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M Harel

Publications and source records attributed to M Harel.

54 records · Page 3Linked to original sources
PubMed1991

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

Alkylation↗
PubMed1991

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

Acetylcholine↗
PubMed1991

Gamma-chymotrypsin is a complex of alpha-chymotrypsin with its own autolysis products.

The determination of three separate gamma-chymotrypsin structures at different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [(1989) Biochemistry 28, 7033-7038]. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both gamma-chymotrypsin and alpha-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 A, respectively) to Ser195 O gamma in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes.

Amino Acid Sequence↗
PubMed1988

Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C.

A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.

Acetylcholinesterase↗
PubMed1988

Prediction of three-dimensional structure of Escherichia coli ribosomal RNA.

A model for the tertiary structure of 23S, 16S and 5S ribosomal RNA molecules interacting with three tRNA molecules is presented using the secondary structure models common to E. coli, Z. mays chloroplast, and mammalian mitochondria. This ribosomal RNA model is represented by phosphorus atoms which are separated by 5.9 A in the standard A-form double helix conformation. The accumulated proximity data summarized in Table 1 were used to deduce the most reasonable assembly of helices separated from each other by at least 6.2 A. Straight-line approximation for single strands was adopted to describe the maximum allowed distance between helices. The model of a ribosome binding three tRNA molecules by Nierhaus (1984), the stereochemical model of codon-anticodon interaction by Sundaralingam et al. (1975) and the ribosomal transpeptidation model, forming an alpha-helical nascent polypeptide, by Lim & Spirin (1986), were incorporated in this model. The distribution of chemically modified nucleotides, cross-linked sites, invariant and missing regions in mammalian mitochondrial rRNAs are indicated on the model.

Binding Sites↗
PubMed1988

Crystallization of halophilic malate dehydrogenase from Halobacterium marismortui.

Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group 12 with cell dimensions a = 113.8 A, b = 122.8 A, c = 126.7 A, beta = 98.1 degrees. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.

Halobacterium↗
PubMed1987

Thyrotropin-releasing hormone potently reverses epinephrine-stimulated hyperglycemia in mice.

Intracerebroventricular microinjection of thyrotropin-releasing hormone (TRH) potently blocked the development of, as well as promptly reversed, epinephrine-stimulated hyperglycemia in mice. The central antihyperglycemic effect was dose-related (0.1-10 micrograms), could be reproduced by an intravenous injection of a large dose of the peptide (100 micrograms), was independent of experimental factors such as stress and age, was effective against other hyperglycemic stimuli, and appeared to be unique to TRH, as it could not be mimicked by many other centrally active peptides known to influence glucoregulation in normoglycemic animals. Moreover, the antihyperglycemic effect of TRH appeared to depend on the structural integrity of the peptide molecule but seemed to be unrelated to the peptide's hypophysiotropic actions or to interaction of the peptide with previously characterized TRH receptors, as it could be mimicked by various analogs devoid of thyrotropin- and prolactin-releasing influences or by peptides resembling TRH in amino acid composition but lacking substantial binding affinity to TRH receptors. Furthermore, the effect of TRH to reverse epinephrine-stimulated hyperglycemia appeared to be mediated by combined action of peripheral sympathetic and parasympathetic mechanisms to stimulate insulin release from the pancreas, since only complete blockade of the central autonomic outflow, but not selective perturbation of the sympathetic or parasympathetic outflow, or depletion of pancreatic insulin could substantially attenuate the antihyperglycemic action. Taken together, these results suggest a new physiologic role of TRH as a central glucoregulatory neuropeptide involved in autonomic modulation of insulin secretion and prevention of hyperglycemia.

Animals↗
PubMed1985

Central thyrotropin-releasing hormone elicits systemic hypoglycemia in mice.

Thyrotropin-releasing hormone (TRH), injected into the central nervous system (CNS) in rats, has been shown to elicit systemic hyperglycemia. In the present study, central TRH administration significantly decreased the plasma glucose in mice. The hypoglycemic response could be blocked by pretreatment with the muscarinic cholinergic antagonist, atropine methyl bromide, or the diabetogenic beta-cytotoxin, alloxan, implicating the involvement of the parasympathetic system and insulin-secreting cells in the endocrine pancreas. The role of TRH in the CNS in the autonomic regulation of glucose homeostasis is discussed.

Animals↗
PubMed1985

Systemic hypoglycemia following central injection of endotoxin in mice.

Intracerebroventricular microinjection of endotoxin in mice resulted in powerful hypoglycemia. The effect was reproduced by the biologically active moiety of endotoxin, lipid A, and prevented by coadministration of the polycationic peptide antibiotic polymyxin B (PMB) or by detoxification of endotoxin by means of mild alkaline hydrolysis. Central treatment with PMB also attenuated the hypoglycemic response to systemic administration of endotoxin or lipid A. These results suggest a direct role of the CNS in the mechanism of endotoxin hypoglycemia.

Animals↗
PubMed1984

Thyrotropin-releasing hormone (TRH) improves survival in anaphylactic shock: a central effect mediated by the sympatho-adrenomedullary beta-adrenoceptive system.

Treatment with thyrotropin-releasing hormone (TRH) significantly improved survival following induction of fatal systemic anaphylaxis in mice. The protective effect was mediated centrally since survival was increased by intracerebroventricular (i.c.v.) administration of TRH at doses which had no effect when given systemically (5-25 micrograms). Acid-TRH, a deamidated metabolite of TRH which lacks hypophysiotropic influences, was as effective as TRH when administered i.c.v., but it was inactive following intravenous (i.v.) administration. The protective effect of TRH in anaphylaxis was reversed by treatments which diminished sympathetic outflow to the adrenal medulla, i.e. ganglionic blockade by chlorisondamine chloride or surgical denervation of the adrenal glands. Destruction of sympathetic nerve endings by the catecholamine neurotoxin 6-hydroxydopamine did not alter the response to TRH. Finally, selective blockade of beta-adrenoceptive sites by propranolol diminished the effect of TRH. Blockade of alpha-adrenoceptors by phentolamine or dopaminergic receptors by domperidone did not alter the protective effect of TRH in anaphylaxis. Collectively, these results indicate that the beneficial effect of TRH in anaphylactic shock involves central nervous system actions which are mediated peripherally through interaction of sympatho-adrenomedullary catecholamines with beta-adrenoceptive effectors. The possibility that TRH exerts its protective actions in shock by acting centrally to functionally antagonize the pathophysiologic effects of endogenous opiate peptides (endorphins) will be discussed.

Adrenal Medulla↗
PubMed1983

Endorphins in endotoxin-induced hyperglycemia in mice.

This study assessed the role of endogenous opiate systems in the hyperglycemic response to endotoxin challenge in mice. Blockade of opiate receptors by administration of the opiate antagonists naloxone (1.0 mg/kg) or naltrexone (1.0 or 5.0 mg/kg) significantly lessened to degree of hyperglycemia cause by endotoxin challenge (80 micrograms). Methyl naltrexone, a peripherally acting opiate antagonist, had no demonstrable effect on endotoxin-induced hyperglycemia. Finally, induction of tolerance to morphine prevented the hyperglycemic response to endotoxin challenge. These results suggest a causative role for central nervous system endorphinergic mechanisms in the hyperglycemic response to endotoxin administration. They support the view that centrally acting opiate antagonist, by blocking the brain opiate receptors, can influence metabolic adaptation to endotoxin shock.

Animals↗
PubMed1983

Role of endorphins in endotoxin-induced hyperglycaemia in mice.

The present study assessed the role of endogenous opiate systems in the hyperglycaemic response to challenge with endotoxin in mice. Blockade of opiate receptors by administration of the opiate antagonists naloxone (1.0 mg/kg) or naltrexone (1.0 or 5.0 mg/kg) significantly decreased the degree of hyperglycaemia caused by challenge with endotoxin (80 micrograms). Naltrexone methyl bromide, a peripherally acting opiate antagonist, had no demonstrable effect on the endotoxin-induced hyperglycaemia. Finally, induction of tolerance to morphine prevented the hyperglycaemic response to challenge with endotoxin. These results suggest a causative role for central endorphinergic mechanisms in the hyperglycaemic response to administration of endotoxin. They support the view that the centrally acting opiate antagonist, by blocking the brain opiate receptors, can influence metabolic adaptation to endotoxin-induced shock.

Animals↗
PubMedDate not supplied

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.

Allosteric Regulation↗