PURIFICATION AND CHARACTERIZATION OF A PROTEASE DEGRADING 30 KDA YOLKPROTEINS OF THE SILKWORM, BOMBYX-MORI

The second major yolk proteins, 30 kDa proteins (30kPs) of the silkwor m, Bombyx mori, which have been provided during oogenesis, are kept co ntinuously unused during embryogenesis and are utilized just before la rval hatching, The crude extracts of newly hatched larvae cleaved 30kP s in an in vitro incubation system, A protease was highly purified fro m newly hatched larvae using ammonium sulfate precipitation, gel filtr ation and ionic exchange chromatography, and non-denaturing-polyacryla mide gel electrophoresis (ND-PAGE), The protease shared the NH2-termin al amino acid sequence conserved in many serine proteases, and the app arent molecular mass was estimated to be approximately 600 kDa by gel filtration column chromatography, The enzymatic activity was strongly inhibited by elastatinal and diisopropyl fluorophosphate (DFP), indica ting that this protease is an elastase-like serine protease, The prote ase selectively hydrolysed 30kP-1 and 30kP-4 between Ser6 and Ala7, bu t could not attack other 30kPs such as 30kP-2, 30kP-3 and 30kP-5, Cons equently, the protease characterized in the present study is a unique protease which may be specialized for the selective degradation of yol k proteins in silkworm eggs, (C) 1997 Elsevier Science Ltd. All rights reserved.