英语翻译请不用机译Measurement of protease activity by azocasein assayThe assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothre

英语翻译请不用机译Measurement of protease activity by azocasein assayThe assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothre
英语翻译
请不用机译
Measurement of protease activity by azocasein assay
The assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothreitol (DTT) (Sigma) at 37°C for 10 min to activate protease activity.100 μl of 5 mg/ml azocasein (Sigma) solution was prepared in PBS (pH 7.4) and incubated with 100 μl of parasitic lysate for 1 h at 37°C.Reaction was stopped by adding 300 μl of 10% trichloroacetic acid and samples were incubated on ice for 30 min.Undigested azocasein was removed by centrifugation (5,000×g,5 min) and the resulting supernatant was transferred to a clean tube containing 500 μl of NaOH (525 mM).Absorbance was measured at 442 nM on a spectrophotometer (Tecan Magellan).For controls,lysates were boiled at 90°C for 15 min to inactivate proteases and 100 μl trypsin (2.5 mg/ml) was used as a positive control.
Analysis of proteases by nondenaturing gelatin-SDS-PAGE
This SDS-PAGE method requires the inclusion of a protein,usually gelatin,in an acrylamide gel.Gelatin is digested by proteases after electrophoresis and clear bands are seen after staining the gel with Coomassie Brilliant blue.Cell lysate (40 μg) of B isolate was analyzed by gelatin-SDS-PAGE for protease activity (Lockwood et al.1987) using the SDS-discontinuous buffer system of Laemmli (1970).The acrylamide concentration of resolving and stacking gel was 12.5 and 5%,respectively.Briefly,0.2% (w/v) gelatin (Sigma) was copolymerized into the resolving gel.Samples were not boiled so that enzymatic activity of the proteases upon renaturation would not be lost.After electrophoresis at a constant current of 30 mA/gel for 1.5 h,gels were immersed with continuous shaking for 1 h in 2.5% (v/v) Triton X-100 to remove the SDS and to allow the proteases to become active.The proteinases bands were developed by immersing the gels in incubation buffer containing 1 mM DTT for 3 h at 37°C.The bands were visualized by staining in 0.12% (w/v) Coomassie Brilliant blue R-250 for 30 min.Cell lysate was also analyzed by 12.5% gelatin-containing native gel (without SDS) to determine the number of proteases present and to investigate whether the proteases were single/multiple subunits.
Inhibition of proteases
To determine the type of proteases that are present in Blastocystis,effects of inhibitors on the activity of Blastocystis proteases were studied by incubating Triton-treated gels in incubation buffer with 1 mM DTT containing one of the following inhibitors:iodoacetamide (IA),EDTA,N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK),8-hydroxyquinoline,phenylmethylsulfonyl fluoride (PMSF) (all 1 mM); pepstatin A,leupeptin or antipain (all 100 μg/ml).All inhibitors were purchased from Sigma except pepstatin A (CHEMICON),IA (MP Biomedicals,LLC) and PMSF (BDH).Gels were stained with Coomassie Brilliant blue.
For azocasein assays,similar concentrations of protease inhibitors (IA,TLCK,pepstatin A,EDTA) were added during a 1-h incubation of parasitic lysate with azocasein.
Determination of pH optimum
The pH dependence of Blastocystis protease activity was investigated by incubating Triton X-100 treated gels in buffers of different pH ranging from 3.0 to 8.5.The following buffers were used:0.1 M glycine/HCl (pH 3.0),0.1 M sodium acetate/acetic acid (pH 4.0 and 5.5),0.1 M sodium phosphate (pH 7.0 and 7.6),and 0.1 M Tris–HCl (pH 8.5).All incubation buffers contained 1 mM DTT and gels were stained with Coomassie Brilliant blue.For azocasein assay,a 1-h incubation with parasitic lysate was performed in buffers of different pH containing 1 mM DTT.
本人仅在此先谢过上回黎明晴空所做的精彩回答!

英语翻译请不用机译Measurement of protease activity by azocasein assayThe assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothre
用固氮酪蛋白分析测定的蛋白酶活性
像之前所说的(Scholze and Tannich 1994)一样再通过一些小小的修饰就可以进行分析了.简单地讲,就是寄生虫溶菌液加入2mMDDT 在37℃保温10分钟,以激活蛋白酶的活性.准备100 μl （5 mg/ml）的固氮酪蛋白溶液,加入100 μl 的寄生虫溶菌液在37℃保温1个小时.加入300 μl 10%的三氯乙酰酸来终止反应,然后将样品在冰中保温30分钟.通过离心(5,000×g,5 min)除去未被消化的固氮酪蛋白,将剩下的上清液转移到已加入500μ(525 mM) NaOH的干净试管中.用分光光度计(Tecan Magellan)测定其在442 nM处的吸光度.为了便于控制,将溶菌液在90℃保温15分钟以灭活酶活性,然后
加入100 μl胰蛋白酶(2.5 mg/ml)用以正调节.
明胶—SDS—PAGE分析蛋白酶
这种SDS—PAGE需要蛋白溶液和明胶,而明胶通常用丙烯酰胺凝胶.电泳之后,蛋白质会侵入明胶,用Coomassie Brilliant蓝染色后会出现多条清晰的带.用—SDS—PAGE分析分离物B的细胞消化液(40 μg)的蛋白酶活性(Lockwood et al.1987)要用到Laemmli (1970).的SDS—非连续缓冲系统.凝胶分解和分层后的丙烯酰胺浓度分别是12.5 and 5%.简而言之,0.2% (w/v)的明胶能聚合成有分解作用的凝胶.这样样品就不用加热,蛋白酶也就不会失去酶活性了.电泳1.5小时(30mA 的直流电)后,加入2.5% (v/v) Triton X-100不断的轻摇凝胶1小时,这样做不但能除去SDS,还可以激活蛋白酶的活性.将凝胶浸入已加了1 mM DTT的缓冲液中在37℃保温3个小时,蛋白酶带将逐渐展开.用 0.12% (w/v) Coomassie Brilliant 蓝 R-250 染色 30分钟这些蛋白酶带就能显现出来了.还可以用12.5%的明胶--天然的凝胶（不含SDS）分析细胞溶菌液,确定蛋白酶的数目,同时研究它有几个亚基.
蛋白酶的抑制作用
可以通过将处理过Triton的凝胶放入含有1 mM DTT的保温的缓冲液保温来研究抑制剂对酵母菌蛋白酶活性的影响,确定酵母菌中蛋白酶的种类,DDT中含有下列抑制剂的一种：IA,EDTA,TLCK,8--羟喹啉 ,PMSF（都是1mM）；胃蛋白酶抑制剂A或者亮肽酶素,抗痛素（都是100 μg/ml）.除了胃蛋白酶抑制剂A,IA和PMSF外所有的抑制剂都可以从Sigma买到.凝胶可以用Coomassie Brilliant蓝染色.
问题补充：分析固氮酪蛋白时,在含有固氮酪蛋白的保温1小时寄生虫溶菌液加入相同浓度的蛋白酶抑制剂（IA,TLCK,胃蛋白酶抑制剂,EDTA）.
最适PH的测定
酵母菌蛋白酶活性与PH的关系可以通过在将处理过Triton X—100的凝胶放在PH（在PH3.0—8.5的范围内）不同的缓冲液中保温来测得.缓冲液的配制如下：0.1 M甘氨酸/HCl (pH 3.0),0.1 M乙酸钠/醋酸(pH 4.0 and 5.5),0.1 M磷酸钠(pH 7.0 and 7.6),and 0.1 M Tris–HCl (pH 8.5).所有的保温缓冲液都含有1 mM DTT,因此可用Coomassie Brilliant蓝染色凝胶.对于固氮酪蛋白的分析,可以用寄生虫溶菌液在含有1 mM DTT不同的PH保温1小时.