Immobilization of luciferase from the firefly Luciola mingrelica — Catalytic properties and stability of the immobilized enzyme

Firefly (Luciola mingrelica) luciferase [Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7] has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5–9.0 and 25°C as well as at pH 7.8 and 20–40°C. The ΔH^≠ and ΔS^≠ values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25°C. A stabilization mechanism for the immobilized luciferase is discussed.     

The Electromotive Protein Prestin as a Sensitive Core of the Fluorescent Voltage Indicator

Russian Journal of Bioorganic Chemistry - In this study, we first used prestin, an electromotive protein of the mammalian auditory analyzer, as a voltage-sensitive core of a genetically encoded...     

Isoquinoline alkaloids from Mahonia aquifolium stem bark are active against Malassezia spp

Bioassay directed fractionation of crude extract fromMahonia aquifolium led to the isolation of fraction A (bisbenzylisoquinoline alkaloid complex, BBI) and a fraction of protoberberine alkaloids, where the major compounds berberine and jatrorrhizine were isolated as their iodides. The antifungal activity of the crude extract, two protoberberine alkaloids and BBI fromM. aquifolium stem bark were evaluated against six strains ofMalassezia spp. The compounds tested were generally found to possess only weak to moderate antifungal properties: the MICs for individual strains were in the range ≤50–≥1000 mg/L.     

Unusual Behavior of Membrane Somatic Angiotensin-Converting Enzyme in a Reversed Micelle System

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.     

Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells

The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.     

ADF proteins are involved in the control of flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis.     

Semax and Selank Inhibit the Enkephalin-Degrading Enzymes of Human Serum

Dose-dependent effect of synthetic heptapeptides Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) on the enkephalin-degrading enzymes of human serum was demonstrated. The inhibitory effects of Semax (IC₅₀10 M) and Selank (IC₅₀20 M) are more pronounced than that of puromycin (IC₅₀10 mM), bacitracin, and some other inhibitors of peptidases. Beside the heptapeptides, their pentapeptide fragments also possessed an inhibitory effect; tri-, tetra- and hexapeptide fragments did not display such an effect. As the above enzymes take part in degradation of not only enkephalins but also other regulatory peptides, it can be assumed that one of the mechanisms of biological activity of Semax and Selank is related to this inhibitory activity of theirs.     

Towards a virtual Arabidopsis plant

A report on the Royal Society Discussion Meeting "The plant cell: between genome and plant", London, 13-14 June 2001.