Mycobacterial adenosine kinase is not a typical adenosine kinase

Adenosine kinase (AK) is only found in eukaryotes. Recently, a Mycobacterium tuberculosis (MTub) protein exhibiting greater sequence similarity to ribokinases (RK) was identified as AK. We have expressed AKs from MTub, human and Chinese hamster (CH) cells in Escherichia coli and also AK from human and MTub in AK-deficient CH cells. While both E. coli and CH cells expressing mammalian AKs efficiently metabolized various adenosine analogs, those expressing MTub-AK were completely inactive. The AK activity of the MTub protein was very low (50-fold lower than E. coli RK) and it was not stimulated by phosphate or inhibited by several AK inhibitors. These results raise questions over MTub protein’s true function and whether it functions as AK in cells.     

Role of adenosine kinase in the control of Streptomyces differentiations: Loss of adenosine kinase suppresses sporulation and actinorhodin biosynthesis while inducing hyperproduction of undecylprodigiosin in Streptomyces lividans

Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.     

Expression and characterization of recombinant human cytochrome c in E. coli

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we expressed human cytochrome c in E. coli and purified it to homogeneity with a yield of 10–15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. On the basis of our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide evidence that human cytochrome c expressed in E. coli possesses a similar function and structure to that of the horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.     

木薯ftsZ基因分离及在原核生物中功能初步鉴定

ftsZ基因是控制细胞分裂的关键基因,其蛋白能够在分裂位点形成一个环状结构而影响细胞分裂.为了研究木薯质体分裂与木薯淀粉品质形成的关系,根据木薯基因组数据库上的预测序列,设计引物,从木薯基因组中分离了与质体分裂相关的ftsZ家族3个新基因(ftsZ1,ftsZ2,ftsZ3).分别将它们与荧光蛋白基因(GFP)融合,构建了3个原核表达载体pET-fisZ1-GFP、pET-fisZ2-GFP、pET-fisZ3-GFP,并转化大肠杆菌BL21(DE3).通过荧光显微镜观察菌体的表型和分裂,初步鉴定了木薯质体分裂相关基因ftsZ家族对细胞分裂的作用.结果显示:尽管木薯与大肠杆菌的亲缘关系较远,ftsZ基因的同源性较低,但是两者表现出相似的功能,木薯ftsZ基因的表达能严重影响大肠杆菌细胞分裂.这一结果为进一步研究木薯ftsZ家族基因的功能奠定了基础.     

鸡大肠杆菌病病原的分离鉴定和部分生物学特性研究

无菌采取某鸡场送检的疑似大肠杆菌病的病、死鸡的心血、肝、脾、肾,进行病原菌的分离、生化鉴定、动物实验和药敏试验。生化鉴定结果显示引起该鸡场鸡只发病的病原为大肠杆菌,并且该菌对小白鼠和鸡有较强的致病力。药物敏感性试验结果显示,该菌对先锋V、头孢呋肟、头孢噻肟敏感,对庆大霉素和氧氟沙星不敏感,而链霉素、新诺明、红霉素、青霉素、氨苄西林、四环素对它完全没有抑制效果。结果表明:该鸡场出现了耐药的致病性大肠杆菌,在以后的肉鸡生产中应慎用抗生素,在使用药物治疗大肠杆菌病时,应根据药敏试验结果,选择敏感药物,且应注意交替用药,按疗程投药。     

Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-kappa B1 p105

Cancer osaka thyroid (COT), a human MAP 3 K, is essential for lipopolysaccharide activation of the Erk MAPK cascade in macrophages. COT 30--467 is insoluble, whereas low levels of COT 30--397 can be expressed, but this protein is unstable. However, both COT 30--467 and COT 30--397 are expressed in a soluble and stable form when produced in complex with the C-terminal half of p105. The k(cat) of COT 30--397 is reduced approximately 47--fold in the COT 30--467/p105 Delta N complex. COT prefers Mn(2+) to Mg(2+) as the ATP metal cofactor, exhibiting an unusually high ATP K(m) in the presence of Mg(2+). When using Mn(2+) as the cofactor, the ATP K(m) is reduced to a level typical of most kinases. In contrast, the binding affinity of COT for its other substrate MEK is cofactor independent. Our results using purified proteins indicate that p105 binding improves COT solubility and stability while down-regulating kinase activity, consistent with cellular data showing that p105 functions as an inhibitor of COT.     

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamadaet al., 1979).By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP^2– and MgADP^–) and (b) the uncomplexed nucleotide substrates (ADP^3– and AMP^2–) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15–25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of AMP.The syntheses are described as a set of peptides corresponding to residues 31–45, 20–45, 5–45, and 1–45, and a set of peptides corresponding to residues 178–192, 178–194, and 172–194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: MgATP/ATP and MgADP/ADP are quantitatively presented in terms of their intrinsic dissociation constants (K_d) and values ofN (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1–44) obtained from rabbit muscle myokinase (Kubyet al., 1984) and with the native enzyme (Hamadaet al., 1979). In addition, the values ofN andK_d are given for the second set of synthetic peptides to the fluorescent ligands AMP and ADP as well as for the peptide fragments MT-XII(172–194) and CB-VI(126–194) (Kuby et al., 1984) and, in turn, compared with the native enzyme.A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., theK _i for AMP and the value of obtained for the native enzyme) (Hamada and Kuby, 1978), and theK'_d measured for Cr³⁺ and the synthetic peptide I_1–45 (Fryet al., 1985b).Paper XVII of this series is Kubyet al. (1983).     

Identification and biochemical characterization of a eukaryotic-type serine/threonine kinase and its cognate phosphatase in Streptococcus pyogenes: their biological functions and substrate identification

A eukaryotic-type signaling system in group A Streptococcus (GAS) was identified and characterized. This system comprises primarily the products of two co-transcribed genes, a eukaryotic-type Ser/Thr kinase (SP-STK) and phosphatase (SP-STP) and their endogenous substrate histone-like protein (SP-HLP). Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). The site on the substrate for reversible phosphorylation by these enzymes was found to be only the threonine residue. Using specific antibodies generated against these proteins, SP-STK was found to be membrane-associated with its N-terminal kinase domain facing the cytoplasm and its C-terminal repeat domain outside the membrane and cell-wall associated. Further, SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. Three isogenic mutants, lacking either the entire SP-STK, or one of its two domains, were found displaying distinct pleiotropic effects on growth, colony morphology, cell division/septation, surface protein/virulence factor expression, bacterial ability to adhere to and invade human pharyngeal cells, and resist phagocytosis by human neutrophils. In addition to these properties, the ability of these three proteins to modulate the expression of the major virulence factors, the M protein and the capsule, indicates that these proteins are structurally and functionally distinct from the kinases and phosphatases described in other microorganisms and play a key role in GAS pathogenesis.     

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl     

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Sequence‐specific interactions of minor groove binders with restriction fragments of cDNAs for H tau 40 protein and MAP kinase 2. A qualitative and quantitative footprinting study

A series of DNA minor groove binders comprising netropsin, distamycin, the bisquaternary ammonium heterocycles SN 6999 and SN 6570, cis‐diammine platinum(II)‐bridged bis‐netropsin, cis‐diammine platinum(II)‐bridged bis‐distamycin and bis‐glycine‐linked bis‐distamycin were investigated for sequence‐specific interactions. The oligonucleotides used were the 154 base pair HindIII–RsaI restriction fragment of cDNA of h tau 40 protein and the 113 base pair NcoI–PvuII restriction fragment of cDNA of MAP kinase 2. Both proteins are believed to be involved in the pathology of Alzheimer's disease. For all these ligands, binding sites were localised at positions 1134–1139 (5′AATCTT3′), 1152–1156 (5′ATATT3′) and 1178–1194 (5′TTTCAATCTTTTTATTT3′) for the former and 720–726 (5′TATTCTT3′), 751–771 (5′AATTGTATAATAAATTTAAAA3′) and 781–785 (5′TATTT3′) for the latter. The AT‐preference of ligand binding was obvious and footprint titration experiments were applied to estimate binding constants (K_a) for each individual binding site mentioned above. The binding strength decreases in the order netropsin > distamycin > SN 6999 ≈ SN 6570>platinum‐bridged netropsin or distamycin≈bis‐glycine‐bridged distamycin and was found independently of the binding sites examined. GC‐base pairs interspersed in short AT‐tracts reduced the K_a‐values by as much as two orders of magnitudes. The dependence of extended bidentate as well as of monodentate binding of netropsin and distamycin derivatives on the length of AT‐stretches has been discussed. Copyright © 1999 John Wiley & Sons, Ltd.     

p185胞外区及其亚区在大肠埃希菌中的表达及与单抗的结合特性分析

目的 旨在获得p185胞外区及其亚区基因在原核系统中表达的多肽,并分析其与单抗结合特性.方法 以pGEX-4T-1为载体,克隆p185胞外区及其亚区基因,并在原核系统中表达,经变性、复性后,以亲和层析法获得纯化的多肽.进一步通过ELISA方法分析了这些多肽与抗p185单抗A18的结合特性.结果 研究获得原核系统表达的p185胞外区及其亚区多肽,GST-E3和GST-E34与单抗A18具有特异结合活性.结论 原核系统表达的p185胞外区及其亚区多肽是可行的,初步确定为与单抗结合的亚区多肽,为进一步研究单抗识别的表位及其抑瘤机制奠定基础.     

Comparative analysis of malate synthase G from Mycobacterium tuberculosis and E. coli: role of ionic interaction in modulation of structural and functional properties

Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persistence through the upregulation of glyoxylate shunt. The malate synthase (MS), an important enzyme of the shunt belongs to the G isoform and expressed predominantly as monomer. Here we did a comparative unfolding studies of two homologous MS from Mycobacterium tuberculosis (MtbMS) and Escherichia coli (ecMS) using various biophysical techniques. Despite having high sequence identities, they show different structural, stability and functional properties. The study suggests that the differences in the stability and unfolding of the two enzymes are by virtue of differential electrostatic modulation unique to their respective molecular assembly.     

The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2α belongs to the CMGC family of the eukaryotic protein kinases. CK2 is an important survival and stability factor in eukaryotic cells: its catalytic activity is elevated in a wide variety of tumors while its down-regulation can lead to apoptosis. Thus, CK2 is a valuable target for drug development and for chemical biology approaches of cell biological research, and small organic inhibitors addressing CK2 are of considerable interest. We describe here the complex structure between a C-terminal deletion mutant of human CK2α and the ATP-competitive inhibitor emodin (1,3,8-trihydroxy-6-methylanthraquinone, International Union of Pure and Applied Chemistry name: 1,3,8-trihydroxy-6-methylanthracene-9,10-dione) and compare it with a previously published complex structure of emodin and maize CK2α. With a resolution of 1.5 Å, the human CK2α/emodin structure has a much better resolution than its maize counterpart (2.6 Å). Even more important, in spite of a sequence identity of more than 77% between human and maize CK2α, the two structures deviate significantly in the orientation, in which emodin is trapped by the enzyme, and in the local conformations around the ligand binding site: maize CK2α shows its largest adaptations in the ATP-binding loop, whereas human CK2α shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite different in this respect.     

A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk,one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.