Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties

High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-surface hydrophobicity of Candida species as determined by the contact-angle and hydrocarbon-adherence methods

Cell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.     

Comparison of inhibitory effects of prolinal-containing peptide derivatives on prolyl endopeptidases from bovine brain and Flavobacterium

The inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in Ki values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with Ki values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with Ki values of nM order, while the Ki values of these compounds for the microbial enzyme were only of microM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.     

Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector

A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosysnthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.