Radish (
Raphanus sativus L.) taproots display high amylolytic activity. Amylase zymograms using gels containing soluble starch and β-limit dextrin have indicated that β-amylase is a major amylolytic ...enzyme. We purified β-amylase with anion-exchange chromatography followed by glycogen precipitation. From the deduced amino acid sequences, a cDNA clone designated
Raphanus sativus β-amylase 1 (RsBAMY1) was isolated. RsBAMY1 closely resembled
Arabidopsis thaliana major β-amylase (At4g15210). Immunoblot analyses performed using an anti-RsBAMY1 antibody that was raised against a peptide sequence found in RsBAMY1 showed that the RsBAMY1 protein accumulated in the taproot. In addition, the transcriptional level and the protein accumulation of RsBAMY1 were enhanced during the taproot growth. These results suggest that RsBAMY1 is a major starch-digestive enzyme in the radish taproot.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We have developed a highly-sensitive leakage current microsensor by using 1,2-dipalmitoyl-sn-glycero-3-phosphocholline (DPPC) liposome entrapping K 4 Fe(CN) 6 solution to detect the existence of ...proteins. In this work, the addition of guanidinium hydrochloride (GuHCl) as protein denaturant successfully resulted in a prominent improvement in sensitivity by 129-folds of magnitude. This is because the modulation of conformation of the target proteins, carbonic anhydrase from bovine (CAB) and lysozyme could contribute to the promotion of the interaction between the liposome and the target protein.
Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of ...hydrogen peroxide (H2O2) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 °C because the H2O2 concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H2O2 produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H2O2 because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H2O2 decomposition. The effect of the glucose oxidation rate, i.e., the H2O2 production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H2O2 formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H2O2 decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.
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Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome ...chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60
°C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using ...stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)−phenyl−poly(ethylene glycol) (PPO n −Ph−PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO−Ph−PEG with a large PPO chain (n = 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50−Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 °C) although the conditions showing the high values of PPO33−Ph−PEG were restricted (more than 0.1 mM and more than 40 °C). It was also found that molecular assemblies of PPO n −Ph−PEG with diameters of 7−18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPO n −Ph−PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPO n −Ph−PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50−Ph−PEG, the reactivation yield of CAB reached approximately 100% at 40−55 °C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions.
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Deep 1.1 mm continuum observations of 1E0657−56 (the ‘Bullet Cluster’) taken with the millimeter-wavelength camera AzTEC on the 10-m Atacama Submillimeter Telescope Experiment (ASTE), have revealed ...an extremely bright (S1.1 mm= 15.9 mJy) unresolved source. This source, MMJ065837−5557.0, lies close to a maximum in the density of underlying mass distribution, towards the larger of the two interacting clusters as traced by the weak-lensing analysis of Clowe et al. Using optical–infrared (IR) colours, we argue that MMJ065837−5557.0 lies at a redshift of z= 2.7 ± 0.2. A lensing-derived mass model for the Bullet Cluster shows a critical line (caustic) of magnification within a few arcsec of the AzTEC source, sufficient to amplify the intrinsic millimetre-wavelength flux of the AzTEC galaxy by a factor of ≫20. After subtraction of the foreground cluster emission at 1.1 mm due to the Sunyaev-Zel'dovich effect, and correcting for the magnification, the rest-frame far-IR luminosity of MMJ065837−5557.0 is ≤1012L⊙, characteristic of a luminous infrared galaxy (LIRG). We explore various scenarios to explain the colours, morphologies and positional offsets between the potential optical and IR counterparts, and their relationship with MMJ065837−5557.0. Until higher resolution and more sensitive (sub)millimetre observations are available, the detection of background galaxies close to the caustics of massive lensing clusters offers the only opportunity to study this intrinsically faint millimetre-galaxy population.
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BFBNIB, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability ...precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (
S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A catalytic reaction of H
2O
2 production by an amyloid β-peptide (Aβ)–Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis–Menten model was applied to the H
...2O
2 production reaction using cholesterol as the substrate catalyzed by the Aβ–Cu complex. The
K
m value for the Aβ–Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine (POPC) liposomes (
K
m=0.436 μM for Aβ(1–40);
K
m=0.641 μM for Aβ(1–42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC) liposomes (
K
m=0.585 μM for Aβ(1–40),
K
m=0.890 μM for Aβ(1–42)). The results imply that membrane properties could play an important role in the interactions of the Aβ–Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H
2O
2-generating activity of the Aβ–Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Oxidative refolding of the denatured/reduced lysozyme was examined in the presence of small unilamellar vesicles (SUVs) essentially composed of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine ...(POPC). SUVs facilitated the recovery of the enzymatic activity of lysozyme like molecular chaperones through the interaction with the refolding intermediate of lysozyme. The highest reactivation yield (87%) was obtained by delaying oxidation time (15–30 s) after dilution of the denaturant concentration was initiated in the presence of SUVs. SUVs supplemented with 1 mol % phosphatidylethanolamine were covalently coupled to gel beads, and then the interactions of SUVs with lysozyme at the various conformations were quantitatively examined with immobilized liposome chromatography (ILC). The reduced lysozyme lacking disulfide bonds was found to have a property similar to that of the molten globule state in terms of its local hydrophobicity, which was determined with the aqueous two‐phase partitioning method. The reduced lysozyme was more clearly retarded on the ILC column than the native and 1 M guanidinium hydrochloride (GuHCl) ‐treated lysozymes with intact disulfide bonds. Similar results were obtained for α‐lactalbumin, which has three‐dimensional structure closely similar to that of lysozyme, regarding the membrane−protein interaction. These results can be interpreted as follows. In the early stage of the oxidative refolding, liposomes bound to the refolding intermediate of lysozyme lacking disulfide bonds. Then, liposomes assisted the formation of the tertiary structure of lysozyme by reducing protein intermolecular interactions, which usually cause the formation of the inactive aggregates. Consequently, the correct formation of the disulfide bonds was promoted. On the basis of the above results, the chromatographic oxidative refolding was also examined with ILC. The denatured/reduced lysozyme (9 mg/mL, 10 μL) was passed through the ILC column with a flow rate of 1 mL/min, which corresponds to the retention of lysozyme about 1 min in the ILC column. Subsequent oxidation of the eluted lysozyme resulted in the almost complete recovery (100%) of the original enzymatic activity of lysozyme.
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