豇豆几丁质酶N端序列测定及与其它植物的比较

通过自动 Edman降解程序 ,测定了经诱导、纯化的豇豆几丁质酶 N端 1 0个氨基酸的序列 ,并将该序列与其它植物几丁质酶 N端相应部分的氨基酸序列进行了比较分析。结果表明 ,该豇豆 ( Vigna sesquipedalis)几丁质酶 N端 1 0个氨基酸的序列为 EQCGSQAGGA,与 类几丁质酶同一部分同感序列同源性高达 1 0 0 % ;而与 、 及 类几丁质酶的相应序列均无同源性。结合考虑此酶的等电点 ( 8.3)及分子量 ( 33k D) ,可推测该豇豆几丁质酶属于 类几丁质酶。其 N端序列的高度保守性提示 ,该段序列可作为 类几丁质酶的一段主要特征序列 ,并可据其合成核酸探针 ,以分离、克隆其它 类几丁质酶编码基因。     

产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

从产气肠杆菌 (Enterobacteraerogenes)突变株EAM Z1中分离出一种具有较高转移酶活性的尿苷磷酸化酶 (UPase)。经测定这种Upase的分子量为 1 2 .8× 1 0 4,亚基分子量为 4 .3×1 0 4,由 3个同型亚基组成。N端氨基酸序列为 :MRMVDLIATKRDGGE。等电点为 4 .46。对尿苷的Km为 0 .2 9mmol L。酶反应的最适pH为 7.8,最适温度为 50℃。该酶能磷酸化尿苷、胸苷、5 氟尿苷、2′ 脱氧 5 氟尿苷及尿嘧啶 β D 阿拉伯呋喃糖 ,且具有较高的转移酶活性 ,能将尿苷和 5 氟尿嘧啶转化成 5 氟尿苷 (一种抗癌药物的中间体 ) ,其转化率为 47%。该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的。     

GL—7ACA酰化酶（C130）β亚基N端氨基酸残基的突变及功能

戊二酰 7 氨基头孢烷酸酰化酶 (即GL 7ACA酰化酶 ,EC .3.5 .1.11)的催化中心通常在 β亚基N端的第一个氨基酸 ,底物亲和标记的研究亦显示N端存在着结合靶点 ,因而该区域的结构可能与酶的功能密切相关。对C130 β亚基N端的 2～ 8位氨基酸残基分别进行了肽段置换和定点突变研究。将N端前 8位肽段置换为来源于Arthrobacterviscosus的青霉素G酰化酶 (PAC)的对应序列后 ,C130酰化酶活力丧失 ;而置换为来源于E .coli的青霉素G酰化酶 (PGA)的对应序列后 ,酰化酶活力仍然保留 ,但Km 值从 0 .44× 10 -3 mol·L-1增大为 0 .5 5× 10 -3mol·L-1,kcat值由 4.92s-1降低为 1.6 4s-1。另对C130 β亚基N端 2～ 4位氨基酸残基作了单点突变 :第 4位的Trp为可能的底物类似物结合位点 ,被变为Tyr后 ,它对底物GL 7ACA的结合能力略为减弱 ,kcat则降低为 2 .2 9s-1;而变为Leu后 ,Km 为 0 .34× 10 -3 mol·L-1,kcat为 3.15s-1;第 3位的Ser变为Met、Ala及Cys后 ,随着Km值逐渐降低 ,kcat也有所降低 ,而S3 M、S3 A突变体的kcat/Km 值比野生型的分别增加了 2 2 .3%和 39.3% ;将活性中心Ser(β1)邻位的Asn(β2 )变为Gln后 ,C130酶活大幅度下降 ,kcat减为 0 .47s-1。上述结果表明 ,C130 β亚基N端的前几个氨基酸残基均可对酶的功能     

光合细菌Chromatium vinosum可溶性氢酶的FTIR谱的研究

光合细菌Chromatium vinosum含有一种可溶性氢酶和一种膜结合态氢酶。氧化态可溶性氢酶在红外光谱区（1860－2140cm^-1）有四个特征吸收峰（2103．7,2086．2,2054．8和1962．5cm^-1）。其中1962．5cm^－1处吸收带的位置与已知的NiFe一氢酶活性中心－CO基团所产生的吸收带位置相近;另外三条吸收带的位置与已知的NiFe一氢酶活性中心－CN基团所产生的吸收带的位置相近。以2,6－二氯酚靛酚（DPIP）氧化可溶性氢酶时,四条吸收谱带的位置基本上没有发生变化。可溶性氢酶被Na2S2O4充分还原时,－CO基团的吸收带移至1946．8cm^-1,而－CN基团的三条吸收带中的两条分别移至2076．8cm^-1和2093．1cm^-1处,另一条则消失了。还原态可溶性氢酶与CO反应后,其红外光谱显示七条吸收带,在－CO基团红外光谱区和－CN基团红外光谱区各产生了两条新的吸收带。研究表明,Cuinosum可溶性氢酶的活性中心的结构类似于其它已知的NiFe－氢酶,但与活性中心金属原子相连的可能包括三个－CN基团和一个－CO基团,结合可溶性氢酶的FPR谱特征,推测C.vinosum可溶性氢酶活性中心的结构可能为Ni(CN)Fe(CN)2(CO).     

假单胞杆菌D-海因酶的纯化及酶学性质

D 海因酶是工业上生产D 型氨基酸的关键酶 ,用热变性 ,硫酸铵沉淀及SepharoseQfastflow ,Phenyl Sepharosefastflow ,Superose 1 2等柱层析步骤从Pseudomonas 2 2 62菌体中分离纯化了该酶 ,纯化倍数约为 60 ,活力回收约为 1 6%。该酶为同源二聚体 ,分子量约为 1 0 9kD ,亚基分子量约为 53 7kD ,反应最适pH为 8 0 ,最适温度为 70℃ ,在pH6.0～ 1 0 0和温度 60℃以下稳定 ,该酶对巯基试剂敏感 ,大多数二价金属离子如镁、锰离子等能促使酶活提高 ,但高浓度锌离子能抑制酶活 ,以二氢尿嘧啶为底物的米氏常数Km =2 .5× 1 0 - 2 mol L。该酶的N末端1 0个氨基酸残基依次为MDKLIKNGTI     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

重组人白细胞介素6部分生化物理学性质的鉴定

采用工程菌 E.coli DH5 α(p IB- h IL- 6 ) ,经过发酵培养、表达和包含体的分离纯化等步骤 ,获得了 N端缺失 5个氨基酸的 rh IL- 6纯品。对其理化生物学性质进行分析。结果表明 ,纯品 N-端氨基酸序列为 MEDSKD… ;在 2 76 nm波长处有一个特异的蛋白吸收峰 ;分子量为 2 1 487.2 ;裂解的肽段分布在理论值的肽段分布范围之内 ;p I为 6 .1 ;能与兔抗人 IL- 6抗体特异性结合 ;比活性达2 .0 5× 1 0 8IU/mg。     

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。     

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。     

N-氨甲酰水解酶的纯化及性质

从Burkholderiacepecianjut1分离纯化N氨甲酰D氨基酸水解酶(NDase)。实验表明,该酶亚基35KD,最适温度为52℃,最适pH为7.2左右。以N氨甲酰D苯丙氨酸作底物,其米氏常数Km为10.22mmol/L,最大反应速度Vmax为0.27mmol/(L·min)。实验表明二价金属离子对酶活有重要影响。     

Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.     

Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum.

The redox behaviour of the Ni(III)/Ni(II) transition in hydrogenase from Chromatium vinosum is described and compared with the redox behaviour of the nickel ion in the F420-nonreducing hydrogenase from Methanobacterium thermoautotrophicum. Analogous to the situation in the oxidised hydrogenase of Desulfovibrio gigas (Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), the C. vinosum enzyme can also exist in two forms: the 'unready' form (EPR characteristics of Ni(III): gx,y,z = 2.32, 2.24, 2.01) and the 'ready' form (EPR characteristics Ni(III): gx,y,z = 2.34, 2.16, 2.01). Like in the oxidised enzyme of M. thermoautotrophicum the Ni(III)/Ni(II) transition for the unready form titrated completely reversible (both at pH 6.0 and pH 8.0). In contrast, the reversibility of the Ni(III)/Ni(II) transition in the ready enzyme was strongly dependent on pH and temperature. At pH 6.0 and 2 degrees C reduction of Ni(III) in ready enzyme was completely irreversible, whereas at pH 8.0 and 30 degrees C Ni(III) in both ready and unready enzyme titrated with E0' = -115 mV (n = 1). Hampered redox equilibration between the ready enzyme and the mediating dyes is interpreted in terms of an obstruction of the electron transfer from nickel at the active site to the artificial electron acceptors in solution. The origin of this obstruction might be related to possible changes in the protein structure induced by the activation process. The E0'-value of the Ni(III)/Ni(II) equilibrium was pH sensitive (-60 mV/delta pH) indicating that reduction of nickel is coupled to a protonation. A similar pH-dependence was observed for the titration of the spin-spin interaction of Ni(III) and a special form of the [3Fe-4S]+ cluster (E0' = +150 mV, pH 8.0, 30 degrees C). Redox equilibration of this coupling was extremely sensitive to pH and temperature. The uncoupled [3Fe-4S]+ cluster titrated pH-independently with E0' = -10 mV (pH 8.0, 30 degrees C).     

Characterization of the iron-sulfur cluster N7 (N1c) in the subunit NuoG of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX(27)C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated Delta N1b, Delta N1c, Delta N4, and Delta N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, Delta N1b, and Delta N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in Delta N4 and Delta N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.     

菜心和水稻绿叶中不同等电点的乙醇酸氧化酶

The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%～20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.     

Purification,substrate range,and metal center of AtzC: the N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism

N-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp. strain ADP, catalyzes the stoichiometric hydrolysis of N-isopropylammelide to cyanuric acid and isopropylamine. The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli, where the expressed enzyme comprised 36% of the soluble protein. AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography. It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000. The K(m) and k(cat) values for AtzC with N-isopropylammelide were 406 micro M and 13.3 s(-1), respectively. AtzC hydrolyzed other N-substituted amino dihydroxy-s-triazines, and those with linear N-alkyl groups had higher k(cat) values than those with branched alkyl groups. Native AtzC contained 0.50 eq of Zn per subunit. The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts. Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Delta epsilon = 84 M(-1) cm(-1)) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g((x)) = 5.18, g((y)) = 3.93, and g((z)) = 2.24. Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g((x)) = 5.11, g((y)) = 4.02, and g((z)) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW. Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s-triazine herbicides.     

车前草粗多糖提取及抗氧化试验

采取热水浸提-醇沉法探讨了车前草中可溶性粗多糖的提取工艺,研究了提取温度、提取时间、提取次数与料液比4因素对多糖提取的影响。单因素试验发现,在80℃,120 min,料液比1:20,提取2次条件下多糖提取较好。正交试验优化提取方案为90 min、80℃和料液比1:25,在此条件下,多糖提取率可以达到29.88 mg.g-1。同时还研究了体外条件下对.OH和O2-.自由基的清除效果。研究发现,多糖与.OH自由基清除呈良好的线性量效关系(y=5.48x+31.34,r=0.9823),与O2-.清除也呈现良好的线性量效关系(y=4.334x+34.134,r=0.9979)。IC.OH-50%为114.57 mg.L-1,ICO2--50%为172.85 mg.L-1,试验结果充分表明车前草多糖在体外有较强的自由基清除能力。     

Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (CXXCXXXCX27C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX(27)C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g(z, y, x) = 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g( parallel, perpendicular) = 2.045, approximately 1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX(27)C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster.     

Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16

The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

树木生长势与松墨天牛种群密度及松材线虫发病程度关系

对不同生长势林区内的松墨天牛种群密度和松材线虫发病程度的研究表明,树木生长势（x）分别与松墨天牛种群密度（y）、松材线虫发病程度（z）呈负相关,其线性回归方程分别为：y=1793.771-16404.47x;z=31.80989-241.9274x,相关系数分别为r=-0.8319和r=-0.8770。而松墨天牛种群密度（y）与松材线虫发病程度（z）呈正相关,其线性回归方程为：y＝－407.0611＋70.51478z;相关系数为r=0.9864。     

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.