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1 (Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-6 ACYL CoA SYNTHETASE [ACS] from Pseudomonas fragi (Acid: CoA ligase(amp forming), EC ) RCOOH + ATP + CoASH RCO SCoA + PPi + AMP Preparation and Specification Appearance : White amorphous powder, lyophilized Specific activity : More than 2 U/mg solid Properties Substrate specificity : See Table Molecular weight : 6 kda(sephadex G 5) Isoelectric point : 5.2 Michaelis constants : Palmitic acid. -5 M ATP.7-4 M CoA M Optimum : Palmitic acid 7.7 Figure : Serum fatty acids 7.7 Figure 2 stability : 6. 8.(37, 2 hr) Figure 3 Thermal stability : Stable at 5 and below :( 7.5, min) Figure 4 Storage stability : At least one year at 2 Figure 5 Effect of various chemicals : See Table 2 and Table 3 Stabilizer : ATP Activator : Triton X Applications for Diagnostic Test This enzyme is useful for enzymatic determination of fatty acid when coupled with Acyl CoA oxidase(t 7). FFA + CoA + ATP Acyl - CoA + O2 2 H2O2 + 4 AA + Phenol FFA: Free fatty acid ACS ACOD POD Acyl - CoA + PPi + AMP Enoyl - CoA + H2O2 Quinoneimine dye + 4 H2O

2 Table. Substrate specificity(fatty acids) Substrate Caproic acid ( 6:) Caprylic acid ( 8:) Capric acid (:) Lauric acid (2:) Myristic acid (4:) Palmitic acid (6:) Stearic acid (8:) Arachidic acid (2:) Myristoleic acid (4:) Palmitoleic acid (6:) Palmitelaidic acid (6:) Oleic acid (8:) Elaidic acid (8:) Linoleic acid (8:2) Linolenic acid (8:3) Arachidonic acid (2:4) Erucic acid (22:) Nervonic acid (24:) Relative activity(%) Km value ( - 5 M) Table 3. Effect of metal ions on ACS activity Metal ion Concentration Relative activity(%) None KCI NaCI LiCI NH4Cl MgCl2 CaCl2 ZnCl2 BaCl2 MnCl2 CuCl2 NiCl2 EDTA mm MgCl2 + CaCl2 mm MgCl2 + ZnCl2 mm MgCl2 + CuCl2 mm MgCl2 + NiCl2 mm MgCl2 + BaCl2 mm MgCl2 + MnCl2.M... mm Table 2. Effect of detergents on ACS activity Detergent(%) Relative activity(%) None Deoxycholate SDS. Cetyltrimethylammoniumchloride Cetylpyridinium chloride Sarcosinate PN Relative Activity (%) 6 4 Relative Activity (%) : 3,3-Dimethylglutarate-NaOH buffer : 3,3-Dimethylglutarate-NaOH buffer 37, 2 hr : Phosphate buffer : Phosphate buffer ,3-Dimethylglutarate- : Tris-HCI buffer : Tris-HCI buffer NaOH buffer Phosphate buffer Tris-HCI buffer 39

3 g T er a Sta ty g Storage yop e po er Temperature ( ) 7.5, min. Phosphate buffer : +2 mm ATP : Non addition Period (month) : 2 : 5 Assay Principle The assay is based on the increase in absorbance at 55 nm as the formation of quinonediimine dye proceeds in the following reactions: ACS Palmitic Acid + CoA + ATP Palmitoyl CoA + AMP + PPi ACOD Palmitoyl CoA + O 2 2 Hexadecenoyl CoA + H 2O 2 POD 2 H 2O AA + MEHA Quinonediimine dye + 4 H 2O ACOD: Acyl CoA oxidase MEHA: 3 Methyl N ethyl N (2 hydroxymethyl) aniline Unit definition One unit is defined as the amount of enzyme which converts μmole of fatty acid to acyl CoA per minute at 37 under the conditions specified in the assay procedure. Reagents. Reaction mixture for the first reaction.2 M KH 2PO 4 K 2HPO 4 buffer ml mm ATP solution 7.5. ml mm MgCl 2 solution. ml mm Palmitic acid 5 %(W/V) Triton X solution 7.5 ).2 ml Distilled water.35 ml mm CoA solution 6.5 2).5 ml ): mm Palmitic acid 5 %(W/V)Triton X solution 7.5 Dissolve 26 mg of palmitic acid with 9 ml of 5% (W/V)Triton X, adjust to 7.5 at 25 with 4 N NaOH, add 5%(W/V)Triton X to make a total of ml. 2): mm CoA solution 6.5 Dissolve 54 mg(purity calculation)of CoA with 5 ml of distilled water, adjust to 6.5 at 25 with 4 N NaOH, and add distilled water to make a total of 2 ml. 2. Reaction mixture for the second reaction.2 M KH 2PO 4 K 2HPO 4 buffer ml 2mM NEM. ml 5mM 4 AA solution.3 ml.3%(w/v)meha solution ml u/ml POD solution 3). ml.5%(w/v)nan 3 solution. ml Distilled water.55 ml 2 U/ml ACOD solution 4). ml NEM: N Ethylmaleimide 3): U/ml POD solution Dissolve, U(PPU)of POD with ml of distilled water. 4): 2 U/ml ACOD solution Dissolve,2 U of ACOD with ml of ACOD dilution buffer ). ): ACOD dilution buffer Dissolve.36 g of KH 2 PO 4 and.82 g of ATP with distilled water, adjust to 7. with 4 N NaOH, add ml of mm FAD, and finally add distilled water to make a total of L. 3. Enzyme dilution buffer mm phosphate buffer( 7.5)containing 2 mm ATP,.5%(W/V)BSA and. %(W/V)Triton X. 4. Reagents: ATP(2Na 3H 2O): Kyowa Hakko Co., Ltd. Palmitic acid : Wako Pure Chemical Industries, Ltd. #69 5 Triton X : Yoneyama Yakuhin Kogyo Co., Ltd. #2 NEM: Wako Pure Chemical Industries, Ltd. # AA: NACALAI TESQUE, INC. Special grade #97 52 MEHA: Wako Pure Chemical Industries, Ltd. (for biochemistry)# 755 POD: Sigma Chemical Co. Type Ⅱ #P 825 ACOD: Asahi Kasei Pharma Corporation #T 7 FAD(2Na): Kyowa Hakko Co., Ltd. 4

4 BSA: Celliane Fraction V 5.2 #322 8 ATP: Adenosine triphosphate FAD: Flavine adenine dinucleotide Enzyme solution Weigh about 2 mg of test sample exactly and add enzyme dilution buffer to make a total of 2 ml. Dissolve it with enzyme dilution buffer to adjust the concentration to within.5. U/ml. Procedure. Pipette accurately. ml of reagent mixture for the first reaction into a small test tube and preincubate at After 5 min, add exactly 5 μl of enzyme solution and mix to start the first reaction at 37. In the case of a test blank, add 5 μl of enzyme dilution buffer in place of enzyme solution. 3. After min, add 2. ml of reagent mixture for the second reaction to stop the first reaction and mix to start the second reaction at After 5 min, measure the absorbance at 55 nm. Absorbance sample : As blank : Ab A =(As Ab).3 Abs 32. : millimolar extinction coefficient of quinonediimine dye at 55 nm(cm 2 / μmole) /2 : a multiplier derived from the fact that 2 mole of H 2O 2 produces mole of quinonediimine dye : reaction time(min) 3.5 : final volume(ml).5 : volume of enzyme solution(ml) X : concentration of ACS in enzyme solution(mg/ml) Storage Storage at 2 in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition. References. Yamada, H., Shimizu, S. and Tani, Y.(98)Vitamin (Japanese), 54, Shimizu, S., Inoue, K., Tani, Y. and Yamada, H.(98) Anal. Biochem., 7, Okabe, H., Uji, Y., Nagashima, K. and Noma, A.(98) Clin. Chem., 26, Shimizu, S., Inoue, K., Tani, Y. and Yamada, H.(979) Anal. Biochem., 98, 34. Calculation A/ 3.5 Activity(U/mg)= 32. /2.5 X ACS 活性測定法 (Japanese) Ⅰ. 試薬液. 第一反応試薬混合液.2M KH 2 PO 4 K 2 HPO 4 緩衝液 ml mm ATP 溶液 7.5. ml mm 塩化マグネシウム溶液. ml mm パルミチン酸 5%(W/V) トリトン X 溶液 7.5 ).2 ml 精製水.35 ml mm CoA 溶液 6.5 2).5 ml ): mm パルミチン酸 5%(W/V) トリトン X 溶液 7.5 パルミチン酸 2 6 m g を 5 %( W / V ) トリトン X- 溶液 9ml で加温溶解した後 4N NaOH で ) に調整し 5%(W/V) トリトン X 溶液で全容 ml とする 2): mm CoA 溶液 6.5 CoA 54mg( 純度換算 ) を精製水 5ml に溶解した後 4N NaOH で 6.5(25 ) に調整し精製水で全容 2ml とする 2. 第二反応試薬混合液.2M KH 2PO 4 K 2HPO 4 緩衝液 ml 2mM NEM 溶液. ml 5mM 4 AA 溶液.3 ml.3%(w/v)meha 溶液 ml U/ml POD 溶液 3). ml.5%(w/v)nan 3 溶液. ml 精製水.55 ml 2U/ml ACOD 溶液 4). ml 3): U/ml POD 溶液 POD, 単位 (PPU) を精製水 ml で溶解する 4): 2U/ml ACOD 溶液 ) ACOD,2 単位 (U) を ACOD 希釈緩衝液 ml で溶解する ): ACOD 希釈緩衝液 KH 2PO 4.36g と ATP.82g を精製水に溶解した後 4N NaOH で 7.(25 ) に調製しさらに mm FAD 溶液 ml を加えて精製水で全容 L とする 3. 酵素溶解希釈用液 2mM ATP と.5%(W/V)BSA 及び.%(W/V) トリトン X を含む mm リン酸緩衝液試薬 ATP( アデノシン三リン酸 2Na 3H 2O): 協和発酵製 CoA( コエンザイム A): 興人製パルミチン酸 : 和光純薬工業製特級 #69 5 トリトン ( トリトン X ): 米山薬品工業製 #2 NEM(N エチルマレイミド ): 和光純薬工業製 # AA: ナカライテスク社製特級 #

5 MEHA[3 メチル N エチル N (2 ヒドロキシエチル ) アニリン ]: 和光純薬工業製生化学用 # 755 POD: シグマ社製 Type Ⅱ #P 825 ACOD( アシル CoA 酸化酵素 ): 旭化成ファーマ製 #T 7 FAD( フラビンアデニンジヌクレオチド 2Na): 協和発酵製 BSA: Celliane 社製 Fraction V Ⅱ. 酵素試料液検品約 2mg を精密に量り酵素溶解希釈用液に溶解して全容 2ml とするその液を酵素溶解希釈用液で.5~.U/ml 濃度となるように適宜希釈する Ⅲ. 測定操作法. 小試験管に第一反応試薬混合液.ml を正確に分注して 37 で予備加温する 2. 5 分経過後酵素試料液 5 μl を加えて混和し 37 で反応を開始する盲検は酵素試料液の代わりに酵素溶解希釈用液 5 μ を加える 3. 分経過後第二反応試薬混合液 2.ml を正確に加えて混和し第一反応を停止させ 37 で第二反応を開始する 4. 5 分経過後 55nm における吸光度を測定する求め Ⅳ. 計算られた吸光度を試料液は As 盲検液は Ab とする ΔA=(As Ab).3 Abs ΔA/ 3.5 活性 (U/mg)= 32. /2.5 X 32.: キノンジイミン色素の 55nm におけるミリモル分子吸光数 (cm 2 / μmole) /2: H 2O 2 2 モルからキノンジイミン色素モルが生成することによる係数 : 反応時間 (min) 3.5: 反応総液量 (ml).5: 反応に供した酵素試料液量 (ml) X : 酵素試料液中の検品濃度 (mg/ml) 42

A.25 Table 1. Substrate specificity(fatty acids) Substrate Relative Km value(1 - activity(%) 5 M) Caproic acid ( 6:) 38 Caprylic acid ( 8:) Cap

A.25 Table 1. Substrate specificity(fatty acids) Substrate Relative Km value(1 - activity(%) 5 M) Caproic acid ( 6:) 38 Caprylic acid ( 8:) Cap A(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T 16 ACYL CoA SYNTHETASE [ACS] from Pseudomonas fragi (Acid: CoA ligase(amp forming), EC 6.2.1.3) RCOOH + ATP + CoASH RCO SCoA + PPi + AMP Preparation and