Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 ºC for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin β chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography

Nucleotide Sequence of Gene PBI Encoding a Protein Homologous to Salivary Proline-Rich Protein P-B 1

The nucleotide sequence of gene PBI, the putative translational product of which i8 homologous to salivary proline-rich protein P-B, has been determined. PBI is 6.4 kb long and contains 3 exons. PBI appears to code for the precursor of a proline-rich protein which we have designated P-Bl. The P-Bl precursor is composed of 134 amino acid residues including 22 residues of signal sequence, 23 residues of N-terminal sequence, five repeating units of 13 to 14 residues and 22 residues of C-terminal sequence. The signal sequence of this precursor is identical with that of the P-B precursor. The N-terminal 61 residue sequence of P-Bl has homology of 75 % with the whole sequence of P-B (57 residues), when deletion of 4 residues is taken into account. P-Bl is 55 residues longer than P-B. The nucleotid