酵母tRNA结合蛋白(43kD蛋白)羧端cDNA的克隆及其序列测定

为了研究酵母分子量为 4 3kD的tRNA结合蛋白的基因来源 ,通过溴化氰部分化学裂解此蛋白 ,产生的 18kD肽段经过蛋白质氨端序列测定 ,测得 18kD肽段氨端部分序列为AFTFKK .针对序列AFTFKK设计简并引物 ,利用简并PCR方法和cDNA的 3′末端的快速扩增方法 (3′RACE) ,成功克隆 4 3kD蛋白mRNA的 3′端序列 .DNA序列测定结果表明 ,该序列位于 3 磷酸甘油酸激酶 (PGK1)基因 10 0 3位— 170 0位 ,长度为 6 98bp ,编码 3 磷酸甘油酸激酶羧基端的 180氨基酸残基以及 3′端非翻译区 .结果证实 ,4 3kD蛋白的基因就是PGK1酶的基因     

An NMR analysis of the binding of inhibitors to yeast phosphoglycerate kinase

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.     

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1–100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.     

Conformational changes in yeast phosphoglycerate kinase upon ligand binding: fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues

The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.     

The soluble sema domain of the RON receptor inhibits macrophage-stimulating protein-induced receptor activation

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one alpha- and one beta-chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of approximately 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSP-responsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer.     

An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation.

This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H-NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3-phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5-triphosphate, 4-amino-6-trichloroethenyl-1,3- benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported. Evidence that binding to the general anion site in the 'basic patch' region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.     

Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through hydrophobic cluster analysis and molecular modelling.

A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain. In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken. We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK). This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method. According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides. Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase. These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane. Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.     

Flexibility and folding of phosphoglycerate kinase

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.     

T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product.

The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E-W-S-cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100-fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5'-kinase and a 3'-phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5'-kinase activity, and possibly also 3'-phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms.     

Immobilized glyceraldehyde-3-phosphate dehydrogenase forms a complex with phosphoglycerate kinase

Yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) covalently attached to CNBr-activated Sepharose 4B was shown to be capable of binding soluble yeast phosphoglycerate kinase (PGK) in the course of incubation in the presence of an excess of 1,3-diphosphoglycerate. The association of the matrix-bound and soluble enzymes also occurred if the kinase was added to a reaction mixture in which the immobilized glyceraldehyde-3-phosphate dehydrogenase, NAD, glyceraldehyde-3-phosphate and Pi had been preincubated. Three kinase molecules were bound per a tetramer of the immobilized dehydrogenase and one molecule per a dimer. An immobilized monomer of glyceraldehyde-3-phosphate dehydrogenase was incapable of binding phosphoglycerate kinase. The matrix-bound bienzyme complexes were stable enough to survive extensive washings with a buffer and could be used repeatedly for activity determinations. Experimental evidence is presented to support the conclusion that 1,3-diphosphoglycerate produced by the kinase bound in a complex can dissociate into solution and be utilized by the dehydrogenase free of phosphoglycerate kinase.     

Polypyrimidine tract binding protein 2 stabilizes phosphoglycerate kinase 2 mRNA in murine male germ cells by binding to its 3'UTR

The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3'-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3'-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3'-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.     

Effect of Cibacron Blue F3GA on phosphoglycerate kinase of Lactobacillus plantarum and phosphoglycerate mutase of Leuconostoc dextranicum

Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U.S. 72, 663--667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273--6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2.7.5.3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2,3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA.     

A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.     

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.     

Human granulocyte phosphoglycerate kinase. Purification by double affinity elution and immunological study.

Human phosphoglycerate kinase has been totally purified from leukemic granulocytes by double 'affinity elution' with ATP and 3-phosphoglycerate. This purification procedure allowed to obtain 19 mg of protein, specific activity of which was 400 IU/mg i.e. a 105-fold purification and an overall yield of 47%. Purified enzyme was homogenous when tested by acrylamide sodium dodecyl sulfate electrophoresis and immunodiffusion. Specific antibodies raised in rabbits totally inactivated phosphoglycerate kinase of crude extracts as well as of the purified preparation. The molecular specific activity (i.e. the ratio enzyme activity/immunological reactivity) was identical in leukocytes, platelets, erythrocytes and was identical in these cells to the value found for the totally purified phosphoglycerate kinase.     

Structural Tightening and Interdomain Communication in the Catalytic Cycle of Phosphoglycerate Kinase

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF₄⁻-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain β-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.     

Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae

The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.     

Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The nine resonance peaks observed in the CIDNP spectrum of yeast phosphoglycerate kinase have been assigned tentatively to five residues: histidines-53 and -151, tryptophan-310, and tyrosines-48 and -195. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR.(ABSTRACT TRUNCATED AT 250 WORDS)