Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.     

Transesterification catalyzed by polystyrene-supported chymotrypsin in toluene: the effect of neutralization of basic or acidic groups attaching to polystyrene resins

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of alpha-chymotrypsin (CT), which catalyzed the transesterification of N-acetyl-L-phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by-product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester-exchange product, AcPheOPr. The pK(a) value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pK(a) values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. (c) 1995 John Wiley & Sons, Inc.     

Enhancement of aromatic amino acid-nucleic acid base stacking interaction by metal coordination to base: fluorescence study on a tryptophan-Pt(II)-guanine ternary complex

In order to investigate the effect of the Pt(II) ion on the stacking interaction between tryptophan and a guanine base, the quenching of Trp fluorescence was monitored for some systems in the absence and presence of the metal ion, and the association constants were obtained by the analysis of Eadie-Hofstee plots. All spectral data suggested that the stacking interaction is enhanced by the Pt(II) coordination to the guanine N7 atom. The result indicates the importance of the metal ion as a bookmark in the specific recognition of a nucleic acid base by an aromatic amino acid residue.     

Block by 5-hydroxytryptamine of neuronal acetylcholine receptor channels expressed inXenopus oocytes

Summary 1. Effects of 5-hydroxytryptamine (5-HT) on neuronal nicotinic acetylcholine (ACh) receptor channels were investigated by expressing cloned channel subunits inXenopus oocytes.2. When channels were expressed with a combination of ₃ and ₄ subunits, 5-HT (10 to 300 µM) reversibly inhibited an inward current activated by 100 µM ACh in a concentration-dependent manner. The inhibition was also observed when ₃ subunit was combined with ₂ subunit instead of ₄ subunit, or ₄ subunit was combined with ₂ or _4–1 subunit instead of ₃ subunit to express channels.3. Compounds known to antagonize at 5-HT receptors (LY53857, metoclopramide and propranolol) exhibited an agonistic effect: they inhibited the ACh-activated current.4. The results suggest that 5-HT inhibits recombinant neuronal nicotinic receptor channels through a binding-site distinct from conventional 5-HT receptors. The binding-site may not be attributed to a unique type of channel subunits.     

Expression of the T antigen on a T-lymphoid cell line, SupT1

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable -N-acetylgalactosaminyl-transferase and 1, 3 galactosyl-transferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with³H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identifiedinter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.     

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.     

Effect of headgroup type on the miscibility of homologous phospholipids with different acyl chain lengths in hydrated bilayer

The miscibility of homologous phosphatidylcholines with different acyl chain lengths in hydrated bilayer was examined through the binary phase diagram constructed by differential scanning calorimetry. By analyzing the phase diagram according to a thermodynamic model based on the Bragg-Williams approximation to evaluate the excess free energy of mixing, the non-ideality parameter of mixing, rho(0), was estimated, which allows one to interpret the mixing behavior of the two lipid components in terms of the difference in the pair-interaction energies between like-pairs and mixed-pairs formed in the mixture. By summarizing the rho(0) values obtained previously for other classes of phospholipids, it was found that rho(0) increases in the order of phosphatidylglycerol (PG) approximately phosphatidylcholine (PC) < phosphatidylethanolamine (PE) < phosphatidic acid (PA). Since the difference in the pair-interaction energies is considered to be determined by the relative contribution of inter-headgroup interaction to the overall intermolecular interaction, this sequence of rho(0) value suggests that the headgroup interaction in hydrated bilayer increases in the order of PA < PE < PC approximately PG.     

Sucrose consumption in mice: Major influence of two genetic Loci affecting peripheral sensory responses

Individual variability in sucrose consumption is prominent in humans and other species. To investigate the genetic contribution to this complex behavior, we conducted behavioral, electrophysiological, and genetic studies, using male progeny of two inbred mouse strains (C57BL/6ByJ [B6] and 129/J [129]) and their F₂ hybrids. Two loci on Chromosome (Chr) 4 were responsible for over 50% of the genetic variability in sucrose intake. These loci apparently modulated intake by altering peripheral neural responses to sucrose. One locus affected the response threshold, whereas the other affected the response magnitude. These findings suggest that the majority of difference in sucrose intake between male B6 and 129 mice is due to polymorphisms of two genes that influence receptor or peripheral nervous system activity. Received: 27 January 1997 / Accepted: 17 March 1997