Purification and characterization of a cysteine proteinase inhibitor (Cystatin) from seeds of foxtail millet, Setaria italica

A cysteine proteinase inhibitor was purified from the extract of seeds of foxtail millet, Setaria italica, to an electrophoretically homogeneous protein by heat treatment, salting-out, ion-exchange chromatography on DEAE-Sepharose CL-6 B, gel filtration on Sephadex G-50, and chromatofocusing on PBE 94. The inhibitor (FMCPI) was a single polypeptide with a molecular weight of 12 000 and an isoelectric point of 5.2. It possessed the amino acid composition characterized by fairly high contents of aspartic acid, glutamic acid, and alanine and by no half-cystine, FMCPI was relatively thermostable since it maintained more than 50% of the original activity after treatment of 100 degrees C for 20 min at pH 2 or 7. However, the inhibitor almost lost its whole activity by the above treatment at pH 10. FMCPI inhibited papain in a 1 : 1 protein molar ratio : the K-i value was 2.4 x 10(-11) M. Complex formation between FMCPI and papain derivatives demonstrated that the inhibitor was capable of combining with even a papain molecule without the catalytic ability.