PROTEOLYTIC-ENZYMES IN YOLK-SAC MEMBRANE OF QUAIL EGG - PURIFICATION AND ENZYMATIC CHARACTERIZATION

Degradation of yolk protein is essential for the early development of the avian embryo. In Japanese quail (Coturnix coturnix japonica), prot eolysis in the surrounding tissue of the yolk, the yolk-sac membrane, can be inhibited by class-specific inhibitors of cysteine proteinases as well as of aspartic proteinases. Purification of the enzymes leads to one cysteine proteinase and one aspartic proteinase with an apparen t molecular mass of 29 kD and 44 kD, respectively. Both enzymes were p urified in a two-chain form, although a single-chain form is also pres ent in the homogenate of yolk sac membrane. The cysteine proteinase wa s identified by NH2-terminal sequence analysis as well as by kinetic s tudies as a new cathepsin B from quail. Like mammalian cathepsin B, th is avian cathepsin B exhibits two different kinds of proteolytic activ ity, an endopeptidase activity and a dipeptidyl carboxypeptidase activ ity. Chicken egg white cystatin, a protein-aceous cysteine proteinase inhibitor, inhibits quail cathepsin B with an equilibrium dissociation constant (K-i) of 3.3 nM. Likewise the aspartic proteinase was identi fied as a new cathepsin D from quail. This avian cathepsin D has a dif ferent processing site to all known mammalian cathepsins D. In quail c athepsin D one NH2-termini is homologous to amino acids 211-230 in mam malian cathepsin D. This is more than 100 amino acids downstream of th e mammalian processing site. Comparison of the enzymatic properties of quail and bovine cathepsin D indicate that the different processing s ite has no influence on the enzymatic properties. (C) 1997 Elsevier Sc ience Inc.