荞麦属植物淀粉酶和甲酸脱氢酶同功酶研究

以聚丙烯酰胺凝胶电泳方法研究了荞麦属植物8个种42个收集系干种子和发芽种子的淀粉酶和甲酸脱氢酶同功酶。结果表明,荞麦淀粉酶在于种子中缺乏活性,但是在发芽种子中活性很强。在供试材料的发芽种子中共发现23个淀粉酶谱带,其中甜荞和苦荞分别有10条和8条。不同荞麦种间淀粉酶谱带差异很大,但是同种内不同收集系间差异较小。谱带聚类分析表明大野荞和毛野荞分别与甜荞和苦荞较近缘,支持它们分别为甜荞和苦荞祖先种的假说。在干种子和发芽种子中,发现所有荞麦种类均只有1条位置一致的甲酸脱氢酶谱带,暗示该酶在进化中具有高度稳定性。     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Use of the addressing sequence of yeast D-lactate dehydrogenase for insertion of CYP11A1p into the inner membrane of yeast mitochondria

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Eur. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYP11A1p by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYP11A1p synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity. Thus, CYP11A1p insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYP11A1p domain in the hybrid protein.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

The crystal structure of seabream antiquitin reveals the structural basis of its substrate specificity

The crystal structure of seabream antiquitin in complex with the cofactor NAD(+) was solved at 2.8A resolution. The mouth of the substrate-binding pocket is guarded by two conserved residues, Glu120 and Arg300. To test the role of these two residues, we have prepared the two mutants E120A and R300A. Our model and kinetics data suggest that antiquitin's specificity towards the substrate alpha-aminoadipic semialdehyde is contributed mainly by Glu120 which interacts with the alpha-amino group of the substrate. On the other hand, Arg300 does not have any specific interaction with the alpha-carboxylate group of the substrate, but is important in maintaining the active site conformation.     

Modified carbon paste electrodes for flow injection amperometric determination of isocitrate dehydrogenase activity in serum

A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1x10(-4)M, with a detection limit of 5x10(-9)M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.     

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.