Kazunari Arima

Cucumisin-like protease from the latex of Euphorbia supina

A protease has been purified from the latex of Euphorbia supina Rafin by two steps of chromatography. The Mr was estimated by SDS-PAGE to be 80 kDa. Its activity was inhibited strongly by diisopropyl fluorophosphate, but not by EDTA, pepstatin, or cysteine protease inhibitors, indicating that the enzyme is a serine protease. The specificity of the protease is broad, but the preferential cleavage sites were C-terminal sites of hydrophobic amino acid residues. The N-terminal sequence of the first fifteen residues was determined and six of the residues match those in cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicate that the E. supina protease is a cucumisin-like serine protease.

Rapid and efficient proteolysis for proteomic analysis by protease-immobilized microreactor

Proteolysis is an important part of protein identification in proteomics analysis. The conventional method of in‐solution digestion of proteins is time‐consuming and has limited sensitivity. In this study, trypsin‐ or α‐chymotrypsin‐immobilized microreactors prepared by a microfluidics‐based enzyme‐immobilization technique were studied for rapid sample preparation in proteomic analysis. The kinetic studies for hydrolysis of substrate by microreactors revealed that immobilized proteases had higher hydrolytic efficiency than those performed by in‐solution digestion. The performance of the microreactors was evaluated by digesting cytochrome c and BSA. Protein digestion was achieved within a short period of time (∼5 min) at 30°C without any complicated reduction and alkylation procedures. The efficiency of digestion by trypsin‐immobilized reactor was evaluated by analyzing the sequence coverage, which was 47 and 12% for cytochrome c and BSA, respectively. These values were higher than those performed by in‐solution digestion. Besides, because of higher stability against high concentration of denaturant, the microreactors can be useful for immediate digestion of the denaturated protein. In the present study, we propose a protease‐immobilized microreactor digestion method, which can utilize as a proteome technique for biological and clinical research.

Efficient Immobilization of Enzymes on Microchannel Surface Through His-Tag and Application for Microreactor

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.

Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai

An endopeptidase has been purified from sprouts of bamboo (Pleioblastus hindsii Nakai) to electrophoretic homogeneity by four purification steps. Its Mr was estimated to be 82 kDa by SDS-PAGE. Enzyme activity was inhibited strongly by diisopropyl fluorophosphate, and weakly by p-chloromercuriphenylsulfonic acid, but not at all by EDTA or pepstatin, indicating that it was a serine protease. The preferential cleavage sites for this protease were found to be large hydrophobic and amide residues at the P1 position. The specificity of the bamboo serine protease differed from that of cucumisin [EC 3.4.21.25], which cleaved the charged amino acid residues at the P1 position.

Crystal structure of cucumisin, a subtilisin-like endoprotease from Cucumis melo L

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin-III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.

A serine endopeptidase from the fruits of Melothria japonica (Thunb.) maxim.

An endopeptidase from the fruits of Melothria japonica (Thunb.) Maxim. has been purified by DEAE-Sepharose chromatography and gel-filtration by a Sephacryl S-300. The enzyme has Mr of 61 kDa. The optimum pH of the enzyme was 8. The enzyme activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonylfluoride, but not by EDTA. Casein was a poor substrate, but angiotensin I was cleaved by the enzyme within 30 min at four different sites. These results indicated that the enzyme was a serine oligopeptidase of broad substrate specificity.

Detection of Hydrolytic Activity of Trypsin with a Fluorescence-chymotryptic Peptide on a TLC Plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.

Isolation and characterization of a cysteine protease of freesia corms.

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.

Selection and Characterization of Human Serum Albumin-specific Porcine scFv Antibodies Using a Phage Display Library

A new single-chain variable fragment (scFv) antibody library was generated and human serum albumin (HSA)-specific clones were characterized to investigate the usefulness of porcine antibodies. Phage libraries were developed from pigs immunized with the model antigen HSA. The library size was 1.5 × 10(7) for kappa (VL) and 1.4 × 10(7) for lambda fragments. Eight HSA-specific clones from the kappa library and one clone from the lambda library were isolated using affinity selection. The binding specificity of these clones was confirmed using a phage enzyme-linked immunosorbent assay (ELISA). The scFvs were expressed in Escherichia coli and purified from the periplasm fraction for further investigation. Based on the results of ELISA and Western blot analysis, four scFv clones with high activity and high yield were selected and purified. The purified scFvs from four of the nine clones exhibited an approximate KD of 10(-8) M. This is the first report describing isolation of HSA-specific porcine scFv antibodies from an antibody phage library and characterization of their binding properties.

Structural Basis of Cucumisin Protease Activity Regulation by Its Propeptide

Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the &agr;–&bgr; sandwich motif with four-stranded antiparallel &bgr;-sheets, two helices and a strand of the C-terminal region. The &bgr;-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.