Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the K_m for NADH is 13 μM, the K_m for CH₂-H₄folate is 0.8 μM, and the turnover number under V_max conditions estimated for the reaction is 1,800 mol of NADH oxidized min⁻¹ (mol of enzyme-bound FAD)⁻¹. NADPH also serves as a reductant, but exhibits a much higher K_m. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

Cloning,Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.     

人脑醛糖还原酶的纯化与性质

联合采用ＤＥＡＥ－纤维素层析、色谱聚焦、ＮＡＤＰ亲和层析与ＳｅｐｈａｄｅｘＧ－１００的凝胶过滤,对人脑醛糖还原酶（ＥＣ１．１．１．２１;ＡＬＲ）进行纯化．现测得该酶的等电点ｐＨ值为５．８５．经聚丙烯酰胺凝胶盘状电泳和Ｗｅｓｔｅｒｎ印迹证实,获得了满意的酶纯度．同葡萄糖,葡糖－６－磷酸与ＮＡＤＰＨ保温后,人脑ＡＬＲ纯品的活性与对照酶组相似,且不被糖酵解途径的一些磷酸化中间产物抑制．苯基硼酸琼脂糖柱层析洗脱谱峰和氢硼化钠还原反应提示,当同葡萄糖保温时,人脑ＡＬＲ（特别是其均一态）可能未被进一步糖基化．在糖尿病并发症和按结构完成药物设计的研究工作中,纯品ＡＬＲ的应用可发挥重要作用     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

The Purification and Properties of a Copper Nitrite Reductase from Haloferax denitrificans

A dissimilatory nitrite reductase from Haloferax denitrificans was purified to apparent electrophoretic homogeneity. The overall purification was 125-fold with about a 1% recovery of activity. The enzyme, which had a molecular mass of 127 kDa, was composed of a 64-kDa subunit as determined by SDS-PAGE. Although maximum activity occurred in the presence of 4 M NaCl, no activity was lost when the enzyme was incubated in the absence of NaCl. The absorption spectrum had maxima at 462, 594, and 682 nm, which disappeared upon reduction with dithionite. Diethyldithiocarbamate (DDC) was inhibitory, and the addition of copper sulfate to DDC-inhibited enzyme partially restored activity. These results suggest this enzyme is a copper-containing nitrite reductase and is the first such nitrite reductase to be described in an Archeon.     

Purification and Properties of 5-Ketogluconate Reductase from Gluconobacter liquefaciens

5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10^?3m). Apparent Km values were 1.8 × 10^?2m for GA, 0.9 × 10^?3m for 5KGA, 1.6 × 10^?5 m for NADP, and 1.1 × 10^?5 m for NADPH₂.     

Purification of streptomycin adenylyltransferase from a recombinant Escherichia coli

Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modification is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni(2+)-IDA-His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The purified fusion protein showed a single band on SDS-PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold purification. The fusion system provided a single step, easy and very rapid purification of SMATase and is suitable for obtaining a highly purified functional protein of interest. The fusion does not affect the functionality of the protein.     

大肠杆菌tRNA~(Leu)的提取、纯化及性质研究

采用高表达大肠杆菌ｔＲＮＡＬｅｕ菌株提取、纯化了亮氨酸等受体转移核糖核酸ｔＲＮＡＬｅｕ１和ｔＲＮＡＬｅｕ２．利用稳态动力学手段研究了ｔＲＮＡＬｅｕ１及脱镁ｔＲＮＡＬｅｕ１在不同稀土离子作用下与纯化亮氨酰－ｔＲＮＡ合成酶的氨酰化作用．ｔＲＮＡＬｅｕ１与亮氨酰－ｔＲＮＡ合成酶的结合及催化效率均受参与稀土离子的影响,表观Ｋｍ值有较明显的变化．结果表明,亮氨酰－ｔＲＮＡ合成酶催化的ｔＲＮＡＬｅｕ１氨酰化反应所需Ｍｇ２＋能够被稀土离子取代,但亲合性能不同．     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Purification of thiamine-binding protein from Escherichia coli

An affinity column coupled with thiamine pyrophosphate quantitatively absorbs the thiamine-binding activity from a partially purified preparation of $\text{Escherichia}\text{coli}$. The thiamine-binding protein can be eluted from the affinity column in high yield by use of 8 M urea-containing buffer. Approximately 90-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogenous.     

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli.

I purified a new dihydrolipoamide dehydrogenase from a lpd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes. The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is different from the lpd gene product. The new dihydrolipoamide dehydrogenase had a molecular weight of 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was expressed in low amounts. It catalyzed the NAD+-dependent reduction of dihydrolipoamide with a maximal activity of 20 mumol/min per mg of protein and exhibited a hyperbolic dependence of catalytic activity on the concentration of both dihydrolipoamide and NAD+. The possible implication of the new dihydrolipoamide in the function of 2-oxo acid dehydrogenase complexes is discussed, as is its relation to binding protein-dependent transport.