PURIFICATION AND CDNA CLONING OF A 4-DOMAIN KAZAL PROTEINASE-INHIBITOR FROM CRAYFISH BLOOD-CELLS

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. I t codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence c onsists of four repeated stretches (45-73% identical to each other), i ndicating that the protein has four domains. The domains have signific ant sequence similarity to serine proteinase inhibitors of the Kazal f amily. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor . A monomeric 23-kDa proteinase inhibitor, which by amino terminal seq uencing of the mature protein was confirmed to be the cloned Kazal inh ibitor, was purified from crayfish blood cells. It inhibited chymotryp sin or subtilisin, but not trypsin, elastase or thrombin. The inhibito r seemed to form a 1:1 complex with chymotrypsin or subtilisin. This p rotein seems to be the first described Kazal inhibitor from blood cell s of any animal and the first one with four domains.