地中海诺卡氏菌丙氨酸脱氢酶的纯化和性质

采用硫酸铵分部沉淀、DEAE纤维素-52柱层析和亲和蓝柱层析的方法,分离纯化了地中海诺卡氏菌(Nocardia mediterranei)U-32丙氨酸脱氢酶(ADH),用聚丙烯酰胺凝肢电泳鉴定为单一组份。以聚丙烯酰胺凝胶梯度电泳测得丙氨酸脱氢酶的分子量为228000,SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为38000,表明地中海诺卡氏菌U-32丙氢脱氢酶由六个相同的亚基组成。ADH加氨反应最适pH为8．5,脱氨反应最适pH为11．5,ADH的pH稳定范围在pH 7．5-11．5。脱氨反应的最适温度为50℃。ADH的米氏常数KM为：丙酮酸4．88×10-4mol／L;NH+44．03×10-3Mol／L;NADH 6．02×10-5Mol／L;L—Ala7．45×10-3mol／L;NAD+6．67×10-5mol／L。 Hill作图法求得ADH的Hill系数n为：ADH对丙酮酸、NADH和NAD+的Hill系数都为1;对L—Ala和NH4+的Hill系数n值分别为0．52和0．51,ADH对L—Ala和NH+4有负协同效应,由此初步推测ADH是一个调节酶。     

尖镰孢青霉素V酰化酶的纯化及性质

通过硫酸铵沉淀、硅藻土吸附、DEAD-纤维素离子交换层析和Sephadex G-200凝胶过滤,由尖镰孢(Fusarium oxysporum)FP941培养滤液中得到了聚丙烯酰胺凝胶电泳均一的青霉素V酰化酶。酶作用最适pH为7.0,最适温度为50℃。酶在pH6.0—8.0和42℃以下稳定。酶作用青霉素V的米氏常数Km为4.65×10~(-3)mol/L;苯氧乙酸是酶的竞争性抑制剂,抑制常数Ki为23.87×10~(-3)mol/L;6-氨基青霉烷酸是酶的非竞争性抑制剂,抑制常数Ki为30.01×10~(-3)mol/L。某些金属离子对酶有抑制作用,Fe~(2+)最强,其次是Hg~(2+)和Cu~(2+)。用SDS凝胶电泳测定酶亚基分子量为77600;用分子筛测定自然酶分子量为148000。     

类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶,用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ,亚基分子量为４７ ｋＤ,提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性,对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃,催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８,在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下,可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上,活力无明显下降,冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．     

大肠杆菌青霉素酰化酶的提纯及其性质的研究

大肠杆菌(Escherichia coli) AS 1.70发酵液经有机溶剂处理,硫酸铵分级,再用聚丙烯酰胺垂直板凝胶电泳进行纯化,得到了聚丙烯酰胺凝肢电泳均一的青霉素酰化酶纯品。纯酶作用的最适温度为45—55℃,最适pH为7．0—7．7,在无NIPAB存在下,纯酶在45℃以下稳定,但在55℃保温一小时,酶活力残存33．58％,纯酶在pH5．0—8.0稳定。酶作用于重排酸的米氏常数为3．33×10-2g／ml。Ag+对酶有抑制作用。用聚丙烯酰胺薄层凝胶等电聚焦测定酶的等电点(pI)为6．7—6．8,用SDS凝胶电泳测酶的亚基分子量分别为14300和58900。纯酶具有水解苯甘氨酸甲酯盐酸盐的作用,反应两小时产生12．74mM苯甘氨酸。     

三角酵母菌D-氨基酸氧化酶的纯化和一些基本性质的研究

1．比较了17株霉菌和36株酵母菌的D-氨基酸氧化酶活力,其中以三角酵母的酶活力最高,比较适宜作为研究D-氨基酸氧化酶的材料。 2．用硫酸铵、聚乙二醇(PEG 6000)分部沉淀,Sephadcx G-200、羟基磷灰石柱层析分离纯化了三角酵母D-氨基酸氧化酶,聚丙烯酰胺凝胶电泳为单一组份。 以聚丙烯酰胺凝股梯度浓度电泳测得D-氢基酸氧化酶的分子量为170,000,sDs凝胶电泳测得其亚基分子量为42,000,表明三角酵母D-氨基酸氧化酶由四个相同亚基组或。 3．三角酵母D-氨基酸氧化酶在以D-丙氨酸为底物时,最适pH为8．3,米氏常数(Km)为3．3mM。三角酵母D-氨基酸氧化酶具有较宽的底物专一性,多种D或DL型氨基酸都可以做它的底物。     

产气气杆菌茁霉多糖酶的研究I．酶的提纯和性质

产气气杆菌(Aerobacter aerogenes) 10016的茁霉多糖酶(pullulanase E．C．3．2．1．41)用Sephadex G100凝胶过滤、聚丙烯酰胺垂直板型凝胶电泳进行纯化,得到了聚丙烯酰胺凝胶电泳均一的纯酶。纯酶作用的最适温度为50℃,最适pH为5．3—5．8,耐热性较差,50℃ 4小时后仅存活力10％。纯酶在pH4．3一8．6稳定。酶作用于糯米淀粉的米氏常数Km为2.0×10-2克／毫升。用聚丙烯酰胺薄屡凝胶等电聚焦测定酶的等电点pI为3．8,用SDS凝胶电泳测定酶的分子量为51,000—52,000;此酶是一种糖蛋白;含糖总量为6．5—7．0％;纯酶能专一性的水解茁霉多糖、糯米淀粉,也能分解糊精,而不作用于糖原、纤维二糖以及环状糊精。     

家蚕后部丝腺GPT的分离纯化与动力学性质

本文用丝腺匀浆抽提液经硫酸铵分级沉淀、ＤＥＡＥ－纤维素柱层析和羟基磷灰石柱层析等步骤分离纯化了家蚕后部丝腺谷丙转氨酶（ＥＣ２．６．１．２,ＧＰＴ）,并研究了它的动力学性质。该酶由两种不同亚基组成,分子量分别是５４０００和２１０００;对丙酮酸、α－酮戊二酸、Ｌ－丙氨酸和Ｌ－谷氨酸的Ｋｍ值分别是２．５×１０－４、４．２×１０－４、９．６×１０－３和１２．５×１０－３ｍｏｌ／Ｌ;最适温度５０℃,最适ｐＨ８．５;Ｋ＋、Ｍｇ２＋和Ｃａ２＋等离子对酶有激活作用,Ｎａ＋、Ｍｎ２＋、Ｃｕ２＋和Ｚｎ３＋等离子对酶有抑制作用;酶的活性中心含有巯基或咪唑基。     

创新霉素产生菌吲哚丙酮酸甲基转移酶的研究

在创新霉素产生菌济南游动披线菌的无细胞提取物中检测到吲哚丙酮酸甲基转移酶活性,并进行了分离提取。该酶能利用S-腺苷-L-甲硫氨酸对吲哚丙酮酸进行甲基化,它可能作用于创新霉素中间体的甲基化。经过硫酸铵分部盐析和DEA F一纤维素柱层析,得到了纯化60倍的甲基转移酶,比活0．66mu／ms。酶的最适底物是吲哚丙酮酸,最适pH7．5,对于底物s一腺苷一L一甲硫氨酸和吲哚丙酮酸的米氏常数(KⅢ)分别是4×10-,mol／L和1．8×10-7moI／L。用Sephadcx G-150凝胶过滤测得分子量是55000士5000道尔顿。     

大肠杆菌噬菌体C2的一些物理化学特性

大肠杆菌噬菌体C2是含RNA的微球形噬菌体,直径约22毫微米。提纯的噬菌体在SSC缓冲液中沉降系数为79 S,在260毫微米比吸收值为7．8／毫克／毫升。纸层析分析表明其核酸碱基组成的克分子百分数为：A22．0、U 27．0、G 26．1和C 24．8。其解链温度为5 7．5℃。聚丙烯酰胺凝胶电泳测得RNA分子量为11×106。SDS-聚丙烯酰胺凝胶电泳测得外壳蛋白和A蛋白的分子量分别是13,200和42,000。     

鸭血清胆碱酯酶的纯化及性质研究

首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 K_m=9.8×10^-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同.     

The effects of ruthenium tetraammine compounds on vascular smooth muscle.

The time course of the relaxation effect induced by a single dose (3 x 10(-6) mol/L) of trans-[Ru(NH3)4L(NO)]3+ (L=nic, 4-pic, py, imN, P(OEt)3, SO(3)(2-), NH3, and pz) species and sodium nitroprusside (4 x 10(-9) mol/L) was studied in aortic rings without endothelium and pre-contracted with noradrenaline (1 x 10(-6) mol/L). All the compounds induced a relaxing effect in the aortic rings, but the intensity and time of relaxation were different. Only the species where L=py, 4-pic, and P(OEt)3 were able to induce 100% (99-100%) of the relaxing effect during the assay. trans-[Ru(NH3)4(L)(NO)]3+ (L=SO(3)(2-) and NH3) showed the lowest relaxing effect (36 and 37%, respectively) when compared with the other compounds. Relationship was observed between the time corresponding to half of the maximum relaxation intensity observed and, respectively, k-NO, E0'[Ru(NO)]3+/[Ru(NO)]2+ in trans-[Ru(NH3)4(L)(NO)]3+ species and E0'Ru(III)/Ru(II) in trans-[Ru(NH3)4(L)(H2O)]3+ ions. These relationships strongly suggested that the NO liberation from the reduced nitrosyl complexes was responsible for the observed relaxation.     

Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2)

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an Mr 41,000 in denaturing conditions and an Mr 70,000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized L-valine and L-alpha-aminobutyric acid efficiently, L-isoleucine and L-leucine less efficiently, and did not act on D-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: L-valine, 10.0 mM; NAD+, 0.17 mM; 2-oxoisovalerate, 0.6 mM; and NADH, 0.093 mM. In minimal medium, VDH activity was repressed in the presence of glucose and NH4+, or glycerol and NH4+ or asparagine, and was induced by D- and L-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.     

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.     

林肯链霉菌谷氨酰胺合成酶活力调节的研究

对不同氮源生长条件下林肯链霉菌无细胞粗提液中谷氨酰胺合成酶 (GS)的研究结果表明 ,高浓度NH+4阻遏了GS的生物合成。从不同氮源生长条件下林肯链霉菌中分离纯化了GS ,其性质没有差别。以受腺苷化调节的产气克雷伯氏菌GS作对照 ,林肯链霉菌GS没有明显的氨休克作用 ,经蛇毒磷酸二酯酶处理后 ,其活力没有变化。这些结果都说明林肯链霉菌GS不存在腺苷化共价修饰这一调节方式。反馈抑制作用是林肯链霉菌GS的一种重要的调节方式 ,这种抑制作用是以累积的方式进行的 ,这表明各种抑制剂对GS作用位点不同 ,各种抑制剂对GS的抑制作用是相互独立的。由此推测 ,林肯链霉菌GS是一种变构酶。     

在林可霉素生物合成过程中铵离子调控作用的酶学研究

在FUS-50L发酵罐内,用林可链霉菌发酵生产林可霉素。研究发现,NH4^＋对林可霉素发酵过程具有显著的调控效应：补入硫酸铵前,发酵液中的NH4^＋浓度由3．0mmol／L消耗至1．0mmol／L以下的控制过程非常关键,这样可能使林可霉素合成酶大量合成,同时,解除了NH4^＋对谷氨酰合成酶（GS）的阻遏效应;20～24h,尽可能提高硫酸铵的平均补入速率,但最高NH4^＋的瞬时浓度应控制在19．0mmol／L以下,一方面可缩短生物量的积累时间,另一方面可避免过高浓度的NH4^＋对GS的抑制作用;24h后逐渐降低NH4^＋的平均补入速率,使主代谢流转入次级代谢;在发酵中期和后期,应将最低NH4^＋浓度控制在3．0～4．0mmol／L的范围内,避免铵离子对GS的阻遏效应,GS比活力与林可霉素的产量呈正相关关系。最终建立了动态的硫酸铵补加工艺。     

Study of the catalytic effect of PAR on the luminol-potassium ferricyanide reaction using a flow-injection chemiluminescence method.

We discovered that 4-(2-pyridylazo) resorcinol (PAR) has a strong catalytic effect on luminol-potassium ferricyanide chemiluminescence (CL). Results indicated that the chemiluminescence intensities at maximum light emission were linearly corrected with the concentration of PAR over the range 1.0 x 10(-5)-1.0 x 10(-7) mol/L. A detection limit of 5.7 x 10(-8) mol/L for PAR was achieved. It was found that some metal ions strongly affected this catalytic reaction. Based on this finding, the luminol-potassium ferricyanide-PAR reaction was developed for the determination of metal ions. The detection limits (S/N = 3) for Ni2+, Cr3+, Zn2+, Co2+ and Mn2+ were determined to be 1.0 x 10(-9) mol/L, 5.0 x 10(-9) mol/L, 5.0 x 10(-8) mol/L, 1.0 x 10(-9) mol/L and 1.0 x 10(-8) mol/L, respectively. In addition, the relative standard deviation values for these metal ion assays were in the range 0.82-2.72% (n = 6).     

Valine dehydrogenase from Streptomyces fradiae: purification and properties

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

Voltage-dependent Vmax blockade in Na+-dependent action potentials after beta 1- and H2-receptor stimulation in mammalian ventricular myocardium

In isolated papillary muscles of guinea pigs, the influence of isoproterenol, histamine, theophylline, and phenylephrine on the maximal rate of rise (Vmax) of Na+-dependent action potentials and on isometric contractile force was studied under rested state conditions. Isoproterenol (1 x 10(-7) mol/L), histamine (2 x 10(-5) mol/L), and theophylline (2 x 10(-3) mol/L) shifted the voltage dependence of Vmax into the hyperpolarizing direction and, consequently, led to a voltage-dependent Vmax blockade. The alpha-adrenoceptor agonist phenylephrine, on the other hand, proved to be ineffective in depressing Vmax. The beta-receptor blocker pindolol (4 x 10(-6) mol/L) or the H2-receptor blocker cimetidine (4 x 10(-5) mol/L) abolished the inhibitory effects of isoproterenol and histamine, respectively, and caused Vmax to return to the initial control value. A concentration-response relationship analysis at -65 mV revealed that isoproterenol exerted only a weak inhibitory effect on Vmax compared with its positive inotropic action. The IC50 value of the former effect amounted to approximately 5 x 10(-6) mol/L, but the EC50 value of the latter effect was 4 x 10(-8) mol/L. It is, therefore, concluded that, in physiologically relevant concentrations, beta-adrenergic agonists are unlikely to significantly modulate Na+-dependent excitability even in partially depolarized myocardium.     

Indirect determination of pyruvic acid by capillary electrophoresis with amperometric detection

A method of indirectly measuring pyruvic acid (PA) by capillary electrophoresis with amperometric detection is proposed for the first time. It is based on the oximation reaction between PA and hydroxylamine (NH(2)OH), and the quantification of PA was performed by direct and sensitive amperometric detection of excessive NH(2)OH after the oximation reaction. This method displayed a good sensitivity, and the detection limits of NH(2)OH and PA are 1.76 x 10(-7) and 3.88 x 10(-7)mol/L, respectively at S/N=3. The linear relationship between the peak current and PA concentration is exhibited over the range from 4 x 10(-6) to 1 x 10(-4)mol/L. This method has been applied to determine PA in rat plasma with satisfactory results.