Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Adaptation to salinity at the plant cell level

Summary Various mechanisms of adaptation of plant cells to salinity are reviewed: (1) protection of enzymes and maintenance of turgor by organic solutes; (2) prevention of ion toxicity by compartmentation; and (3) energization of solute transport by the proton pump. All these mechanisms seem to play a role in adaptation. The particular advantages of using salt-adapted cells in suspension culture to identify mechanisms of adaptation are pointed out.     

Rapid group separations of nucleotides and related compounds on silica columns

Nucleosides, bases, and nucleotides can be separated from one another rapidly (10–15 min) on 1-ml silica cartridges. Samples adjusted to 4 mm ammonium borate, 90% acetonitrile are loaded onto 1-ml columns equilibrated with the same solvent. Bases do not absorb to the silica under these conditions. Nucleosides are eluted with 16 ml of 0.5 m acetic acid in 90% acetonitrile. Nucleotides are then eluted with water. The 1-ml silica columns have performed well with samples up to 10 ml in volume. We have found the procedure to be quantitative and the gels to have high capacity (61 μmol Cyd/ml silica). Acid extracts from a large number of cells (10⁸) have been processed on a single cartridge.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Changing behavior of surface immunoglobulin on mouse B lymphocytes during immune development: I. LPS-induced changes in the lateral mobility of B lymphocyte surface immunoglobulins on two populations which express different surface immunoglobulin phenotypes

The redistribution of surface membrane immunoglobulin molecules (sIg) was studied in two functionally distinct populations of mouse splenic B lymphocytes, namely, those bearing membrane IgM(IgG^?) and those bearing IgG. Brief exposure to mitogenic doses of bacterial lipopolysaccharide (LPS) produced direct but differential effects on the subsequent ability of specific antibodies to induce this redistribution on each cell type. Studied as a function of temperature, antibody-induced redistribution of sIgM on cells previously exposed to LPS was observed to occur at temperatures lower than the temperatures required for similar sIgM redistribution on lymphocytes not exposed to LPS. In contrast, mitogen-treated sIgG⁺ cells demonstrated an opposite and long-lasting effect (at least 40 hr), requiring higher temperatures to allow sIgG movement comparable to that seen on untreated sIgG-bearing lymphocytes. Thus, we conclude that LPS interacts with both IgM⁺(IgG^?) and IgG⁺ lymphocytes, but that such interactions produced different membrane effects on each B-cell subset. This membrane change can therefore be useful as a quasi-functional differentiation marker. Furthermore, differences in sensitivity to cellular activation by LPS seen between sIgM-bearing (sIgG^?) and sIgG-bearing B cells may be a reflection of such direct, although different, membrane effects.     

Carnitine transport by rat kidney cortex slices: stimulation by dibutyryl cyclic AMP+.

The transport of carnitine by rat kidney cortex slices against a concentration gradient has been demonstrated. Similarities to other transport systems included a linear period of uptake, as well as indications of saturability of the system with increasing concentrations of substrate. The transport of carnitine was inhibited by anoxia, and carbonyl cyanide-m-chloro-phenylhydroxazone (CCC1P), an uncoupler of oxidative phosphorylation. Carnitine uptake was stimulated approximately 50% when kidney slices were treated with dibutyryl cAMP.     

A semiautomated fluorometric determination of 5-hydroxyindoles in the nanogram range

Automated determinations of 5-hydroxytryptamine and its main metabolite, 5-hydroxyindoleacetic acid, have been described (Technicon autoanalyzer). The determinations are based on an extraction procedure from deproteinized tissue extracts or cerebrospinal fluid by means of butanolheptane mixtures. The indoles are transferred from the organic phase to a water phase and determined fluormetrically with the cysteine-o-phthaldialdehyde method. The method is highly sensitive: solutions containing 1 ng/ml can be reproducibly determined. Twenty samples per hour can be passed through the system. The determination of both 5-hydroxyindoles is performed with the same manifold.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.