Grosfeld H

General

Full name : Grosfeld Haim

First name : Haim

Mail : Dept. of Biol. Chemistry, Israel Inst. for BioI. Res.,P.O.B. 19, 70450 Ness-Ziona

Zip Code :

City :

Country : Israel

Email :

Phone : (972) 8 381525

Fax : (972) 8 401094

Website :

Directory :

References (13)

Title : Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability - Clery-Barraud_2002_Eur.J.Biochem_269_4297
Author(s) : Clery-Barraud C , Ordentlich A , Grosfeld H , Shafferman A , Masson P
Ref : European Journal of Biochemistry , 269 :4297 , 2002
Abstract : The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
ESTHER : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedSearch : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedID: 12199708

Title : Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives - Grosfeld_1996_Mol.Pharmacol_50_639
Author(s) : Grosfeld H , Barak D , Ordentlich A , Velan B , Shafferman A
Ref : Molecular Pharmacology , 50 :639 , 1996
Abstract : Diethylphosphoryl conjugates of human acetylcholinesterase (AChE) and selected mutants, carrying amino acid replacements at the active center and at the peripheral anionic site, were subjected to reactivation with the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1'-pyridinium),propane dibromide (TMB-4(Trimedoxime)) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2 - oxapropane dichloride (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of Trp86, the anionic subsite in the active center, lowered the affinity of the free enzyme toward all three reactivators, but in the corresponding diethylphosphoryl conjugate, only affinity toward TMB-4(Trimedoxime) was affected. Replacement of other constituents of the hydrophobic subsite (Tyr337, Phe338) had no major effect on either affinity to the free enzymes or rates of reactivation. Substitution of residues of the acyl pocket (Phe295, Phe297) lowered the affinities toward reactivators except for the 20-fold increase in affinity of F295A toward HI-6. Replacement of the acidic residues in the active center (Glu202, Glu450) affected mainly the rates of nucleophilic displacement of the phosphoryl moiety. The effect of substituting residues constituting the peripheral anionic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyridinium chloride and HI-6, mainly the nucleophilic reaction rate constants were affected, whereas for TMB-4(Trimedoxime), the affinities of the phosphorylated enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for only some of the observed effects on the reactivation rates suggests that the binding modes of oxime to the phosphorylated and nonphosphorylated enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.
ESTHER : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedSearch : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedID: 8794905

Title : Recombinant human acetylcholinesterase - Enzyme engineering -
Author(s) : Shafferman A , Velan B , Barak D , Kronman C , Ordentlich A , Flashner Y , Leitner M , Segal Y , Grosfeld H , Stein D , Ariel N
Ref : Medical Defense Bioscience Review , 3 :1097 , 1993
PubMedID:

Title : Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding - Shafferman_1992_J.Biol.Chem_267_17640
Author(s) : Shafferman A , Kronman C , Flashner Y , Leitner M , Grosfeld H , Ordentlich A , Gozes Y , Cohen S , Ariel N , Barak D , Harel M , Silman I , Sussman JL , Velan B
Ref : Journal of Biological Chemistry , 267 :17640 , 1992
Abstract : Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
ESTHER : Shafferman_1992_J.Biol.Chem_267_17640
PubMedSearch : Shafferman_1992_J.Biol.Chem_267_17640
PubMedID: 1517212

Title : Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center - Shafferman_1992_EMBO.J_11_3561
Author(s) : Shafferman A , Velan B , Ordentlich A , Kronman C , Grosfeld H , Leitner M , Flashner Y , Cohen S , Barak D , Ariel N
Ref : EMBO Journal , 11 :3561 , 1992
Abstract : Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
ESTHER : Shafferman_1992_EMBO.J_11_3561
PubMedSearch : Shafferman_1992_EMBO.J_11_3561
PubMedID: 1396557

Title : Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit - Kronman_1992_Gene_121_295
Author(s) : Kronman C , Velan B , Gozes Y , Leitner M , Flashner Y , Lazar A , Marcus D , Sery T , Papier Y , Grosfeld H , Cohen S , Shafferman A
Ref : Gene , 121 :295 , 1992
Abstract : To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
ESTHER : Kronman_1992_Gene_121_295
PubMedSearch : Kronman_1992_Gene_121_295
PubMedID: 1446827

Title : The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant - Velan_1991_J.Biol.Chem_266_23977
Author(s) : Velan B , Grosfeld H , Kronman C , Leitner M , Gozes Y , Lazar A , Flashner Y , Marcus D , Cohen S , Shafferman A
Ref : Journal of Biological Chemistry , 266 :23977 , 1991
Abstract : Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.
ESTHER : Velan_1991_J.Biol.Chem_266_23977
PubMedSearch : Velan_1991_J.Biol.Chem_266_23977
PubMedID: 1748670

Title : Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme - Velan_1991_Cell.Mol.Neurobiol_11_143
Author(s) : Velan B , Kronman C , Grosfeld H , Leitner M , Gozes Y , Flashner Y , Sery T , Cohen S , Ben-Aziz R , Seidman S , Shafferman A , Soreq H
Ref : Cellular Molecular Neurobiology , 11 :143 , 1991
Abstract : 1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.
ESTHER : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedSearch : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedID: 1849451

Title : Serologically defined linear epitopes in the E2 envelope glycoprotein of Semliki Forest virus - Ariel_1990_Arch.Virol_113_99
Author(s) : Ariel N , Lehrer S , Elhanaty E , Sabo T , Brodt P , Lachmi B , Katz D , Levin R , Grosfeld H , Velan B , et al.
Ref : Arch Virol , 113 :99 , 1990
Abstract : A set of 41 overlapping peptides, representing the complete sequence of SFV-E2 protein were synthesized and analyzed in the ELISA test against murine anti-SFV sera. No single peptide was recognized by all antisera. Eight peptides were found to be highly reactive with hyperimmune anti-SFV sera. Six out of the eight peptide sequences coincide with the most hydrophilic regions of SFV-E2. Out of these, four peptides (amino acid positions 16-35, 61-80, 166-185, 286-305) that contain the least number of alphavirus conserved residues were selected. This panel constitutes the minimal number of peptides necessary and sufficient for specific recognition of hyperimmune mouse anti-SFV sera.
ESTHER : Ariel_1990_Arch.Virol_113_99
PubMedSearch : Ariel_1990_Arch.Virol_113_99
PubMedID: 1696808

Title : Cloning, expression and biological activity of a new variant of human interferon alpha identified in virus induced lymphoblastoid cells - Cohen_1985_Dev.Biol.Stand_60_111
Author(s) : Cohen S , Velan B , Grosfeld H , Shalita Z , Leitner M , Shafferman A
Ref : Developmental Biology Stand , 60 :111 , 1985
Abstract : A synthetic oligonucleotide complementary to a highly conserved sequence in the IFN-alpha gene family, was used to screen a Namalva cDNA library. Among the cDNA clones having typical IFN-alpha traits, one was distinct from previously characterized IFN-alpha cDNAs. E. coli cells carrying this recombinant cDNA plasmid express an alpha-interferon activity. The sequence of this IFN-cDNA is extremely homologous (99.5%) to that of the IFN-alpha J gene and is designated IFN-alpha J1. Several E. coli trp expression plasmids were constructed for efficient transcription and translation of the mature IFN-alpha J1. The maximal level of expression (5 X 10(3) molecules/cells) was obtained from plasmid pJ1-4. A synthetic consensus translation initiation sequence coupled to the trp p/o region (in pJ1-5) proved to be 10 times less effective in promoting metIFN production in bacteria, than the in-vitro mutated trpL initiation sequence carried on pJ1-4. The bacterial IFN-alpha J1 was purified (to over 90% purity) to a specific activity of 1.3 X 10(8) units/mg. The antiviral activity of the purified IFN-alpha J1 was compared with other highly purified IFN-alpha species (bacterial IFN alpha A and alpha C, leukocyte IFN-alpha 1, leukocyte IFN mixture and Namalva IFN preparation) on a large panel of mammalian cell cultures. IFN-alpha J1 exhibits a distinct antiviral activity.
ESTHER : Cohen_1985_Dev.Biol.Stand_60_111
PubMedSearch : Cohen_1985_Dev.Biol.Stand_60_111
PubMedID: 2995168

Title : Cloning of a bovine interferon-alpha gene subfamily and comparisons between genetically engineered and leukocyte bovine interferons - Velan_1985_Dev.Biol.Stand_60_355
Author(s) : Velan B , Cohen S , Grosfeld H , Shalita Z , Shafferman A
Ref : Developmental Biology Stand , 60 :355 , 1985
Abstract : Bovine peripheral leukocytes were virally induced for interferon production, and an acid stable, SDS stable, antiviral activity was detected in the preparation. This bovine interferon (BoIFN) was tested for its ability to induce an antiviral state in various mammalian cells and was found to be specific to cells from bovine origin. The BoIFN cross reacts with antibodies against human IFN-alpha but these antibodies do not neutralize the bovine IFN activity. Leukocyte BoIFN exhibits polymorphism upon Affi-Gel Blue chromatography and SDS-PAGE (16k and 24K). The virally induced leukocytes produce a 13S mRNA which upon translation in oocytes yields an active IFN molecule. Bovine genomic library was constructed and screened for BoIFN-alpha sequences, using human IFN-alpha probes. From the clones isolated, five were found to represent distinct genes. Sequence analysis indicate that these genes are closely related (94% homology). One of these genes was expressed in E. coli under the control of trp promoter operator. The physicochemical and biological properties of the bacterial BoIFN-alpha product resemble those of a subpopulation of natural BoIFN.
ESTHER : Velan_1985_Dev.Biol.Stand_60_355
PubMedSearch : Velan_1985_Dev.Biol.Stand_60_355
PubMedID: 3899795

Title : Mutations not altering the symmetrical sequences in the trp operator yield a constitutive phenotype - Grosfeld_1984_Mol.Gen.Genet_195_358
Author(s) : Grosfeld H , Cohen S , Velan B , Shalita Z , Shafferman A
Ref : Molecular & General Genetics , 195 :358 , 1984
Abstract : An E. coli trp promoter operator mutant was constructed, having two base pair alterations at position -4 and -1 relative to the transcription initiation site (+1). Expression of chloramphenicol acetyltransferase gene under this trp promoter operator suggests that it is almost fully constitutive. This trp Oc in vitro derived mutant differs from previously isolated Oc mutants in that its twofold symmetry sequence is identical to that of the wild type trp operator. The base substitution in the operator does not affect the functionality of the trp promoter. The trp Oc promoter DNA fragment is engineered so that it can be manipulated conveniently for efficient expression of various genes in E. coli.
ESTHER : Grosfeld_1984_Mol.Gen.Genet_195_358
PubMedSearch : Grosfeld_1984_Mol.Gen.Genet_195_358
PubMedID: 6092859

Title : Membrane associated cytoplasmic mRNA in Artemia salina\; functional and physical changes during development - Grosfeld_1977_Nucleic.Acids.Res_4_2109
Author(s) : Grosfeld H , Soreq H , Littauer UZ
Ref : Nucleic Acids Research , 4 :2109 , 1977
Abstract : The physical and functional properties of the mRNA population from developing embryos of the brine shrimp Artemia salina were characterized. About 20% of the total poly(A)-rich mRNA in these embryos appears to be specifically associated with the membrane fraction throughout early development, and physically differs markedly from the free cytoplasmic mRNA. The membrane-associated mRNA fraction consists of two well-defined populations of molecular weight of 5.2x10(5) and 3.6x10(5), whose relative amount changes during the various stages of embryo development. The size of the poly(A) tail at the 3'-end of the mRNA molecules, as estimated by processive phosphorolysis, was found to consist of 180 and 210 adenosine residues for the two respective mRNA species. The in vitro translation products of the membrane-bound mRNA molecules are apparently similar to those of the free mRNA molecules.
ESTHER : Grosfeld_1977_Nucleic.Acids.Res_4_2109
PubMedSearch : Grosfeld_1977_Nucleic.Acids.Res_4_2109
PubMedID: 909766