Thiamine-Binding Protein of Escherichia coli

The ability to transport thiamine in Escherichia coli was reduced by osmotic shock treatment with a concomitant release of a thiamine-binding protein; its formation was repressed by thiamine added to the growth medium.     

Coimmobilization of malate dehydrogenase and formate dehydrogenase in polyethyleneglycol(#4000)diacrylate gel by droplet gel-entrapping method

A new immobilization technique suitable for coupled enzymes requiring cofactors was established. This is a droplet gel-entrapping method in which many small droplets including the enzymes are fixed in the gel. The first emulsion was prepared by mixing of a solution containing thermostable malate dehydrogenase (MDH) and formate dehydrogenase (FDH) with benzene containing a surfactant. The first emulsion was added to a solution containing polyethyleneglycol(#4000)diacrylate and N,N'-methylenebisacrylamide to prepare the second emulsion (w/o/w). After the second emulsion was gelled by addition of potassium persulfate and 3-dimethylaminopropionitrile, the benzene was removed. The expressed MDH and FDH activities of the MDH-FDH immobilized gel were 7.1 and 13.9% of the initial activities, respectively. The K(m) values of the gel were 0.60mM for formate and 1.5muM for NAD, respectively. The K(m) for formate and NAD were found to be extremely low. By using the column packed with 30 g gel having the MDH activity of 41.7 units and the FDH activity of 11.1 units, 13.8mM oxalacetate was completely converted to malate at 30 degrees C. The malate production rate was not affected by the concentration of more than 50mM formate, more than 2mM oxalacetate, and more than 0.1 mM NAD, respectively. Long-term malate production was demonstrated at 30 degrees C by passing the substrate solution containing the two substrates and NAD through the column. The maximum conversion ratio (7.8%) was obtained at the fifth day, and 83% of maximum productivity was maintained even after 3 weeks. The expressed FDH activity at the fifth day was calculated to be 20.5% of the initial activity.     

Distribution of lipoxygenase and hydroperoxide lyase in the leaves of various plant species

The enzyme activity responsible for volatile C₆-aldehyde formation was accompanied by lipoxygenase and hydroperoxide lyase in the green leaves of 28 plant species tested, but the level of each enzyme's activity varied. Lipoxygenase activity rather than hydroperoxide lyase activity appears to affect the overall C₆-aldehyde formation. There was a positive correlation (r = 0.712) between hydroperoxide lyase activity and the chlorophyll content of the green leaves; no correlation was found between lipoxygenase activity and chlorophyll content.     

Cloning, sequence analysis and transcriptional expression of a ras gene of the edible basidiomycete Lentinus edodes

In the edible basidiomycete, Lentinus edodes, the presence of a high level of intracellular cyclic AMP (cAMP) is closely related to the onset of fruiting and/or primordium formation. Since a close relationship between intracellular cAMP levels and expression of ras genes was reported for organisms such as Saccharomyces cerevisiae and Dictyostelium discoideum, we have cloned and sequences a ras gene homologue from L. edodes (Le.), and analyzed its expression during development of the fungus. This gene, named Le.ras, has a coding capacity of 217 amino acids (aa) interrupted by six small introns. The deduced Le.Ras protein exhibited the highest homology to the Schizosaccharomyces pombe RAS protein (219 aa): 86% homology in the N-terminal 80-aa sequence and 74% homology in the next 80 aa. The Le.ras gene was transcribed at similar levels during mycelial development in fruiting-body formation, suggesting no direct correlation of Le.ras expression with intracellular cAMP levels in this organism.     

Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).     

Inhibition of Thiamine Transport by Chloroethylthiamine in Escherichia coli

Chloroethylthiamine was found to inhibit an entrapment of thiamine as thiamine monophosphate by blocking thiamine monophosphokinase in the cytoplasm after thiamine was taken up by the cells of Escherichia coli.     

Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels

Genetic analyses have revealed an association between the gene encoding the Rab3A-interacting molecule (RIM1) and the autosomal dominant cone-rod dystrophy CORD7. However, the pathogenesis of CORD7 remains unclear. We recently revealed that RIM1 regulates voltage-dependent Ca(2+) channel (VDCC) currents and anchors neurotransmitter-containing vesicles to VDCCs, thereby controlling neurotransmitter release. We demonstrate here that the mouse RIM1 arginine-to-histidine substitution (R655H), which corresponds to the human CORD7 mutation, modifies RIM1 function in regulating VDCC currents elicited by the P/Q-type Ca(v)2.1 and L-type Ca(v)1.4 channels. Thus, our data can raise an interesting possibility that CORD7 phenotypes including retinal deficits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents.