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Biomedical subjects

I Silman

Publications and source records attributed to I Silman.

At least 91 records · Page 5Linked to original sources
PubMed1990

Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata.

In Torpedo electric organ much of the acetylcholinesterase is a 'globular' dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and solubilized by a bacterial phosphatidylinositol-specific phospholipase C. This suggested that selective solubilization with phosphatidylinositol-specific phospholipase C, coupled with immunocytochemistry, might be used to localize G2 acetylcholinesterase in excitable tissues of Torpedo. Cryostat sections of electric organ, electromotor nerve, electric lobe and back muscle from Torpedo ocellata were labelled, using three different antibody preparations to Torpedo acetylcholinesterase, followed by a fluorescent second antibody, before and after exposure to the phospholipase. Sites of innervation on electrocytes and myofibers were labelled selectively, as were motor and electromotor nerves. In all these cases labelling was substantially diminished by prior exposure to the phospholipase. The results support our previous assignment, based on biochemical evidence, for a neuronal and synaptic localization of the G2 acetylcholinesterase in Torpedo. Electric lobe acetylcholinesterase appears insensitive to the phospholipase treatment and lacks certain epitopes present in both electric organ and electromotor nerve enzyme. This suggests that substantial processing of the G2 form occurs concomitantly with its movement from the electric lobe into the electromotor nerve.

Acetylcholinesterase↗
PubMed1990

Torpedo acetylcholinesterase is inactivated by thiol reagents.

A number of sulphydryl reagents inhibit AChE of Torpedo california with pseudo-first-order kinetics, and inhibition can be retarded by quaternary ligands which bind at either the catalytic or peripheral anionic binding sites. Colorimetric determination with one of the inhibitory sulphydryl agents, 5,5'-dithiobis (2-nitrobenzoic acid), reveals the presence of a single thiol group per catalytic subunit; our data thus suggest that inhibition is achieved by reaction with the single free sulphydryl group of Cys231.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Aged and non-aged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin. Preparation and comparison by 31P-NMR spectroscopy.

Homologous pairs of non-aged and aged pyrene-containing phosphoryl conjugates of chymotrypsin were prepared in order to characterize by NMR and optical spectroscopy putative differences in the conformation of non-aged and aged organophosphoryl conjugates of serine hydrolases. Pyrenebutyl-O-P(O)(OC2H5)F and pyrenebutyl-O-P(O)(OC2H5)Cl were used to obtain the non-aged form pyrenebutyl-O-P(O)(OC2H5)-Cht, whereas pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, and pyrenebutyl-O-P(O)(p-nitrophenoxy)2 were used to produce the aged conjugate pyrenebutyl-O-P(O)(O )-Cht. These ligands bind covalently to the active site of serine hydrolases. The absorption spectra of both the non-aged and aged conjugates fitted approximately a 1:1 stoichiometry of bound organophosphate and enzyme in the non-aged and aged conjugates. Pyrenebutyl-O-P(O)(OC2H5)-Cht could be reactivated by pyridine-3-aldoxime methiodide, whereas no reactivation was observed for the similarly treated pyrenebutyl-O-P(O)(O-)-Cht. The 31P-NMR and reactivation data taken together strongly support the hypothesis that the aged form of the OP-Cht conjugate contains a P--O- bond. These results provide a partial interpretation for the known resistance of the aged conjugates of serine hydrolases to reactivation.

Chymotrypsin↗
PubMed1989

Conformational differences between aged and nonaged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin as detected by optical spectroscopy.

Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.

Chymotrypsin↗
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

A membrane-associated dimer of acetylcholinesterase from Xenopus skeletal muscle is solubilized by phosphatidylinositol-specific phospholipase C.

The susceptibility to phosphatidylinositol-specific phospholipase C of the membrane associated acetylcholinesterase (AChE) forms of Xenopus laevis skeletal muscle was examined. This treatment released almost all the detergent-soluble AChE species from muscle homogenates. Sucrose gradient analysis showed that the released acetylcholinesterase form corresponds to a hydrophilic G2 dimer, indicating that this dimer has a glycolipid anchoring domain which contains phosphatidylinositol.

Acetylcholinesterase↗
PubMed1988

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue.

Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14 + 18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.

Acetylcholinesterase↗
PubMed1988

Acetylcholinesterase in Schistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol.

Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.

Acetylcholinesterase↗
PubMed1987

Modes of attachment of acetylcholinesterase to the surface membrane.

Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by post-translational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagen-like fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparin-like proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxyl-terminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.

Acetylcholinesterase↗
PubMed1987

Microtiter assay for acetylcholinesterase.

A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.

Acetylcholinesterase↗
PubMed1987

Removal of covalently bound inositol from Torpedo acetylcholinesterase and mammalian alkaline phosphatases by deamination with nitrous acid. Evidence for a common membrane-anchoring structure.

Our earlier evidence suggested that both acetylcholinesterase and alkaline phosphatase are anchored to the cell surface via covalently-attached phosphatidylinositol [Low, Futerman, Ferguson & Silman (1986) Trends Biochem. Sci. 11, 212-215]. We now present chemical data, based upon a nitrous acid deamination reaction, showing that in both proteins the phosphatidylinositol moiety is attached through a glycosidic linkage to a sugar residue bearing a free amino group.

Acetylcholinesterase↗
PubMed1987

Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adult Torpedo ocellata.

The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G2 form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G2 form in back muscle is sensitive to PIPLC, whereas the G4 tetramer of Torpedo brain is completely resistant.

Acetylcholinesterase↗
PubMed1986

Acetylcholinesterase of Schistosoma mansoni: purification and characterization.

Larval acetylcholinesterase (acetylcholine acetylhydrolase) EC 3.1.3.7 of the trematode Schistosoma mansoni was characterized and purified by affinity chromatography. The enzyme was solubilized from sonicated cercarial tissue and showed a Km value of 1.83 mM and a Vmax value of 102 U/mg protein. It was characterized as a true AChE since it hydrolyses acetylthiocholine more than seven times faster than butyrylthiocholine, and since it was inhibited by high concentrations of substrate. The enzyme was purified by affinity chromatography on a Sepharose column of the inhibitor [N-(6-aminocaproyl-6-aminocaproyl)-m-aminophenyl] trimethyl ammonium. The purified enzyme eluted from the column by decamethonium bromide migrated as a single band of 500 kD on nondenaturing polyacrylamide gel electrophoresis (PAGE), whether stained for proteins or for enzymatic activity. Analysis by SDS-PAGE revealed two major polypeptide bands of 76 kD and 30 kD. By labeling the enzyme with 3H-DFP (di-isopropyl-fluorophosphate), the 30-kD polypeptide was shown to contain the active site of the enzyme, with an additional labeled band of 110 kD also being detected. On the basis of our data we suggest that the principal species of S. mansoni AChE is a tetramer of four subunit polypeptides each of MW ca. 110 kD which are not linked by disulfide bonds, and which are further cleaved into two fragments, one of MW 76,000 and one of MW 30,000, the latter bears the active site.

Acetylcholinesterase↗
PubMed1985

Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C.

The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

Acetylcholinesterase↗
PubMed1985

Identification of covalently bound inositol in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase.

The hydrophobic, membrane-bound form of Torpedo acetylcholinesterase is specifically solubilized by a phosphatidylinositol-specific phospholipase C, suggesting that acetylcholinesterase is bound to the membrane via a direct and specific interaction with phosphatidylinositol (Futerman et al., Biochem. J. (1985) 226, 369-377). Here we demonstrate the presence of covalently bound inositol in the membrane-anchoring domain of purified Torpedo acetylcholinesterase. Upon removal of this domain, levels of inositol are reduced to only 15-20% of those found in the intact enzyme. The results presented strongly support our suggestion that phosphatidylinositol is indeed involved in anchoring acetylcholinesterase to the plasma membrane.

Acetylcholinesterase↗
PubMed1985

Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C.

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

Acetylcholinesterase↗