Ken-ichi Yoshino

Sensitive Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2-(Diethylamino)ethyl Ester by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028−4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637−640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50−200 fmol of sample; in the PSD analyses from the spots contained 500 fmol−1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the...

Some more speract derivatives associated with eggs of sea urchins Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina

1. Fourteen peptides were isolated from the egg jelly of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina and their amino acid sequences were determined. 2. The peptides stimulated H. pulcherrimus sperm respiration one half-maximally at about 8-60 pM. 3. Addition of speract to intact spermatozoa of P. depressus, H. pulcherrimus and A. crassispina resulted in the appearance of a newly stained protein (Mr 128,000 for P. depressus, Mr 128,000 for H. pulcherrimus and Mr 131,000 for A. crassispina) on sodium dodecyl sulfate-polyacrylamide gels.

Purification and characterization of a novel superantigen produced by a clinical isolate of Yersinia pseudotuberculosis

A superantigen designated as Yersinia pseudotuberculosis‐derived mitogen (YPM) was purified in an equal manner from both the culture supernatant and cell lysate of a clinical isolate (KUR‐1) of Y. pseudotuberculosis serotype 4b. A significant proliferative response of human peripheral blood mononuclear cells to purified YPM was detectable even at a concentration of 1 pg/ml. The N‐terminal sequence of YPM which included 23 amino acid residues was determined, by automated Edman degradation, as Thr‐Asp‐Tyr‐Asp‐Asn‐Thr‐Leu‐Asn‐Ser‐Ile‐Pro‐Ser‐Leu‐Arg‐Ile‐Pro‐Asn‐Il e‐Ala‐Thr‐Tyr‐Thr‐Gly‐. This sequence differed from not only all the, hitherto, reported superantigens but also known proteins. While molecular weights of known bacterial superantigens are more than 22,000, electrospray ionization mass spectrometry showed that the molecular weight of YPM was 14524.4. These results indicate that YPM comprises a novel superantigen with substantial structural differences from other bacterial superantigens produced by Gram‐positive cocci.

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm‐activating peptide I (SAP‐I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP‐I analogue [GGGY(125I)‐SAP‐I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP‐I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(125I)‐SAP‐I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)‐binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30‐residue amino‐terminal signal peptide, followed by the same sequence as the N‐terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.

Characterization of a highly toxic, large molecular size heat-stable enterotoxin produced by a clinical isolate of Yersinia enterocolitica

A novel heat‐stable enterotoxin (ST) designated as Y‐STc was purified to homogeneity from the culture supernatant of a pathogenic strain of Yersinia enterocolitica serotype O3 and its amino acid sequence was determined. The mature Y‐STc was found to consist of 53 amino acid residues, which includes the putative pro‐sequence. The molecular weight of Y‐STc was 5683 and constituted the largest molecular size in the family of currently known STs. The minimum effective dose of purified Y‐STc in the suckling mouse assay was 0.6 ng (0.1 pmol), indicating that, despite the long sequence, Y‐STc is the most toxic in the ST family.

Purification and sequence determination of heat-stable enterotoxin elaborated by a cholera toxin-producing strain of Vibrio cholerae O1

Four molecular species of heat‐stable enterotoxins elaborated by a cholera toxin‐producing strain of Vibrio cholerae O1 were isolated from its culture supernatant. The amino acid sequence of one of the enterotoxins was determined to be Phe‐Ile‐Lys‐Gln‐Val‐Asp‐Glu‐Asn‐Gly‐Asn‐Leu‐Ile‐Asp‐Cys‐Cys‐Glu‐Ile‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Phe‐Gly‐Cys‐Leu‐Asn with three intramolecular disulfide linkages. The other enterotoxins had shorter amino acid sequences in the N‐terminal regions, but possessed the same sequence in their C‐terminal regions including the three disulfide linkages. The enterotoxins with the shorter N‐terminal sequences showed more potent toxicities, and the minimum effective dose of the longest one with 28 amino acid residues was 10‐folds of that of the shortest one.

Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.

Determination of the amino acid sequence of an intramolecular disulfide linkage-containing sperm-activating peptide by tandem mass spectrometry

A sperm‐activating peptide (SAP) was isolated from the egg jelly of the sea urchin Stomopneustes variolaris. The presence of an intramolecular disulfide linkage in the peptide was demonstrated by fast atom bombardment (FAB) mass spectrometry with the intact and reduced peptides. The amino acid sequence of the reduced peptide was determined to be Lys‐Phe‐Cys‐Pro‐Glu‐Gly‐Lys‐Cys‐Val by tandem mass spectrometry from the spectrum produced by a collision‐induced decomposition method. Furthermore, it was also demonstrated that SAPs obtained from sea urchins Arbacia punctulata and Glyptpcidaris crenularis are cyclic peptides containing one cystine residue by FAB mass spectrometry.

A novel group of sperm activating peptides from the sea urchin Glyptocidaris crenularis.

1. Six new sperm activating peptides were purified from the egg jelly of the sea urchin Glyptocidaris crenularis and their amino acid sequences were determined as follows: Ser-Ala-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Ser-Phe-Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val and Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val. 2. The peptides were specific for G. crenularis spermatozoa and caused significant increases of sperm respiration rates and sperm cyclic nucleotide concentrations at concentrations as low as 10(-10) M. 3. The addition of the peptides to intact spermatozoa resulted in the change of the apparent mol. wt of a sperm protein from 195,000 to 200,000.

A species-specific sperm-activating peptide from the egg jelly of the sea urchin Diadema setosum

1. A species-specific sperm-activating peptide was isolated from the egg jelly of the sea urchin Diadema setosum and the amino acid sequence was determined as follows: (formula; see text). 2. The peptide caused significant increases of respiration rates and cyclic nucleotide concentrations in D. setosum spermatozoa as low as 10(-9) M. 3. The addition of the peptide to D. setosum spermatozoa resulted in the appearance of a newly stained protein (mol. wt 128,000) on sodium dodecyl sulfate-polyacrylamide gels.