Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite

A procedure for the simultaneous preparation of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. The yield was 95% for S-sulfo-l-cysteine and 91% for l-alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. Separation of the reaction product, S-sulfo-l-cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 (M. K. Gaitonde, 1967, Biochem. J.104, 627–663). The recovery was 96.8 ± 0.3%.     

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.     

Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies.

Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles.     

Species-specific differences in heterogonic development of serially transferred free-living generations of Strongyloides planiceps and Strongyloides stercoralis

The direction of free-living development (homogonic vs. heterogonic) in Strongyloides stercoralis and Strongyloides planiceps was examined by successive transplantation of the uterine eggs of free-living females into a test tube culture system containing fresh feces. The eggs from the first-generation free-living females of S. stercoralis did not develop into second-generation free-living adult worms, but all developed into filariform larvae. However, the majority of S. planiceps eggs from the first-generation free-living females developed into second-generation free-living adults. By successive transfer of uterine eggs of each generation, 9 generations developed, and in every cycle more adult worms developed than filariform larvae. However, the number of free-living generations was not infinite; in experiments repeated twice, the number of worms developing from the eggs of eighth or ninth generations was too small to continue further culture. These findings indicate that the pattern of free-living development is different between the 2 species.     

Inhibitory effects of pectic substances on activated hyaluronidase and histamine release from mast cells.

In this paper, we report the effect of pectic substances and D-galacturonic acid, the main constituent of pectic substances, on activated hyaluronidase and histamine release from mast cells. Although D-galacturonic acid itself showed no inhibition, IC50 values of hyaluronidase inhibition were correlated with the D-galacturonic-acid content of pectic substances. It was thought that the polymerization of D-galacturonic acid was necessary for inhibition of activated hyaluronidase. This type of inhibition was suggested to be non-competitive by the Lineweaver-Burk plot. Furthermore, pectic substances, including those purified from Gymnema sylvestre, inhibited histamine release from isolated rat peritoneal mast cells, which had been induced by the antigen. These results suggest that pectic substances may have anti-allergic activities.     

Mechanism of adenylate kinase. Are the essential lysines essential?

Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

Structural studies of the antigenic polysaccharide of Eubacterium saburreum, strain S29

The antigenic polysaccharide produced by Eubacterium saburreum, strain S29, is composed of (1→6)-linked β-d-glycero-d-galacto-heptopyranosyl residues, all of which are substituted with 6-deoxy-α-d-altro-heptofuranosyl groups at O-2.     

Effect of fatty acids and monoglycerides on permeability of lipid bilayer

The effect of fatty acids and monoglycerides on barrier properties of liposomal membranes prepared from egg phosphatidylcholine was investigated. The incorporation of these lipids as liposomal membrane components induced the alteration of the permeability to less permeable liposomally entrapped drugs, sulfanilic acid and procainamide ethobromide (PAEB). Monoolein caused greatly increased permeability of both drugs and unsaturated fatty acids markedly enhanced the release rate of PAEB, while saturated fatty acids caused a small increase in the release rate.Electron spin resonance (ESR) investigation with 5-nitroxide stearic acid showed that fatty acids disordered the hydrophobic region of the lipid bilayer and the disordering effect of unsaturated fatty acids was greater than that of saturated ones. It was demonstrated that the incorporated fatty acids and monoglycerides interacted with the polar region of the membranes by ESR study with cholestane label and ¹H-NMR study. These results indicated that the increase in the membrane permeability caused by fatty acids and monoglycerides associated with the disorder in the membranes' interior and the interaction of the incorporated lipid with the polar head group of phospholipid.     

Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS.

The xylDEGF operon and the regulatory gene xylS of the TOL plasmid found in Pseudomonas putida mt-2 were cloned onto Escherichia coli vector plasmids. A 9.5-kilobase fragment, derived from the TOL segment of pTN2 deoxyribonucleic acid, carried the xyl genes D, E, G, and F, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. The enzymes were noninducible unless a 3-kilobase PstI fragment, derived also from the TOL segment, was provided in either cis or trans. The PstI fragment appeared to contain the regulatory gene xylS, which produced a positive regulator. The regulator was activated by m-toluate or benzoate, but not by m-xylene or m-methylbenzyl alcohol. the map positions of xylG and xylF were also determined.