richarz, R.; Wuthrich, K. High-field 13C nuclear magnetic resonance studies at 90.5 MHz of the basic pancreatic trypsin inhibitor. Biochemistry 1978, 17, 2263-2269. Spitz, H. D. A new approach for sample preparation of protein hydrolyzates for amino acid analysis. Anal. Biochem. 1973, Staswick, P. E.; Hermodson, M. A.; Nielsen, N. C. Identification of the acidic and basic subunit complexes of glycinin. J. Biol. Chem. 1981,256, 8752-8755. Staswick, P. E.; Hermodson, M. A,; Nielsen, N. C. Identification of the cystines which link the acidic and basic components of the glycinin subunits. J. Biol. Chem. 1984,259, 13431-13435. Thanh, V. H.; Shibasaki, K. Major proteins of soybean seeds. A straightforward fractionation and characterization, J. Agric. Food Chem. 1976,24, 1117-1121. Utsumi, S.; Inaba, H.; Mori, T. Heterogeneity of soybean glycinin. Phytochemistry 1981, 20, 585-589. Van Binst, G.; Biesemans, M.; Barel, A. 0. Comparative carbon-13 nuclear magnetic resonance study at 67.9 MHz on lysozyme 56, 66-73. (Human and Egg-white) and a-lactalbumin (Human and Bovine) in their native and denatured state. Bull. SOC. Chim. Belg. Van Kleef, F. S. M. Thermally induced protein gelation: Gelation and rheological characterization of high concentrated ovalbumin and soybean protein gels. Biopolymers 1986, 25, 31-59. Vold, R. L.; Waugh, J. S.; Klein, M. P.; Phelps, D. E. Measurements of spin relaxation in complex systems. J. Chem. Phys. 1968, 48, 3831-3832. Wittebort, R. J.; Rothgeb, T. M.; Szabo, A.; Gurd, F. R. N.Aliphatic groups of sperm whale myoglobin: W-NMR study. Briggs, D. R. Studies on the cold-insoluble fraction of the water-extractable soybean proteins. 11. Factors influencing conformation changes in the 11s component. Arch.The effects of frozen storage temperatures on the formation of disulfides and the denaturation of myosin, extracted from milkfish (Chanos chanos) dorsal muscle, were investigated. The activities of Ca-ATPase and Mg(Ca)-ATPase and solubility in 0.6 M KCl decreased, and the total NaF3H4-soluble and -insoluble proteins increased a t a much higher rate a t -20 "C than at -35 "C. During freezing, the total SHs of samples a t -20 "C decreased significantly, but not a t -35 "C. The decreasing rate of total SHs a t -20 "C was significantly faster than a t -35 "C during storage.