Zeolites as new chromatographic carriers for proteins--easy recovery of proteins adsorbed on zeolites by polyethylene glycol

Zeolites are able to adsorb proteins on their surface and might be suitable as a new type of chromatographic carrier material for proteins and for their conjugates (Matsui et al., Chem. Eur. J. 7 (2001) 1555-1560). Interestingly, maximum adsorption was observed at the isoelectric point (pI) of each protein. The current study was performed to investigate the desorption of proteins from the zeolites at pI. Proteins adsorbed to zeolites could be desorbed at pI by polyethylene glycol (PEG), but not by conventional eluents. The eluted proteins still retained their activities. The zeolite Na-BEA was an especially good composite for desorption by PEG. Using this method for the adsorption and desorption of proteins at pI, we succeeded in separating various proteins. The application of zeolites to biochemistry and biotechnology is also discussed.     

Higher plant RecA-like protein is homologous to RadA

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.     

Discovery of nigerose phosphorylase from Clostridium phytofermentans

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.     

Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.     

Relative Contributions of Cystathionine ��-Synthase and ��-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H₂S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H₂S generation. CBS from both organisms shows a marked preference for H₂S generation by β-replacement of cysteine by homocysteine. The alternative H₂S-generating reactions, i.e. β-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H₂S production by CBS would account for ∼25–70% of the total H₂S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H₂S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H₂S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H₂S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.Hydrogen sulfide (H₂S)² elicits an array of physiological effects, including modulation of blood pressure and reduction of ischemia reperfusion injury (1, 2). Other novel effects of H₂S include induction of a state of suspended animation in mouse by decreasing oxygen consumption and drastically reducing the metabolic rate (3) and synchronizing ultradian metabolic oscillation in yeast (4). Under conditions of metabolic cycling in yeast, H₂S production is catalyzed by sulfite reductase in the sulfur assimilation pathway (4). Inhibition of sulfite reductase reduces H₂S production and in turn perturbs metabolic oscillations. H₂S is a specific and potent inhibitor of cytochrome c oxidase in the electron transport chain (3).Although concentrations of H₂S have been reported to range from 50 to 160 μm in brain (5–7) and 30–50 μm in the peripheral system (8), these appear to be grossly overestimated (9). Significantly lower H₂S concentrations of 17 and 14 nm in liver and brain, respectively, have been reported recently (9). The very significant discrepancy between these and the previous estimates of H₂S levels presumably derives from the earlier use of acidic conditions that led to the release of acid-labile sulfur from iron-sulfur centers.In mammals, the primary catalysts for H₂S generation are reported to be the two pyridoxal phosphate (PLP)-dependent enzymes involved in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (10, 11). The trans-sulfuration pathway operates in the reverse direction in mammals serving to convert homocysteine to cysteine (Fig. 1), although in yeast and bacteria the pathway is involved in sulfur assimilation from sulfate to cysteine. CBS is widely assumed to be the major contributor to H₂S production in the brain because of its relatively high expression in this organ (10). However, a recent study reported that 3-mercaptopyruvate sulfurtransferase together with cysteine aminotransferase might also generate H₂S in brain (12). The relative contributions of these enzymes and of CSE, which is also present in brain (13, 14), to H₂S production remain to be assessed. Genetic disruption of CSE in mouse leads to cardiac deficits, including pronounced hypertension and reduced endothelium-dependent vasorelaxation, consistent with a major role for CSE in the peripheral system (1). However, brain H₂S levels are reportedly unchanged in CSE^−/− mice.Open in a separate windowFIGURE 1.Diversity of reactions catalyzed by the trans-sulfuration pathway. The turnover numbers (v/[E]) estimated at physiological substrate concentrations, i.e. 10 μm homocysteine, 100 μm cysteine, 560 μm serine, and 5 μm cystathionine, are shown in parentheses for each reaction. The thick arrows highlight reactions that are sensitive to elevated levels of homocysteine. The fold change represents the fold increase in the turnover number of a given reaction under conditions of severe hyperhomocysteinemia (200 μm homocysteine).Despite the growing recognition of the varied physiological effects of H₂S, our understanding of its regulation and mechanism of its biosynthesis is poor. We have recently reported on the complex kinetics of H₂S generation by human CSE (15). The profligacy of the human enzyme affords H₂S generation by a multiplicity of routes involving cysteine and/or homocysteine as substrates. Kinetic simulations predict an increasingly important contribution of homocysteine to H₂S generation with increasing grade of hyperhomocysteinemia, a risk factor for cardiovascular and neurodegenerative diseases (16–18). In addition to H₂S, a variety of products is generated in these reactions, including two novel sulfur metabolites, lanthionine and homolanthionine, which represent the condensation products between 2 mol of cysteine and homocysteine, respectively. Although the steady-state kinetic parameters for H₂S generation from cysteine and homocysteine have been reported for human CBS (hCBS) (19), a comparable detailed kinetic analysis of H₂S generation by CBS by multiple pathways and their sensitivity to the grade of hyperhomocysteinemia is not known. Furthermore, the relative contributions of CBS and CSE to H₂S and lanthionine generation at physiologically relevant concentrations of substrate are not known.Human CBS is a unique heme containing PLP-dependent enzyme (20) that catalyzes the β-replacement of serine by homocysteine to produce cystathionine. The latter is further metabolized by CSE in an α,γ-elimination reaction to produce cysteine. Although yeast and human CBS are highly homologous and catalyze the same chemical reaction with similar kinetic parameters, the yeast enzyme lacks heme and is not allosterically regulated by S-adenosylmethionine (AdoMet) (21).In this study, we have elucidated the kinetics of H₂S biogenesis by yeast and human CBS and used simulations to estimate the relative contributions of CBS and CSE to H₂S production at physiologically relevant concentrations of substrate. We find that CBS and CSE share a common feature, i.e. catalytic promiscuity. However, in contrast to CSE, which is proficient at catalyzing reactions at the β- and γ-carbons of substrates (15), CBS activity is confined to chemical transformations at the β-position. Our studies provide new insights into the existence of alternative trans-sulfuration reactions that can be a source of diverse sulfur metabolites, viz. H₂S, lanthionine, and homolanthionine increasing the diversity of the sulfur metabolome.     

Novel urinary metabolite of d-delta-tocopherol in rats

A novel metabolite of d-delta-tocopherol was isolated from the urine of rats given d-3,4-[3H2]-delta-tocopherol intravenously. The metabolite was collected from the urine of rats given d-delta-tocopherol in the same manner as that of the labeled compound. It was found that the metabolites consisted of sulfate conjugates. The portion of the major metabolite released with sulfatase was determined to be 2,8-dimethyl-2-(2'-carboxyethyl)-6-chromanol by infrared spectra, nuclear magnetic resonance spectra, and mass spectra. The proposed structure was confirmed by comparing the analytical results with those of a synthetically derived compound. As a result of the structural elucidation of this novel metabolite, a pathway for the biological transformation of delta-tocopherol is proposed which is different from that of alpha-tocopherol. A characteristic feature of the pathway is the absence of any opening of the chroman ring throughout the sequence.     

DNA topoisomerase II interacts with Lim15/Dmc1 in meiosis

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein–protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.     

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.