Acyl migrations in diacyl derivatives of 2-methylmercaptobenzimidazole : A model of biotin

Diacyl derivatives of 2-methylmercaptobenzimidazole undergo the tautomerization 2 1 2′. Thermodynamic predominancy of one isomer over the others depends on the substituents on carbonyl groups. It has been found that electron-withdrawing substituents tend to favor 2-type compounds, whereas electron-releasing substituents make 1-type compounds more stable. The migration has been extended to include the carboethoxy group, and the results are discussed in relation to the mechanism of biotin-dependent enzymic carboxylation.     

Optimal control of a semibatch fermentation

This work is concerned with the optimization study of the semibatch fermentation by which an amino acid is produced. The particular fermentation studied is the synthesis of lysine by the auxotrophic mutant. Applying Green's theorem to the maximization problem was proposed, and it succeeded in determining the feed rate of the substrate that maximized the production rate of the desired product.     

Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium.

Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.     

Incorporation of [14C]glucose from UDP[14C]glucose into a phosphoglycolipid by cell-free particulate systems of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423.

A glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme preparation from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP[U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol.     

Kinetic study of alpha-chymotrypsin catalysis with regard to the interaction between the specificity-determining site and the aromatic side chain of substrates.

In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in alpha chymotrypsin [EC 3.4.21.1]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of alpha-chymotrypsin at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-0Me, Ac-Tyr(3-NO2)-OMe, and Ac-m-Tyr-OMe were much higher than those of the corresponding specific substrates, indicating that derivatives with a substitute as large as a formyl, nitro or hydroxyl group at the xi-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual parameters for Ac-Dopa-OMe, however, were comparable to those for Ac-Tyr-OMe.     

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes

 It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells. Accepted: 7 May 1997