The effect of organic cryosolvents on actin structure: studies by small angle X-ray scattering

Small-angle X-ray scattering was used to probe the structure of actin in the presence of cryosolvents: 1,2-propanediol, glycerol, or a mixture of both solvents. In media devoid of polymerizing salts, a radius of gyration of 23 Å is measured, as expected from the literature. In the presence of 1,2-propanediol alone, the scattering pattern begins to exhibit the characteristic slope of elongated objects with a non-negligible thickness, such as actin filaments polymerized in 40 mM KCl and 1 mM MgCl₂. However, only short fragments (radius of gyration 40 Å) are generated. We infer that in a medium of low ionic strength containing 15% 1,2-propanediol, actin assumes a structure closer to that of filamentous actin. 1,2-propanediol apparently induces nucleation of oligomers, as with polymerizing salts, but no propagation occurs. Glycerol and/or propanediol induce no alteration in the structure of individual salt-polymerized actin filaments. Aggregation occurs with propanediol, even in the presence of glycerol. Glycerol alone has no such effect. No shortening is detected within the scale covered, with either solvent, although 1,2-propanediol is known to shorten actin filaments. We suggest that in the absence of salts, 1,2-propanediol induces a conformational change in monomeric actin that is necessary for nucleation. This could correlate with a conformational change of actin protomers within microfilaments observed in the presence of 1,2-propanediol by other authors using different techniques.Abbreviations SAXS small-angle X-ray scattering - G-actin globular monomeric actin - F-actin filamentous polymerized actinCorrespondence to: E. Pajot-Augy     

一株以甘油为底物产二羟丙酮(DHA)微生物菌种的筛选及鉴定

甘油为微生物可利用的理想碳源, 从自然界筛选出21株以甘油为唯一碳源产二羟丙酮(DHA)的菌株, 经初步发酵测定发酵液中DHA含量, 其中菌株6-8 DHA产量最高达6.4 g/L。对其进行常规生理生化鉴定实验, 并结合16S rDNA基因分析, 比对结果表明, 菌株6-8与Acinetobacter sp. 相似性最高, 达99.7%, 在细菌分类学上属于假单胞菌目莫拉菌科不动杆菌属。将其命名为Acinetobacter sp.6-8。     

Purification of triosephosphate isomerase and isolation of its gene from the mosquito Culex tarsalis

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5′-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5′ flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.     

一株以甘油为底物产二羟丙酮(DHA)微生物菌种的筛选及鉴定

甘油为微生物可利用的理想碳源,从自然界筛选出21株以甘油为唯一碳源产二羟丙酮（DHA）的菌株,经初步发酵测定发酵液中DHA含量,其中菌株6-8DHA产量最高达6.4g／L。对其进行常规生理生化鉴定实验,并结合16S rDNA基因分析,比对结果表明,菌株6-8与Acinetobacter sp.相似性最高,达99．7％,在细菌分类学上属于假单胞茵目莫拉茵科不动杆菌属。将其命名为Acinetobactersp.6-8。     

Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40–160 eV). The results established the order of dimer stability as TWT > T75S > Q64E ∼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies.     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.     

酵母细胞对高渗环境的适应与胞内甘油累积

甘油是包括酿酒酵母在内的许多种酵母细胞中的主要相容性溶质。为适应在高渗环境下的生存,酵母细胞将在胞内累积甘油。胞内甘油累积的增加可由甘油合成的增强,甘油利用的减弱,细胞膜通透性下降导致的胞内甘油流失的减少以及从环境中吸取更多的甘油而产生。本文综述了酵母细胞对环境渗透压变化的信号传导,高渗诱导的基因表达,环境渗透压升高时酵母细胞内甘油的累积以及甘油合成的限速步骤。     

Different catalytic properties of two highly homologous triosephosphate isomerase monomers

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.     

Antibodies to triosephosphate isomerase in patients with neuropsychiatric lupus

Sera from patients with neuropsychiatric lupus (NP lupus) were screened for antibodies to mouse choroid plexus cell line ECPC-4 by Western blotting. A 29-kDa protein band detected in NP lupus sera was identified as triosephosphate isomerase (TPI). Using Western blotting with TPI, TPI was confirmed as the reactive molecule in sera (6 of 14 samples) and in cerebrospinal fluids (1 of 2 samples) of patients with NP lupus. Enzyme-linked immunosorbent assay with TPI showed that the serum anti-TPI antibody index of 89.8 (SD, 70.1) in NP lupus group was significantly higher than in systemic lupus erythematosus without NP manifestations, 34.6 (29.6); scleroderma, 38.2 (39.9); polymyositis/dermatomyositis, 42.1 (51.5); and control, 31.7 (27.4) groups (p<0.02). Sensitivity, specificity, and positive and negative likelihood ratios of serum anti-TPI antibody index for NP lupus were 42.9%, 94.7%, 8.1%, and 0.6%, respectively. These results suggest that anti-TPI antibodies are closely associated with NP lupus.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

Hominoid triosephosphate isomerase: Characterization of the major cell proliferation specific isozyme

Summary Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35^S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [³⁵S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains an blocked NH₂-terminus and length heterogenity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

蜜蜂TPI基因克隆与生物信息学预测

利用电子克隆方法获得蜜蜂(Apis mellifera)磷酸甘油醛异构酶(triosephosphate isomerase,TPI)基因,并采用生物信息学方法对该基因编码蛋白从等电点、疏水性/亲水性、二级结构等进行了预测,以及试验验证,结果表明蜜蜂TPI基因全长为1 768 bp,具有完整的开放阅读框架(ORF),并得到了试验证实.     

腐生型眼虫Astasia longa两种TIM同工酶cDNA的克隆和分析

利用 3′ RACE和 5′ RACE技术 ,从腐生型眼虫长变胞藻 (Astasialonga)克隆了磷酸丙糖异构酶(TIM)的两个同工酶cDNA全序列。分析表明 :它们分别编码定位于细胞质的胞质型TIM (cTIM)和定位于质体的质体型TIM (pTIM) ;后者的N端具有引导该酶定位到质体中去的典型“前导序列”。根据这些事实我们推测腐生型眼虫A .longa质体中可能存在功能性的TIM ,并进一步认为该质体可能不只是一般意义上的“叶绿体退化的残迹” ,而仍是一种至少有TIM参与其代谢活动的功能性细胞器     

1, 2-propanediol from the Pacific Ocean fish Ctenochaetus striatus

(±) 1, 2-propanediol has been isolated from the fish Ctenochaetus striatus. Obtained a first time as the free alcohol with a high yield, it has been found in smaller amount and esterified, in specimens of a different location. The possible origin of this compound is discussed.     

Structural considerations for the rational design of selective anti-trypanosomal agents: the role of the aromatic clusters at the interface of triosephosphate isomerase dimer

Seven benzothiazoles were successfully docked into the interface of both human and trypanosomal triosephosphate isomerases, and the binding free energies of each complex were calculated using the program AutoDock. Structural and energetical analysis of the complexes showed that large benzothiazoles could form more stable complexes with trypanosomal triosephosphate isomerase than with human triosephosphate isomerase. Thus, we hypothesize that the distribution of the residues forming the aromatic clusters at the enzyme's interface and the size of the inhibitors might play a crucial role in the selective inhibition of TcTIM. Following the findings here presented, it is possible to better determine the structural elements involved in the origin of the selectivity at the trypanosomal triosephosphate isomerase interface, and to enable efficient anti-trypanosomal drug design strategies.     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。     

The relation between the assimilation of methanol and glycerol in yeasts

Fifteen yeast strains of the genera Candida, Lodderomyces, Endomycopsis, Saccharomyces, Hansenula, Pichia and Torulopsis were investigated with respect to their ability to grow on methanol, glycerol and glucose as sole carbon and energy source. Eight of them can grow on both methanol and glycerol.Methanol is assimilated via triosephosphate (dihydroxyacetone) pathway. The dihydroxyacetone kinase is a key enzyme in methanol metabolism.The assimilation of glycerol can take place in bacteria via a phosphorylative or/and oxidative pathways. In general, the phosphorylative pathway is found in eucaryotes. In the present paper it is shown that in yeasts, which can utilize methanol and glycerol, too, glycerol is assimilated via an oxidative pathway, Dihydroxyacetone is a central intermediate in the assimilation of methanol as well as glycerol. It is metabolized by means of the dihydroxyacetone kinase.The enzyme formed during growth of Candida methylica on methanol does not differ from that of Candida valida H 122 after growing on glycerol as far as the regulatory properties are concerned.