Partial purification of glycerophosphate acyltransferase from Escherichia coli.

Glycerophosphate acyltransferase, a membrane-bound enzyme catalyzing the initial step of phospholipid biosynthesis in Escherichia coli, has been extracted with Triton X-100, a nonionic detergent, and purified 20- to 40-fold. This preparation is free from lysophosphatidate acyltransferase. Glycerophosphate acyltransferase is inactive in detergent extracts, but can be reconstituted by the addition of phospholipid. Under such conditions, the enzyme is associated with phospholipid. The sole product of the reaction with acyl coenzyme A as substrate is 1-acyl-sn-glycero-3-phosphate. Furthermore, the enzyme shows a marked preference for saturated fatty acyl conenzyme A, implying that this enzyme is responsible for the predominance of saturated moieties in position 1 of E. coli phospholipids. Acyltransferase from two mutants, plsA and plsB, was partially purified and characterized. Results support the view that plsB is a structural gene for the acyltransferase, but suggest that the plsA gene product is not directly involved in phospholipid biosynthesis.     

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.

The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.     

Hypoxanthine-DNA glycosylase from Escherichia coli. Partial purification and properties

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.     

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12

Four hitherto undescribed endodeoxyribonucleases, temporarily designated A₁, A₂, A₃, and B, have been isolated from E. coli K-12. Each requires Mg⁺⁺ and is not stimulated by ATP or S-adenosylmethionine. A₃ is strongly inhibited by Fe⁺⁺⁺ and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3′-phosphatase. A₁ (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3′-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A₂ (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3′-hydroxyl- and 5′-phosphoryl termini. A₃ (molecular weight approximately 38,000) cleaves fd DNA to form 3′-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A₃. B appears to form 3′-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.     

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.     

Partial purification and properties of diglyceride kinase from Escherichia coli.

Diglyceride kinase (diacylglycerol kinase, E.C. 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.     

Partial purification and characterization of S-adenosylhomocysteine hydrolase isolated from Saccharomyces cerevisiae

S-Adenosylhomocysteine (SAH) hydrolase was purified 25-fold from bakers' yeast by chemical methods and column chromatography. The purified enzyme could readily synthesize SAH from adenosine and homocysteine, but could hydrolyze only negligible amounts of SAH. The purified enzyme showed no activity towards S-adenosylmethionine, methylthioadenosine, or adenosine. Several nucleotides, sulfhydryl compounds, and ribose could not replace adenosine or homocysteine in the reaction mixture. SAH could be hydrolyzed by SAH hydrolase if commercial adenosine deaminase was included in the reaction mixture. Under these conditions l-homocysteine could act as a product inhibitor. A number of compounds structurally similar to adenosine and homocysteine were found to inhibit synthesis of SAH from adenosine and homocysteine. The strongest inhibitors were adenine, adenosine-3'-monophosphate, adenosine-2'-monophosphate, adenosine diphosphate, adenosine triphosphate, and adenosine-5'-monophosphate. The biosynthetic and hydrolytic activity of SAH hydrolase in yeast cell ghosts was similar to the activity of the enzyme in vitro.     

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.     

Partial purification and characterization of an acetylcarnitine hydrolase from bovine epididymal spermatozoa

We previously reported that intact epididymal spermatozoa from bulls and hamsters oxidize [1-14C]acetyl-L-carnitine to 14CO2 at about the same rate as they oxidize [1-14C]acetate. In addition, we showed that acetylcarnitine is hydrolyzed by a hydrolase present in the plasma membrane and that the carnitine moiety does not enter the cell. Here we report the partial purification of the acetylcarnitine hydrolase from bovine spermatozoa and describe some of its properties. The detergent-extracted enzyme was purified by FPLC using an anion-exchange Mono-Q column. The hydrolase activity eluted from the column with the application of 0.22 to 0.30 M NaCl and was separated from acetylcholinesterase activity, which eluted with 0.35 to 0.40 M NaCl. Specific inhibitors of acetylcholinesterase had little effect on acetylcarnitine hydrolase but p-hydroxymercuriphenylsulfonate was a potent inhibitor of the hydrolase. Kinetic studies of the hydrolase yielded a K'm of 6-10 mM for acetylcarnitine and a V'max of 0.16 nmol min-1 mg protein-1. Similar studies with the acetylcholinesterase yielded a K'm for acetylcholine of about 300 microM and a V'max of 165 nmol min-1 mg protein-1. Acetylcarnitine was a poor substrate for the acetylcholinesterase. Several acyl-L-carnitines were tested as substrates for the hydrolase and the preferred substrate was acetylcarnitine. The role of acetylcarnitine hydrolase in the metabolism of acetylcarnitine by epididymal spermatozoa is discussed.     

Partial purification and properties of D-desthiobiotin synthetase from Escherichia coli

d-Desthiobiotin synthetase, an enzyme that catalyzes the synthesis of d-desthiobiotin from dl-7,8-diaminopelargonic acid and HCO(3) (-), was purified 100-fold from cells of a biotin mutant strain of Escherichia coli. Adenosine triphosphate and Mg(2+) were shown, especially in purified extracts, to be obligatory for enzyme activity, although concentrations higher than 5 mm caused severe inhibition of the reaction with unpurified cell-free extracts. Adenosine diphosphate and adenosine monophosphate were shown to inhibit the reaction, but fluoride (up to 50 mm) had no detectable effect. The product of the enzyme reaction was identical to d-desthiobiotin on the basis of biological activity and chromatography. Furthermore, when H(14)CO(3) (-) was used as a substrate, the radioactive product was shown to be (14)C-desthiobiotin labeled exclusively in the ureido carbon.     

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.     

K5裂解酶在大肠杆菌中的表达、纯化及酶学性质分析

K5多糖裂解酶(Elma)能够裂解半合成肝素的底物-K5多糖,裂解产物是半合成法生产低分子量肝素的底物。利用PCR方法扩增elma,构建表达载体pET-28a-Elma,将构建好的质粒转化至大肠杆菌BL21中,以0.2 mmol/L的IPTG在16℃诱导5 h实现了高效表达,SDS-PAGE分析表明Elma表达量可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法和G-75分子筛层析纯化目的蛋白,其纯度大于95%。通过PAGE多糖电泳发现裂解前后的K5多糖分子量有明显的减小。根据Elma裂解产物产生双键从而在232 nm处有吸光度的变化来测Elma的酶活。其最适反应温度为37℃,反应的最适pH值为7.0。底物特异性分析发现Elma除K5多糖外对肝素和透明质酸也有降解作用。     

血小板第四因子在原核中的高效表达、分离纯化及活性测定

目的：构建重组表达质粒pET-32c/PF4,并在原核中进行表达,以探讨其对白血病细胞系（HEL）细胞增殖的作用。方法：通过PCR方法从含有PF4基因的PQE-60/PF4质粒中扩增PF4,用NcoⅠ/HindⅢ双酶切,克隆到原核表达质粒pET-32C中,使之在BL21表达,Ni-Chelating Sepharose亲和柱纯化,用细胞集落形成方法研究重组PF4对HEL的抑制作用。结果：菌株筛选后在大肠杆菌中获得可溶性高效表达,重组PF4占菌体总蛋白的22%,肠激酶酶切除去端啧合部分,获得了高纯度的重组人血小板第四因子（rh-PF4),纯度为95%以上,活性实验发现重组PF4可抑制HEL细胞集落的形成,抑制率为47%,结论：原核表达质粒pET-32C可高效可溶性表达PF4,重组PF4对HEL细胞的增殖有抑制作用。