PTable. Substrate specificity Lecithin Lysolecithin Sphingomyelin Substrate Relative activity(%) Table 2. Effect of detergents on PLDP activity

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1 PT 39 (Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T 38 PHSPHLIPASE D [PLDP(T-39),PLDPV(T-38)] (Glycerophospholipid specific) from Streptomyces sp. (Phosphatidylcholine phosphatidohydrolase, EC ) H 2 C C R H 2 C C R HC C R 2 H H2 HC C R 2 H HCH 2 CH 2 N (CH 3)3 H 2 C P choline Phosphatidylcholine H 2 C P H Phosphatidic Acid Choline Preparation and Specification PLDP(T-39) Appearance : Light brownish amorphous powder, lyophilized Specific activity : More than U/mg solid PLDPV(T-38) Appearance : White to brownish lyophilized powder Specific activity : More than 3 U/mg solid Animal derived material free. Properties Substrate specificity : See Table Molecular weight : 46 kda(gel filtration) Isoelectric point : 4.2 ptimum : Figure stability : (37, 6 min,.5% BSA) Figure 2 Storage stability : At least one year at -2 Figure 3 Effect of various chemicals : See Table 2 and Table 3 Transphosphatidylation Catalyzed by Phospholipase D 3 Figure 4 Figure 5 Figure 6 Figure 7

2 PTable. Substrate specificity Lecithin Lysolecithin Sphingomyelin Substrate Relative activity(%) Table 2. Effect of detergents on PLDP activity Additive Concentration(%)Relative activity(%) None Triton X SDS Deoxycholate Table 3. Effect of metal ions on PLDP activity Metal ion Concentration (mm) None CaCl2 MgCl2 MnCl2 ZnCl2 CoCl2 BaCl2 CuCl2 EDTA EDTA Relative activity (%) Relative Activity (%) 5 Residual Activity (%) Residual Activity (%) : 3,3-Dimethylglutarate-NaH buffer : Phosphate buffer : Tris-HCI buffer : Glycine-NaH buffer , 6 min. : 3,3-Dimethylglutarate-NaH buffer : Phosphate buffer : Tris-HCI buffer : Glycine-NaH buffer Period (month) R R P Transphosphatidylation Catalyzed by Phospholipase D N Me3 X-H H N Me3 R R P X Yield(%) rganic phase : Solvent(.5ml)Contained mg Dipalmitoylphosphatidylcholine Aqueous phase :.25M Cytidine HCl buffer 4.5(.ml)and.5ml of water contained.9 Unit of PLDP Chloroform Diethylether Ethylacetate Benzene n-hexane THF Reaction Temp : 35, Reaction : 9 min. 3

3 P Uracil Nucleoside Donor : Uridine 25 mm 4-6 : Acetate buffer Cytosine Nucleoside 6-7 : MES-NaH buffer Deoxycytidine (Donor:dipalmitoylphosphatidylcholine, Acceptor: Cytidine, 4.5) Time (hr) : Glycine-HCl buffer : Acetate buffer Adenine Nucleoside Cytidine Cytidine, Deoxycytidine : 25mM Product (%) Phosphatidylcytidine Phosphatidic acid Adenosine Deoxyadenosine Cytidine ratio vs phospholipid rganic solvent : mm Dipalmitoylphosphatidylcholine in Chloroform(ml) Aqueous solvent :. M Glycine HCl(4.5).5ml containing.8 Units of PLDP : Glycine-HCl buffer 4-6 : Acetate buffer Adenosine : 5mM Deoxyadenisine : 5mM Reaction : at 35, with continuous stiiring, for 8min. Phospholipid acceptor : Dipalmitoylphosphatidylcholine in Chloroform(ml) Buffer :.5ml contained.8 Units of PLDP Reaction Temp : 35 Reaction period : 9 min. Assay Principle The assay is based on the increase in absorbance at 5 nm as the formation of quinoneimine dye proceeds in the following reactions: PLDP Phosphatidyl choline H2 Phosphatidic acid Choline CD Choline 2 2 H2 Betaine 2 H22 2 H22 Phenol 4 AA CD: Choline oxidase PD Quinoneimine dye 4 H2 Unit definition ne unit is defined as the amount of enzyme which hydrolyzes μmole of phosphatidylcholine to phosphatidic acid and choline per minute at 37 under the conditions specified in the assay procedure. 32

4 P Reagents. Reaction mixture for the first reaction.2 M DMG NaH buffer 5.5. ml mm CaCl2.5 ml 3%(W/V)Triton X solution.5 ml mm Substrate solution ). ml Distilled water.5 ml DMG: 3, 3 Dimethylglutarate ): mm Substrate solution Dissolve 78.6 mg of L α phosphatidylcholine, dioleoyl with ml of 5% Triton X (W/V). 2. Reaction mixture for the second reaction 5 mm 4 AA.5 ml.2%(w/v)phenol.5 ml M Tris HCl buffer, 8..5 ml 5 U/ml PD 2).5 ml 5 U/ml CD 3).5 ml Distilled water.25 ml 2): 5 U/ml PD Dissolve 5 U(PPU)of PD with ml of distilled water. 3): 5 U/ml CD Dissolve 5 U of CD with ml of mm Tris HCl buffer, Reaction stopper M Tris HCl buffer containing mm EDTA and %(W/V)Cetyltrimetylammonium chloride EDTA: Ethylenediamine tetraacetic acid 4. Reaction dilution solution %(W/V)Triton X 5. Enzyme dilution buffer mm DMG NaH buffer, 5.5 containing.% Triton X 6. Reagents: DMG : Tokyo Kasei Kogyo Co., Ltd. #D322 Triton X : The Dow Chemical Company,2 Dioleoyl sn glycero 3 phosphocholine: Sigma Chemical Co. #P 6354 EDTA(2Na 2H2): KISHIDA CHEMICAL Co., Ltd. # CD: Asahi Kasei Pharma Corporation #T 5 4 AA: NACALAI TESQUE, INC. Special grade #97 52 Cethyltrimethylammonium chloride: Wako Pure Chemical Industries, Ltd. # PD: Sigma Chemical Co. Type Ⅱ #P 825 Enzyme solution Weigh about 2 mg of test sample exactly and add enzyme dilution buffer to make a total of 2 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required. Procedure. Pipette.45 ml of reaction mixture for the first reaction into a small test tube and preincubate them at After 5 min, add exactly 5 μl of enzyme solution and mix to start the first reaction. In the case of a test blank, add 5 μl of enzyme dilution buffer in place of enzyme solution at this point. 3. After min, add.5 ml of reaction stopper and mix. n stopping the first reaction, add.5 ml of the reaction mixture for the second reaction immediately to start the second reaction at After 2 min, add.5 ml of reaction dilution solution and mix. 5. After min, measure the absorbance at 5 nm. Absorbance sample :As blank :Ab A =(As-Ab).4 Abs Calculation A/ 3. Activity(U/mg)= 2.2 /2 2.5 X 2.2 : millimolar extinction coefficient of quinoneimine dye at 5 nm(cm 2 / μmole) 2 : the multiplier derived from the fact that mole of phosphatidyl choline produces 2 mole of H22 /2 : the multiplier derived from the fact that 2 mole of H22 produces mole of quinoneimine dye : reaction time(min) 3. : final volume(ml).5 : volume of enzyme solution(ml) X : concentration of the sample 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(figure 3). References. Satosi S., Shigeyuki I., Hideo S., and Jun-ichi M.,(987) Chem. Pharm. Bull., 35., Satoshi S., Shigeru U., Shigeyuki I., Kiyofumi F., Akira M., and Tohru U.,(987)Tetrahedron, 28, 2, Satoshi S., Hiromichi I., Shigeru U., Shigeyuki I., Kiyofumi F., Masatoshi T., Akira M., and Tohru U., (988)Chem. Pharm. Bull., 36,, Satoshi S., Shigeyuki I., Kiyofumi F., and Tohru U.,(988) Chem. Pharm. Bull., 36, 2, Satoshi S., Hiromichi I., Atsushi S., Keishi N., Shigeyuki I., and Akira M.,(5)Bioorg. Med. Chem., 3, 3,

5 PPLDP/PLDPV 活性測定法 (Japanese) Ⅰ. 試薬液. 第一反応試薬混合液.2M DMG NaH 緩衝液 5.5. ml mm 塩化カルシウム溶液.5 ml 3%(W/V) トリトン X 溶液.5 ml ) mm 基質溶液. ml 精製水.5 ml ): mm 基質溶液 ( フォスファチジルコリンジオレオイル溶液 ) L α フォスファチジルコリンジオレオイル 78.6mg を 5%(W/V) トリトン X 溶液 ml で溶解する 2. 第二反応試薬混合液 5mM 4 AA 溶液.5 ml.2%(w/v) フェノール液.5 ml M トリス HCl 緩衝液 8..5 ml 2) 5U/ml PD 溶液.5 ml 3) 5U/ml CD 溶液.5 ml 精製水.25 ml 2): 5U/ml PD 溶液 PD5 単位 (PPU) を精製水 ml で溶解する 3):5U/ml CD 溶液 CD5 単位 (U) を mm トリス -HCl 緩衝液 8. ml で溶解する 3. 反応停止液 %(W/V) 塩化セチルトリメチルアンモニウムと mm EDTA を含む M トリス HCl 緩衝液反応用希釈液 %(W/V) トリトン X 溶液 5. 酵素溶解希釈用液.% トリトン X を含む mm DMG NaH 緩衝液試薬 DMG(3,3 ジメチルグルタール酸 ): 東京化成製 #D322 トリトン X :Dow Chemical 社製,2 ジオレオイル sn グリセロ 3 ホスホコリン : シグマ社製 #P 6354 EDTA( エチレンジアミン四酢酸 2Na 2H2): キシダ化学社製 # CD( コリン酸化酵素 ): 旭化成ファーマ製 #T 5 4 AA: ナカライテスク社製特級 #97 52 塩化セチルトリメチルアンモニウム : 和光純薬工業製 # PD: シグマ社製 Type Ⅱ #P 825 Ⅱ. 酵素試料液検品約 2mg を精密に量り酵素溶解希釈用液で全容 2ml とするその液を酵素溶解希釈用液で適宜希釈する Ⅲ. 測定操作法. 小試験管に第一反応試薬混合液.45ml を正確に分注し 37 で予備加温する 2. 5 分経過後酵素試料液 5 μl を正確に加えて混和し 37 で第一反応を開始する盲検はこの時点で酵素試料液の代わりに酵素溶解希釈用液 5 μl を加える 3. 分経過後反応停止液.5ml を加えて混和し第一反応を停止すると共に直ちに第二反応試薬混合液.5ml を加えて混和し 37 で第二反応を開始する 4. 2 分経過後反応用希釈液.5ml を加えて混和する 5. 分経過後 5nm における吸光度を測定する求められた吸光度を試料液は As 盲検液は Ab とする ΔA =(As-Ab).4 Abs Ⅳ. 計算 ΔA/ 3. 活性 (U/mg)= 2.2 /2 2.5 X 2.2 : キノンイミン色素の 5nm におけるミリモル分子吸光係数 (cm 2 / μmole) 2 : フォスファチジルコリンモルから H22 2 モルが生成することによる係数 /2 : H22 2 モルからキノン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. : 反応総液量 (ml).5 : 反応に供した酵素試料液量 (ml) X : 酵素試料液の検品濃度 (mg/ml) 34

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