A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-G Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heatstable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.Enterotoxigenic Escherichia coli produces two types of enterotoxins that are responsible for diarrhea : a high-molecular-weight heat-labile enterotoxin (LT) and a low-molecularweight heat-stable enterotoxin (ST) [l]. It has been reported that several distinct ST molecules are produced by enterotoxigenic E. coli. Burgess et al. [2] reported two types of ST: the first, ST,, is methanol-soluble and active in neonatal piglets and infant mice, but inactive in weaned pigs; the second, STb, is methanol-insoluble and active in weaned pigs, but inactive in infant mice. This finding was confirmed by several investigators [3,4]. Moreover, recent reports by So and her coworkers [5,6] purified the STh by chromatography on cation and anion exchangers and high-performance liquid chromatography (HPLC) on a reverse-phase column and determined its amino acid sequence by a combination of fast atom bombardment (FAB) mass spectrometry and carboxypeptidase digestion and Edman degradation. The sequence was found to be the same as that deduced from the nucleotide sequence encoding STh 191, except for the C-terminal amino acid residue. The amino acid sequence of STh determined clearly differed from that of ST reported by Chan and Giannella [8]. MATERIALS A N D METHODS Materials and ApparatusThe chemicals used were reagent grade from several suppliers and were purified before use. SP-Sephadex C-50 and DEAE-Sephadex A-25 were purchased from Pharmacia Japan. Carboxypeptidase B and (Staphylococcus uureus) protease V 8 were from Boehringer (Mannheim, FRG) and Miles (Indiana, USA), respectively. Zorbax ODS columns (5 pm, 4 . 6~ 250 mm) were obtained from DuPont (USA). The high-performance liquid chromatography equipment consisted of a Shimadzu HPLC LC-3A (Japan) fitted with a solvent programmer (GRE-2B) and a data processor chromatopac C-RIA. Reversed-phase columns were packed with Lichrosorb RP-8 (5 pm, Merck) in our laboratory. Column eluates were monitored for absorbance at 220 nm using a variable-wavelength flow-through spectrophotometer and fractions were collected in silicon-coated glass tubes. Bacterial Strain and Culture ConditionsEscherichia coli SK-1 was used throughout. This strain was obtained by genetic labeling of E. coli 53402 A-1, an STproducing strain from a human patient, by Kan' transposon (Tn5) [12]. The cells were grow...

Several analogues of beat-stable enterotoxins (ST, and ST,) produced by enterotoxigenic Escherichia cdi were synthesized. Peptides (ST&-181 and ST&171) consisting of 13 amino acid residues from the Cys residue near the N-terminus to the Cys residue near the C-terminus and linked by three disubide bonds had the same biological and immunological properties as native ST, and ST,, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

A heat-stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ionexchange and reversed-phase high-pressure liquid chromatography. The amino acid sequence of the purified toxin was determined by Edman-degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-ProAla-Cys-Ala-Gly-Cys-Tyr.Enterotoxin Isolation Amino acid sequence FAB mass spectrometry

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or 13 C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay. Heparin and heparan sulfate (HS)1 are known as glycosaminoglycan (GAG) components of extracellular matrix-forming connective tissues of animals. The former GAG, heparin, is exclusively distributed in mast cells, and the latter GAG, HS, is widely distributed in animal tissues. With the accumulation of the information concerning biological roles of heparin and HS, it has been revealed that their biological functions mostly depend upon interaction between polysaccharides and physiologically active molecules, although the biological roles of heparin and HS are highly diverged. For instance, they interact with lipoprotein lipase (1, 2), anti-thrombin III (3, 4), basic fibroblast growth factor (bFGF) (5-8), etc. Furthermore, minimum structures of heparin and/or HS necessary for binding with antithrombin III and/or bFGF have been determined (9, 12). Chemical modification of heparin has been undertaken by several researchers, focusing on the elucidation of the mechanism underlying interaction between heparin and the physiologically active molecules as described above. Specific removal of major sulfate groups of heparin such as 2-O-sulfate, 6-Osulfate, and N-sulfate groups would be useful in order to clarify the backbone structures of oligosaccharides bearing specific array of sulfate groups responsible for the interactions with physiologically active molecules. For instance, selective removal of 6-O-sulfate groups from glucosamine residues of heparin is of great importance in order to evaluate the involvemen...

The amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows: Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176; Tib S F N R V I T V; Tic E. The amino acid sequences of Pro(60)-Ser(61) and Asp(154)-Asp(155)-Gly(156)-His(157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser(60)-Pro(61) and His(154)-Asp-Asp-Gly(157), respectively.

Treatment of the pyridinium salts of heparin with N-methyltrimethylsilyl-trifluoroacetamide (MTSTFA) in pyridine for 2 h at various temperatures caused specific 6-O-desulfations from trisulfated disaccharide units to various degrees without detectable depolymerization or other chemical changes. In order to assess the importance of 6-O-sulfate groups in N-sulfated glucosamine (GlcNS) residues to promote FGF-1 and FGF-2 activities, various 6-O-desulfated (6-O-DS-) heparins were quantitatively examined for activity as enhancers or inhibitors of specific FGF-1- and FGF-2-induced proliferation of BALB/c3T3 clone A31 (A31) cells and the chlorate-treated cells. The present results suggested that a high content of 6-O-sulfate groups in GlcNS residues was required for activation of FGF-1, but not FGF-2. However, complete 6-O-desulfation of trisulfated disaccharide units in heparin resulted in loss of the ability to activate FGF-2, although the desulfated product bound strongly to FGF-2.

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K a ‫؍‬ 6.54 ؋ 10 8 M ؊2 ). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S ‫؍‬ 1 ⁄2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.