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Benzyloxycarbonyl-Leu-Leu-Leucinal (Z-Leu-Leu-Leu-H), a leupeptin derivative, stimulates neurite outgrowth of PC12 cells in a manner different from NGF and also inhibits thiol-protease such as calpain. In this study, we aimed at isolation and identification of target protein(s) for this drug by means of a newly designed affinity resin, to which Z-LeuLeu-Leu-OH was coupled at the C-terminal carboxyl group of the peptide. Our affinity chromatography system showed a unique protein retained and eluted in a Ca“-dependent manner. This protein was purified from bovine brain extract by DEAE ion-exchange chromatography and reversed phase chromatography on the basis of its affinity character. The amino acid sequencing of peptides obtained under various degradation conditions of native or pyridylethylated protein has revealed that the target protein agrees with S-1006’ subunit in 86% of its amino acid sequence. Neurite outgrowth of PC12 cells or other cultured cells is induced by inhibiting protease activity (4, 16). Out of various protease inhibitors (leupeptin, leupeptin analogue, pepstatin etc.) tested for PC12 cells (19), only Ac-Leu-Leu-Leu-Nle-H, a leupeptin analogue, induced neurite outgrowth at a low concentration. Ito er al. have synthesized several derivatives of Ac-Leu-Leu-Nle-H, and examined the effect of their chemical structures on neurite outgrowth (8). Benzyloxycarbonyl-Leu-Leu-Leucinal (Z-Leu-LeuLeu-H) has been found to be 50 times more active than Ac-Leu-Leu-Nle-H, but loses almost all activity by the substitution of C-terminal aldehyde. It has so far been known that Z-Leu-Leu-Leu-H Abbreviations: Z-Leu-Leu-Leu-H, benzyloxycarbonylLeu-Leu-Leucinal; WSC, water-soluble carbodiimide; PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate; DMSO, dimethylsulfoxide; EDTA, ethylenediamine tetraacetic acid 3Correspondence to: H. Kasai at the above address. Tel: (+81) 3-3819-1211; Fax: (+81) 3-3819-1406 inhibits thiol-protease such as calpain and proteasome (24), DNA polymerase (22), and enhances morphogenetic processes of osteoblast (21). Saito er al. have isolated clathrin from PC12 cell, using an affinity resin, to which Leu-Leu-LeuH was immobilized on the N-terminal amino group of the peptide (18, 20, 21). On the assumption that the moiety of Z-LeuLeu-Leuis required for the recognition of protein by Z-Leu-Leu-Leu-H, we prepared a newly designed affinity resin, to which Z-Leu-Leu-LeuOH is immobilized on the C-terminal carboxyl group of the peptide. Using the affinity resin as a prove, we attempted to isolate and identify a target protein for Z-Leu-Leu-Leu-H. MATERIALS AND METHODS

ISOLATION AND IDENTIFICATION OF A TARGET PROTEIN FOR Z-Leu-Leu-Leu-H, A NEURITE OUTGROWTH FACTOR, FROM BOVINE BRAIN