Journal of Life Sciences 6 (2012) 952-956
bloom是什么意思Stability and Refolding of Prophenol Oxida Protein with 2-Propanol in Drosophila melanogaster
Eri Sato1, 2, Kotomi Mita1 and Nobuhiko Asada1
1. Department of Zoology, Faculty of Science, Okayama University of Science, Kita-ku, Okayama 700-0005, Japan
2. Graduate School of Systems Life Sciences, Kyusyu University, Higashi-ku, Fukuoka 812-8581, Japan
Received: February 13, 2012 / Accepted: April 07, 2012 / Published: August 30, 2012.
没问题英语怎么说Abstract: Phenol oxida in Drosophila melanogaster occurs as folded pha precursors designated as prophenol oxida A1 and A3, and prophenol oxida is activated with alcohol, especially 2-propanol, within a few minutes as unfolded-pha in vitro. To clarify a common effect of alcohols on proteins and peptides, the extract containing prophenol oxida protein was prepared. Phenol oxida activity activated with 2-propanol has been maintained stable at least 24 hours remains as it is. Protein of prophenol oxida was not denatured opposite hypnos known as the instability of protein with alcoho
颜色英语l. Activated prophenol oxida with 2-propanol remain enzyme activity with no aggregation, stable, renaturation, and the refolding phenomena occurred around the active pha within the catalytic active center of prophenol oxida protein in Drosophila melanogaster. This study is important to induce the wide range applications of the effect in many fields for rational drag design.
Key words: Stability, 2-propanol, refolding, prophenol oxida, Drosophila melanogaster.
1. Introduction
Inct phenol oxida (designated tyrosina in mammals) is a copper-containing enzyme that catalyzes two reactions: oxygenation of monophenol
to o-diphenol and oxidation of diphenol to o-quinone using catechol and catecholamine as the substrates. The two reactions are key steps to synthesis of black pigment melanin. Detailed analys has been performed extensively on the activation systems of prophenol oxida (folding pha) with innate activating system, designated AMM-1, in vivo (unfolding pha) [1] and with organic cationic and anionic detergents, chymotrypsin, fatty acid, and various kinds of alcohol including 2-propanol as the induced fit type (refolding pha) in vitro [2]. Phenol oxida activity (unfolding pha) was applicable to the Ping-pong model in Drosophila melanogaster.
Corresponding author: Nobuhiko Asada, Ph.D., professor, rearch field: population genetics. E-mail: **************.ac.jp.Purified fragment of prophenol oxida shows homodimer in vivo and binds to di-copper bonds at the active center of the prophenol oxida protein. The copper-binding active center with a short Cu(II)-Cu(II) distance of 2.9 Հ is coordinated by the evolutionary highly conrved in the given prophenol oxida protein in invertebrate species [3].
Effects of alcohols on proteins and peptides of prophenol oxida have been studied [2], since the studies are important on the wide range applications of the alcohol effects on many fields [4, 5]. This application implies, focus, and new hypothesis on that a conformational change and refolding pha of prophenol oxida protein without the previous showing danaturation of rigid native state proteins. This article shows and discuss the properties and effects on that prophenol oxida became active and stable state in prence of the alcohol, especially 2-propanol, in Drosophila melanogaster.
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Stability and Refolding of Prophenol Oxida Protein with 2-Propanol in Drosophila melanogaster953
2. Materials and Methods
2.1 Flies and Chemicals
Late third instar larvae of the Drosophila melanogaster were reared on a standard cornmeal yeast medium at 25 °C. Laboratory strain, Oregon-R, was rved as the standard. Pefabloc (4-(2- aminoethyl)-benzenesulfonyl fluoride, hydrochloride) SC (AEBSF) was purchad from Roche Diagnostics GmbH, (Mannheim, Germany), dopamine was from Nakarai Tesque Inc., (Kyoto, Japan). Pefabloc SC from Boehringer Mannheim Biochemicals, GmbH (Mannheim, Germany), Protein Assay Kit was from Bio-Rad Laboratory (Hercules, CA), FPLC system from Pharmacia LKB Biotechnology (Uppsala, Sweden).
2.2 Preparation of Prophenol Oxida
The following procedures were performed at 0-4°C, unless otherwi specified. Centrifugation was performed at 30,000 g (16,000 rpm), for 2 min using a Sakuma M-150 (Tokyo, Japan). Prophenol oxi
da was collected in the supernatant after centrifugation and ud as the starting material. The larvae were homogenized with sample buffer containing 25 mM Tris-HCl, 5 mL 2-mercaptoethanol (Nakarai Tesque, Inc., Kyoto, Japan), 10 mL glycine, 2 g sodium dodecyl sulfate, bromophenol blue (Nakarai Tesque, Inc., Kyoto, Japan), powder and distilled water up to 50 mL by glass bar. After centrifugation at 16,000 rpm, for 2 minutes, at 4 °C using a Sakuma Model M-150 (Tokyo, Japan), supernatant preparation including prophenol oxida A1 and A3 was collected for characterization.
2.3 Activation of Prophenol Oxida, Phenol Oxida Activity, and Circular Dichrois Spectroscopy
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Activation of prophenol oxida with 50% 2-propanol and determination of the phenol oxida activity were performed as described in our previous report [1]. Dopamine in 200 mM Tris-maleate buffer, pH 6.0 at room temperature was ud as the substrate of phenol oxida. Circular dichroism (CD) spectra of prophenol oxida protein with 50% 2-propanol had been measured in the range of 195-245 nm as the previous paper [6]. Distilled water against 2-propanol was ud as the control. Five times were run and calculate the average values.
2.4 Refolding Reaction
Refolding reaction of prophenol oxida protein was performed after our previous paper [2]. 50% 2-p
ropanol, dopamine in 200 mM Tris-maleate buffer, pH 6.0 at room temperature, Spectrophotometer using a Hitachi U-2000 (Tokyo, Japan), block-heater, using a Dry Thermo Unit, Taitec (Tokyo, Japan) were ud for this rearch.
3. Results and Discussion
考研数学二3.1 Stability of Prophenol Oxida Protein with 2-Propanol
Suspend including prophenol oxida was activated with 50% 2-propanol with small amounts of Pefabloc powder to inhibit the activation of prophenol oxida with innate activating factor, AMM-1. Phenol oxida activities were detected at least 24 hours after addition of 50% 2-propanol to prophenol oxida protein at both temperatures (Fig. 1). After incubated the samples at 4 °C or 30 °C, value of phenol oxida activity was measured at both temperatures. Special phenomenon was that no agglutination and polymerization was occurred against the commonn protein agglutination with alcohol [4]. The data will be applied to make new medicine prevent amiroid-agglutination in the Altsheimer dia. Values of phenol oxida activity calculated by spectrophotometer showed approximately same as 0.20 between 4 °C and 30 °C, or without 2-propanol (control) and with 2-propanol. The values of the phenol oxida activity tend to prent hig
her with time, however, no statistical differences with or without 50% 2-propanol were shown. The chain
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Stability and Refolding of Prophenol Oxida Protein with 2-Propanol in Drosophila melanogaster
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Fig. 1 Change in rate in activation of prophenol oxida (folding pha) with 50% 2-propanol and phenol oxida activity was determined after incubation with 10 mM dopamine as the substrate in Drosophila melanogaster . Distilled water against 2-propanol was ud as the control. Activation velocity is fast (unfolding pha), maintained at least 24 hours the enzyme activity showing the prophenol oxida protein is stable. Data point are at 4 °C (above) and 30 °C (below). Dots indicate the average values (n = 5).
reactions can be attached enzyme to the Ping-pong Bi Bi reaction. The reason why the effect is no statistical difference between distilled water and 2-propanol is not clear. The data shows that prophenol oxida protein is not unstable but stable with alcohol, especially 2-propanol. Since the authors’ previous study prented activation of prophenol oxida with alcohols, methanol, ethanol, 1-butanol, 2-butanol, 1-propanol, 2-propanol, and glycerol unless melanization in the solution [1, 6], the stability of prophenol oxida protein with 2-propanol is gives emphasis. No significant evidence was detected showing the oxygena activity during time in this study.
3.2 Structural Redundancy at the Active Center of Prophenol Oxida Protein
Depend on the Ribbon-model of higher-order
structure and CD spectra of prophenol oxida cleared the condary structure at the active center of prophenol oxida protein, that is, the active center was located almost not at the peripheral site of the protein, but at the central site of the protein molecule of prophenol oxida. The significance of the prence of di-copper was estimated as the strong magnetic binding with the two oxygen-binding Cu(II) atoms paration by 2.9 Հ [3] surrounded by α-helices and three His residues per single Cu(II) atoms (total six His residues, Fig. 2). After addition of 2-propanol to prophenol oxida protein, redundancy of the condary structure, especially α-helix, was occurred (unfolding pha) immediately, substrate can enter from outside to the active center by the narrow canal of prophenol oxida protein. The chain reaction can be attached enzyme to the Ping-pong Bi Bi model in enzyme reaction.
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Stability and Refolding of Prophenol Oxida Protein with 2-Propanol in Drosophila melanogaster
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Fig. 2 Refolding phenomena of prophenol oxida protein activated with 2-propanol. Activation velocity of prophenol oxida (unfolding pha) is fast, however, reactivation velocity after dilution is slow like the Origami model. 3.3 Refolding of Prophenol Oxida Protein with 2-propanol
After our previous study [7], effect of veral ionic concentrations from 0 to 200 mM KCl on the specific activity of phenol oxida was examined using FPLC gel filtration chromatography. Considering the salt concentration in Drosophila hemolymph, the results indicate that prophenol oxida exists as a homodimer in vivo, and the higher-order structure (folding pha) of prophenol oxida can be activated and changed the structure fast velocity (unfolding pha), then reversibly slow velocity (refolding pha) by change salt concentration ud in vitro.
勺子的英文At a temperature of 80 °C, prophenol oxida was
Fig. 3 Ribbon model diagram at the active center of prophenol oxida A1 isoform in Drosophila melanogaster. Dicopper, Cu(II), domain of prophenol oxida is shown to be sorrowed by six His residues at the center. Helical region is omitted in this figure.
partially denatured, however, it showed reversible recovery (refolding pha) by renaturation (refolding pha) at 30 °C. The prophenol oxida protein involved in 2-propanol would offer an ideal system for studying the difference of higher-order structures between active and inactive states in the inct phenol oxida. The diversification of melanin particle process in Drosophila melanogaster reflects evolutionary change, biodiversity, is primarily driven by effect of mutations and stochasticity [8] (Fig. 3).
brasrieThe protein of prophenol oxida shows reversible dissociation and reassociation in the higher-order structure. The reactions can be applied the Origami (Japane typical paper craft) model and the Anfinn dogma on behalf of β-lacto albumin to prophenol oxida, and of 2-mercaptoethanol to 2-propanol. In this study, velocity dimension was first discusd on refolding of the protein.
4. Conclusions
After prophenol oxida activated with 2-propanol, the protein was not unstable, but stable until 24 hours and maintained the phenol oxida activity. The findings are consistent with the Origami model and uful in designing new medicines to prevent protein agglutination in Alzheimer’s dia.
Acknowledgments
We thank Drs. Yuji Goto and Hisashi Yagi of the Laboratory of Protein Folding, Division of Protein Structural Biology, the Institute for Protein Rearch, Osaka University, for their cordial advice. This work
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Stability and Refolding of Prophenol Oxida Protein with 2-Propanol in Drosophila melanogaster 956
was supported in part by the Ryobi-Teien Foundation, Kita-ku, Okayama, Japan.
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