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Biomedical subjects

I Silman

Publications and source records attributed to I Silman.

At least 109 records · Page 6Linked to original sources
PubMed1985

Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C.

Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.

Acetylcholinesterase↗
PubMed1985

Butyrylcholinesterase: inhibition by arsenite, fluoride, and other ligands, cooperativity in binding.

Arsenite is a quasi-irreversible inhibitor of human serum butyrylcholinesterase with a dissociation constant of 0.129 mM at pH 7.4, 25 degrees, 0.067 M phosphate, mu = 0.17 M. The inhibition process is second order with a rate constant of 340 M-1 min-1. The first order rate of dissociation, 0.044 min-1, is unaffected by fluoride but is decreased by substrate. The binding of arsenite and fluoride, as determined by the effect of fluoride on the apparent arsenite-enzyme dissociation constant, is highly anticooperative and may be mutually exclusive. The fluoride-enzyme dissociation constant determined from these experiments is 0.90 mM. The binding of a number of other substances, such as dibucaine, is markedly anticooperative with arsenite binding. The binding of some of these substances is positively cooperative with fluoride binding. The effect can be large; procainamide binds 17 times more strongly in the presence of fluoride. Similarly, the mutual binding of benzoylcholine as substrate and fluoride is cooperative, 30-fold, butyrylthiocholine and fluoride, 21-fold, propionylthiocholine and fluoride, 8.3-fold, and acetylthiocholine and fluoride, only 1.8-fold.

Arsenic↗
PubMed1984

Molecular forms of acetylcholinesterase in synaptic and extrasynaptic regions of avian tonic muscle.

Molecular forms of acetylcholinesterase and pseudocholinesterase were analyzed directly in the micro-dissected individual endplates of a slow-tonic chicken muscle. The major form in the endplate is the L2(6.5 S) form, while the collagen-tailed H2c (20 S) form, normally considered to be the synaptic form, is a very minor component, in contrast to its predominance at the chicken fast-twitch fibre endplate. The same is true for pseudocholinesterase at these endplates. Outside the tonic fibre endplates the same forms occur as at the endplates, but at a very much lower concentration. The enzyme at the tonic fibre endplate cannot be attached to the basal lamina by a collagen tail, but appears to have a hydrophobic attachment. Acetylcholinesterase is functional at tonic fibre endplates, but the absence of the collagen-tailed form may account for the lower efficiency of the enzymic removal of acetylcholine there.

Acetylcholinesterase↗
PubMed1984

Characterization of activities and forms of cholinesterases in human primary brain tumors.

The activities and molecular forms of cholinesterases were studied in a collection of primary brain tumors consisting of primarily gliomas and meningiomas, together with samples of forebrain taken postmortem from patients suffering from diseases unrelated to the nervous system. Both types of tumors, as well as normal forebrain, contained substantial amounts of cholinesterase activity and some gliomas contained exceptionally high levels. In both normal forebrain and meningiomas, acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) accounted for almost all the cholinesterase activity, but in almost all gliomas elevated pseudocholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) could be detected. The cholinesterase activity of both normal forebrain and gliomas migrated on sucrose gradients as a major component of 10-11 S together with a minor component of 4-5 S. In meningiomas a light (4.5 S) form was the principal component.

Acetylcholinesterase↗
PubMed1983

A hydrophobic dimer of acetylcholinesterase from Torpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C.

A dimeric form of acetylcholinesterase from the electric organ of Torpedo californica was solubilized by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The solubilized enzyme had a sedimentation coefficient of 7.3S which was not modified by detergents. The high salt-soluble asymmetric forms of acetylcholinesterase were not solubilized by the phospholipase. Our data suggest that the hydrophobic dimer of acetylcholinesterase may be associated with the plasma membrane through a specific interaction involving phosphatidylinositol.

Acetylcholinesterase↗
PubMed1983

The induction by protons of ion channels through lipid bilayer membranes.

The appearance of ion channels was induced in phospholipid bilayers by acidification of the bulk solution on one side Of the bilayer, by addition of HCl, acetic acid or by hydrolytic production of protons using purified acetylcholinesterase. Further acidification below an apparent critical pH range led to restoration of a low conductance state similar to that seen at neutral pH. Such experiments were performed with a heterogeneous soybean lecithin extract, with homogeneous synthetic diphytanoylphosphatidylcholine, and with a mixture of cholesterol and synthetic dioleoylphosphatidylcholine. It is proposed that the physical mechanism for this phenomenon involves fluctuations of lipid order induced by fluctuations in protonation of phospholipid head groups within a critical pH range; these, in turn, create conductive defects in the two-dimensional lattice of the lipid bilayer.

Electric Conductivity↗
PubMed1982

Molecular forms of acetylcholinesterase and pseudocholinesterase in chicken skeletal muscles: their distribution and change with muscular dystrophy.

Chicken muscles offer several significant advantages for the use of cholinesterase as a marker of nerve-muscle interactions. A series of molecular forms of chicken muscle acetylcholinesterase (AChE), and likewise of pseudocholinesterase (psi ChE), has been defined. The form of AChE inside the endplates of fast-twitch muscle is H2c (20 S), with a collagenous tail. The same is true for psi ChE. The changes in these forms in the muscle with embryonic development, with muscle fibre-type composition and under the influence of inherited muscular dystrophy, are described quantitatively.

Acetylcholinesterase↗
PubMed1982

Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.

A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.

Acetylcholinesterase↗
PubMed1982

Comparison of the molecular forms of the cholinesterases in tissues of normal and dystrophic chickens.

The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (psiChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and psiChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or psiChE in a slow-tonic muscle. In the pectoral of PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the pesChE pattern remains abnormal. Muscle psiChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of psiChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or psiChE were found in cardiac or neural tissues from dystrophic chickens.

Acetylcholinesterase↗
PubMed1981

In vivo modulation of the number of muscarinic receptors in rat brain by cholinergic ligands.

Administration of the muscarinic agonist oxotremorine led to a decrease in the number of muscarinic receptors, as determined by specific binding of [3H]quinuclidinyl benzilate ([3H]QNB), in several rat brain regions both during development and at maturity. In contrast, administration of the muscarinic antagonist scopolamine led to an increase in the number of [3H]QNB-binding sites in various brain regions. Scopolamine also prevented the decrease in the number of [3H]QNB-binding sites induced by administration of an organophosphorus drug. The results are compatible with the hypothesis that the number of brain muscarinic receptors, or at least of a sub-class of them, is regulated by their transmitter.

Acetylcholinesterase↗
PubMed1981

Molecular forms of the cholinesterases inside and outside muscle endplates.

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic, were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.

Acetylcholinesterase↗
PubMed1980

Electric eel acetylcholinesterase: a multisubunit enzyme containing a collagen tail.

Molecular forms of acetylcholinesterase (AChE) in fresh electric organ tissue are elongated structures in which a multisubunit head containing the catalytic sites is attached to a fibrous tail. The principal form, 18S AChE, is of MW ca. 1,100,000 and aggregates reversibly at low ionic strength. Trypsin converts it to an 11S globular tetramer devoid of the tail and lacking the capacity to aggregate reversibly in low salt. Amino acid analysis, collagenase and pepsin digestion and immunological techniques were utilized to demonstrate that the fibrous tail of the elongated forms of AChE is a collagen triple helix. The distal portion of the tail contains a region responsible for the capacity for aggregation at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring AChE to the fibrillar matrix of the basal lamina.

Acetylcholinesterase↗
PubMed1980

Fluorescent organophosphates: novel probes for studying aging-induced conformational changes in inhibited acetylcholinesterase and for localization of cholinesterase in nervous tissue.

Aging of acetylcholinesterase (AChE) inhibited by certain organophosphates such as diisopropylfluorophosphate apparently involves dealkylation of the bound organophosphoryl moiety; this renders the inactive enzyme resistant to reactivation by quaternary oximes such as 2-pyridinealdoxime methiodide (2-PAM) which are used in therapy of organophosphate intoxication. The fluorescent pyrenyl organophosphates synthesized in this study were designed to detect putative conformational changes which might explain this resistance. The following inhibitors: 1-pyrenebutyl phosphorodichloride (PBPDC), 1-pyrenebutyl ethylphosphorochloridate (PBEPC), and 1-pyrenebutyl ethylphosphorofluoridate (PBEPF), react specifically with purified electric eel AChE (ki = 10(6)-10(7) M-1 min-1). AChE inhibited by PBEPC and PBEPF was readily reactivated by 2-PAM, while enzyme inhibited PBPDC could not be reactivated. Conjugates were prepared of both PBEPC and PBPDC with AChE, each containing one molecule of florophore per catalytic subunit. Thus two stoichiometric conjugates, PBEP-AChE (non-aged) and POBP-AChE (aged), were obtained. The two complexes exhibited identical absorption spectra, but differed in their steady-state fluorescence spectra. Although the wave-lenths of the excitation and emission spectra were similar, the pyrene fluorescence of the non-aged conjugate was ca. 50% quenched relative to the aged conjugate. Nanosecond fluorescence decay studies revealed two principal lifetime components of pyrene fluorescence. Both were longer for the aged (PBP-AChE) than for the non-aged (PBEP-AChE) conjugate and revealed a ca. 50% lower quantum yield for the non-aged as compared to the aged conjugate. A possible interpretation for these results is that in the aged conjugate the organophosphoryl moiety is less acessible to the external medium. Measurement of quenching of pyrene fluorescence in the aged and non-aged conjugates by the peripheral anionic site ligand propidium also indicated marked conformational differences between the two conjugates, and circular polarization of luminescence measurements revealed that propidium itself induced a substantial conformational change in both conjugates. Fluorescence lifetime measurements revealed that whereas propidium had little effect on the decay parameters for the non-aged conjugate it caused a decrease in lifetime and in relative quantum yield for the aged conjugate. PBEPF virtually eliminated cholinesterase activity in dissociated cord and brain cultures. Fluorescence microscopy reveals fine green fluorescent grains distinctly located throughout many neurons and glia. Labelling is much more pronounced in larger and older neurons. No specific fluorescence could be detected in cultures preincubated with nonfluorescent organophosphates.

Animals↗