原文我只想翻譯分光光度計(jì)法Acetyl-CoACarboxylasefromRat

1、原文：我只想翻譯分光光度計(jì)法Acetyl-CoA Carboxylase from Rat LiverEC 641.2 Acetyl-CoA: carbo n-dioxide ligase (ADP-formi ng)By TADASHI TANABE, SHIGETADA NAKANISHI, TAKASHI HASHIMOTO, HIDEO OGIWARA, JUN-ICHI NIKAWA, and SHOSAKU NUMAATP+HCO 3 +acetyl-CoAADP+Pi+malonyl-CoAAssay MethodsThe prin ciples un derly ing the

2、 various assays of acetyl-CoA carboxylase have bee n1-4described in previous articles in this series. Most conveniently, the enzyme activity14is determined by CO2 fixation assay or by the spectrophotometric assay in comb in ati on with the pyruvate kin ase and lactate dehydroge nase reacti ons. The1

3、4CO2 fixation assay can be used for enzyme preparations from all steps, whereas the spectrophotometric assay is applicable to preparation from the DEAE-cellulose chromatography step and subseque nt steps.14CO2 Fixation MethodReage ntsTris-HCl buffer, 0.5M, PH 7.5Potassium citrate, 0.1MMgCl2, 0.1MRed

4、uced glutathio ne, 0.1M, PH 7.5Bovine serum albumin, 3%ATP, 0.5MAcetyl-CoA, 10mMKH14CO3 (0.25 卩 Ci/ 卩 mol), 0.2MHCl, 5MSci ntillator soluti on: 4g of 2,5-diphe nyl oxazoleand 0.1g of 1,4-bis2-(4-methyl-5-phe nyloxazolyl)be nzene in 1 liter of tolue ne plus o.5 liter of Trit on X-100Procedure: Whe n

5、the crude extract is assayed, it is passed through a Sephadex G-50 colu mn to remove en doge nous substrates. Because rat liver acetyl-CoA carboxylase requires preincubation with citrate to attain its full activation,5 the enzyme is first preincubated at 37 °C for 300 min in a mixture containin

6、g 50mM Tris-HCl buffer, PH 7.5, 10mM potassium citrate, 10mM MgCl2, 3.75mM glutathione, and 0.75mg of bovine serum albu min per milliliter. The react ion is the n in itiated by add ing an aliquot of the prein cubated en zyme (up to 0.2mU) to an assay mixture (final volume, 0.8ml) con tai ning 50mM T

7、ris-HCl buffer, PH 7.5, 10mM postassium citrate, 10mM MgCl2,143.75mM ATP, 0.125mM acetyl-CoA, and 12.5mM KH CO3(0.25 卩 Ci/ 卩 mol). After incubation at 37 C for 10 min, the reaction is terminated with 0.2ml of 5M HCl. Thereact ion mixture is allowed to sta nd in a vacuum desiccator for 30 min to remo

8、ve the14un reacted H CO3 and is cen trifuged at 1500g for 10 min to elimi nate the in soluble material. A 0.5-ml aliquot of the supernatant is taken to dryness at 60°C in a countingvial in a vacuum desiccator. After addition of 0.5ml of distilled water and 10 ml of the scintillator solution, th

9、e radioactivity is determined with the use of a liquid scintillation spectrometer. Un der the assay con diti ons described the reacti on follows Zero-order kin etics, and the in itial rate of react ion is proport ional to en zyme concen trati on.Spectrophotometric MethodReage nts KHCO3, 1M Potassium

10、 phosphoe no Ipyruvate, 40mM NADH, 5mM, PH 8Pyruvate kin ase (rabbit muscle; Boehri nger), 10mg/mlLactate dehydroge nase (rabbit muscle; Boehri nger), 5mg/ml14Other reagents, as for the CO2 -fixation methodProcedure: The assay mixture con tai ns 50mM Tris-HCl buffer, PH 7.5, 10mM potassium citrate,

11、10mM MgCl2, 3.75mM glutathione, 0.75mg of bovine serum albumin per milliliter, 3.75mM ATP, 0.125mM acetyl-CoA, 25mM KHCO 3, 0.5mM potassium phosphenoIpyruvate, 0.125mM NADH, 15 卩 g or pyruvate kinase and 6 卩 g of lactate dehydroge nase per milliliter, and en zyme (up to 5 mU) in a final volume of 0.

12、8ml. A mixture (0.76ml) con tai ning all i ngredie nts except ATP and KHCO 3 is preincubated at 37 C for 10 min in a cuvette with 1cm light path. The oxidation of NADH is followed at 37 C with a recording spectrophotometer at 340nm (or at 334nm). After addition of ATP, the con sumption of NADH is fo

13、llowed for 1 min, and the react ion is the n started by additi on of KHCO3. I nitial velocities are obta ined fromthe initial slopes of the recorder traces. Under the assay conditions described, the react ion follows zero-order kin etics for at least 3 min, and the in itial rate of reacti on is prop

14、orti onal to en zyme concen trati on.分光光度計(jì)法檢測 ACC活性【原理】Acetyl-CoA+ATP+HCO 3 AC（Malpnyl-CoA+ADP+Pi該催化反應(yīng)過程會(huì)消耗 NADH HCO、ATP生成Malonyl-CoA、ADP等。目前檢測ACC的方法有兩種：1、放射法：一般采用14C標(biāo)記，放射計(jì)數(shù)檢測HCO轉(zhuǎn)化情況，判斷ACCS活性。2、分光光度計(jì)法（本方法）：檢測NADH的消耗量，判斷ACC的活性。【活性單位定義】在37T的檢測條件下，反應(yīng)體系中每分鐘催化生成1卩molMalonyl-CoA或ATP的酶定義為1個(gè)活性單位（1U）o【試劑】Tri

15、s-HCl檸檬酸鉀ATPKHCOMgCl2乙酰CoANADH還原性谷胱甘肽丙酮酸激酶磷酸烯醇式丙酮酸鉀牛血清白蛋白乳酸脫氫酶1、底物緩沖液Tris-HCl50mM/LPH7.5檸檬酸鉀10mM/LMgCl210mM/L谷胱甘肽3.75mM/L牛血清白蛋白0.75mg/ml乙酰CoA0.125mM/L磷酸烯醇式丙酮酸鉀0.5mM/LNADH0.125mM/L丙酮酸激酶15 卩 g/ml乳酸脫氫酶6 卩 g/ml2、啟動(dòng)液ATP3.75mM/LKHCO325mM/L3、待測酶液每個(gè)反應(yīng)管酶最大濃度不超過 5mU。本文獻(xiàn)為肝臟勻漿。勻漿液用量及勻漿稀釋度視具體情況而定；血清用量也要視實(shí)際情況調(diào)整【操

16、作步驟】空白管測定管底物緩沖液0.76ml0.76ml待測酶液(血清/勻漿)a mla ml混勻，37 T水浴，10min啟動(dòng)液0.04ml0.04m注：啟動(dòng)液應(yīng)先預(yù)溫好，以保證啟動(dòng)液加入后反應(yīng)即可進(jìn)行。迅速加入啟動(dòng)液混勻立即 計(jì)時(shí)?！緳z測】用紫外可見分光光度計(jì)在340nm處檢測吸光度，石英比色杯，光徑 1cm;雙蒸水調(diào)零。 讀取1min時(shí)各管的吸光度?！净钚杂?jì)算】1、血清：ACC( U/L) = '：A 340nm1000(ml/L)名 g(ml)AA2、血漿：O ACC (U/L)="000(ml/L)gxa(ml)O考馬斯亮蘭法測定蛋白含量P (mg/L)ACC( U/mgpro) = ACC(U /L)P(mg/L) A340nm=A (測定管)一A (空白管)&為吸光度相關(guān)系數(shù)，即1卩mol的NADH寸應(yīng)的習(xí)光度值A(chǔ),通過下列NADH標(biāo)準(zhǔn)曲線測得?！綨ADH標(biāo)