Terrone L. Rosenberry

Large scale isolation of human erythrocyte membranes by high volume molecular filtration

A molecular filtration procedure for preparing large quantities of human erythrocyte ghost membranes is presented. Hemolysate ghost membranes are rapidly cycled in the retentate channel of the filtration apparatus, while hemoglobin is removed as it passes through Pellicon filters into the filtrate channel. Several-liter quantities of washed packed erythrocytes can be processed in a few hours with this system, and the filtration procedure does not appear to alter intact erythrocyte or ghost membranes. Intact erythrocytes in isotonic solution can be circulated through the retentate channel for 16 h with only 3% hemolysis and with preservation of their original morphology in scanning electron microscopy. Ghost membranes isolated by the procedure are virtually identical in morphology, polypeptide composition and acetylcholinesterase content to membranes isolated by conventional centrifugation techniques.

Subunit heterogeneity of acetylcholinesterase

Abstract Several different preparations of purified 11 S acetylcholinesterase have been examined for structural heterogeneity. While no contaminant protein was observed in any of the preparations, minor isozymic forms with catalytic activity were observed in addition to the major component both in polyacrylamide gel electrophoresis and in isoelectric focusing. Major differences in the relative composition of the disulfide-reduced polypeptides among the preparations were found by gel electrophoresis in sodium dodecyl sulfate. Several characteristics of these differences strongly suggest that they derive from a proteolytic fragmentation of a single subunit species. In particular, the apparent fragmentation in the crude enzyme solution is inhibited by benzethonium chloride, an inhibitor of proteolysis which also prevents the conversion of 18, 14, and 8 S acetylcholinesterase species to the 11 S form in fresh electric tissue extracts. No significant differences in the enzyme specific activity are observed among the preparations, an observation which indicates that fully active native enzyme molecules are composed of subunits which are heterogeneous with respect to discrete points of polypeptide cleavage.

Identification of discrete disulfide-linked oligomers which distinguish 18 S from 14 S acetylcholinesterase.

Abstract Acetylcholinesterase (EC 3.1.1.7) purified by affinity chromatography from 1.0 m ionic strength extracts of electric organ from the eel Electrophorus electricus consists of a mixture of 18 and 14 S enzyme forms. When examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate without exposure to disulfide reducing agents, these purified preparations show two major high molecular weight bands (>300,000), labeled oligomers A and B, in addition to a major band corresponding to catalytic subunit dimers (150,000 M r ). All these major bands reflect intersubunit disulfide bonding. The 18 and 14 S forms in purified preparations were separated by extensive sucrose gradient centrifugation. Gel analyses of the isolated 18 and 14 S pools indicated that the larger oligomer A derives from the 18 S pool, while oligomer B is found primarily in the 14 S pool. These observations support a previous model for 18 S acetylcholinesterase ( T. L. Rosenberry and J. M. Richardson (1977) Biochemistry , in press ) which considers this molecule to consist of one oligomer A unit, composed of three pairs of catalytic subunits disulfide-bonded to a collagen-like tail structure, and three catalytic subunit dimers. Proteolytic cleavage of the tail structure in the 18 S form can occur to release an 11 S enzyme tetramer containing a residual tail fragment and to leave a 14 S form. We propose this 14 S form to consist of one oligomer B unit, composed of two pairs of catalytic subunits disulfide-bonded to the remaining tail structure, and two catalytic subunit dimers.

Electrophysiological studies of thymectomized and nonthymectomized acetylcholine receptor-immunized animal models of myasthenia gravis

Abstract White New Zealand rabbits were immunized with acetylcholine receptor (AChR) purified from the electric organs of either the Electrophorous electricus (eel) or Torpedo californica (torpedo). Approximately 4 days after a second injection of 100 μg AChR in complete Freunds adjuvant, the immunized animals developed weakness of the peripheral muscles which was aggravated by repetitive activity and was partially alleviated by anticholinesterases such as edrophonium or pyridostigmine. Electromyograms taken from the anterior tibialis muscle demonstrated a decrement of the compound action potentials at a stimulation frequency of 5 Hz which was reversed by the intravenous administration of 1 mg pyridostigmine. If the animals were not treated with anticholinesterases they died within a couple of days. Biopsies of the intercostal muscle were done under Nembutal anesthesia, and intracellular recordings revealed subliminal end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) of greatly diminished amplitudes. At some neuromuscular junctions no MEPPs were detected, and in other junctions MEPPs were seen but no EPP was evoked upon nerve stimulation. Muscles from two immunized rabbits showed spontaneous muscle action potentials occurring at 1 to 2 Hz. Serum from the rabbits immunized with torpedo or eel AChR, when applied to in vitro preparations of the eel electroplaque or frog cutaneous pectoris muscle, caused a decrease in postjunctional membrane sensitivity to carbamylcholine and reduced the MEPP and EPP amplitudes even after heat inactivation of the serum complement (56°C for 30 min). In addition to a decrease in chemosensitivity, an enhancement of receptor desensitization occurred in the neuromuscular junctions of serum-treated frog muscles. Thymectomies on adult rabbits prior to the immunization had the effect of delaying the onset of myasthenia in two of six rabbits, and in three of six rabbits only mild myasthenia occurred. The blood antibody titers to AChR were similar in both the thymectomized and nonthymectomized rabbits.

Modification of electroplax excitability by veratridine

Veratridine influences membrane-potential changes arising both from the action potential and from the application of external cholinergic agonists in the isolated monocellular electroplax preparation. The action potential shows a long depolarizing after-potential in the presence of veratridine. The effects of various pharmacological agents and of external ion changes on this after-potential are similar to those reported for other nerve and muscle fibers and are consistent with the view that veratridine acts chiefly to increase the Na+ conductance. Membrane depolarizations by cholinergic agonists are inhibited by veratridine at pH 7 but strikingly amplified at pH 9. The former effect appears to involve interaction with the cholinergic receptor at the surface of the membrane, while the latter potentiation parallels the increase in the spike after-potential at pH 9 and presumably arises from a Na+ conductance increase. Veratridine appears to interact with the component involved in the Na+ conductance in the interior membrane phase. The possible localization of this component in both the conducting and synaptic membrane is discussed.

Functional identity of catalytic subunits of acetylcholinesterase

11 S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the electric eel Electrophorus electricus essentially consists of four catalytic subunits which appear to be identical structurally but to be assembled with slight asymmetry. During isolation and storage of the enzyme, proteolysis cleaves a portion of the subunits into major fragments containing the active site and minor fragments containing no active sites without change in the enzyme molecular weight. A previous report (Gentinetta, R. and Brodbeck, U. (1976) Biochim. Biophys. Acta 438 437--448) indicated that the intact and the fragmented subunits reacted with diisopropylfluorophosphate at different rates and that the reaction rate in the presence of excess phosphorylating agent was not strictly first order. Those findings could not be reproduced in this report. Intact and fragmented subunits were observed to react at the same rate with diisopropylfluorophosphate. In addition, the overall reaction kinetics both of 11 S and 18 S plus 14 S acetylcholinesterase were found to be strictly first order in the presence of an excess of diisopropylfluorophosphate throughout the course of reaction. These results are consistent with several previous reports that only one type of active site can be detected in acetylcholinesterase. The proteolysis which fragments a portion of the catalytic subunit has no apparent effect on the catalytic properties of the enzyme.

Inactivation of Electrophorus electricus acetylcholinesterase by benzenemethane sulfonylfluoride

Abstract Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation.

Vedatridine enhancement of agonist-induced synaptic membrane depolarization in electroplax

Abstract Veratridine in low concentrations (20 μ M ) and at high pH (pH 9) acts as a synergist for carbamylcholine-induced depolarizations in the electroplax of electric eel. This potentiation is not sensitive to tetrodotoxin, but is significantly reduced by d-tubocurarine. Veratridine alone does not depolarize this preparation at the concentration used (20 μ M ). The increased carbamylcholine depolarization arising in the presence of veratridine does not simply sum with the carbamylcholine depolarization; the fractional contribution of veratridine to the total depolarization decreases as the carbamylcholine concentration is increased, and at 50 μM carbamylcholine no significant difference is apparent between groups with and without veratridine. Depolarization with increased external K + , unlike carbamylcholine depolarization, is not potentiated by veratridine.