Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases.

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase-6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. california AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Kinetic investigations into the interactions of aprophen with cholinesterases and a carboxylesterase.

Acetylcholinesterases, butyrylcholinesterases, and carboxylesterases appear to form kinetically a homologous enzyme series with respect to many substrates and inhibitors. The present paper evaluates the interaction of aprophen with acetylcholinesterases, butyrylcholinesterases, and carboxylesterases with respect to protecting the enzyme from organophosphate and carbamate inhibition, accelerating pralidoxime iodide (2-PAM) regeneration of the diisopropylphospho-enzyme, and comparing the inhibition and regeneration kinetics of a soluble mammalian acetylcholinesterase with that of bovine erythrocyte acetylcholinesterase. The irreversible inhibition kinetics of diisopropyl fluorophosphate (DFP) and eserine inhibition of fetal bovine serum acetylcholinesterase were typical of other acetylcholinesterases as indicated by the bimolecular inhibition rate constants, ki, of 7.7 +/- 1.3 X 10(4) M-1 min-1 and 2.9 +/- 1.7 X 10(6) M-1 min-1, respectively. Similarly, the bimolecular regeneration rate constant, kr, for 2-PAM regeneration of the diisopropylphospho-acetylcholinesterase was 14.7 M-1 min-1. The bimolecular rate constants, ki and kr, were not statistically perturbed when the reaction was monitored in the presence of aprophen with the fetal bovine serum acetylcholinesterase. Human serum butyrylcholinesterase was partially protected from DFP inhibition by aprophen with no detectable change in the bimolecular inhibition rate constant, ki. The regeneration of the diisopropylphospho-butyrylcholinesterase by 2-PAM was accelerated in the presence of aprophen by a factor of 2.7 over that of 2-PAM alone (8.4 +/- 2.2 M-1 min-1 to 23.1 +/- 2.6 M-1 min-1 respectively). Neither the inhibition (DFP) nor the regeneration (2-PAM) kinetics observed for the carboxylesterase was perturbed by the presence of aprophen.

Aprophen: a substrate and inhibitor of butyrylcholinesterase and carboxylesterases.

Aprophen, alpha-methyl-alpha-phenylbenzeneacetic acid-2-(diethylamino) ethyl ester, is a potent reversible inhibitor and a poor substrate of human serum butyrylcholinesterase (BuChE). Complex mixed competitive noncompetitive inhibition kinetics were observed; an apparent competitive inhibition constant was estimated to be 3.7 X 10(-7) M. BuChE hydrolysis of aprophen to diphenylpropionic acid and diethylaminoethanol did not appear to follow Michaelis-Menten kinetics. The BuChE turnover number for aprophen was 2.0 X 10(-3) sec-1. Rabbit liver oligomeric and monomeric carboxylesterases (CE) also hydrolyzed aprophen with a similar turnover number that varied from 1.4 X 10(-3) sec-1 to 4.3 X 10(-4) sec-1 respectively. Comparison of the catalytic rate of aprophen hydrolysis with butyrylthiocholine (BTC) and the neutral aromatic substrate, phenylthiobutyrate (phi TB), indicated that BuChE hydrolyzed BTC and phi TB 3.2 X 10(5) and 3.1 X 10(5) times more rapidly than aprophen respectively. Similarly, the CEs also hydrolyzed BTC and phi TB 17.6 and 1.9 X 10(5) times rapidly than aprophen. Acetylcholinesterases from bovine erythrocyte and electric eel were not inhibited by aprophen nor was aprophen hydrolyzed by these enzymes. The hydrolysis and inhibition reactions may best be described by a complex reaction scheme involving multiple binding sites for both the substrate and the inhibitor as well as positive cooperative ligand binding.

Multiple molecular forms of rat brain enkephalinase.

Rat brain enkephalinase has been partially purified by ion exchange chromatography, chromatofocusing, and affinity chromatography on immobilized lectins. Ion exchange chromatography resolved two principle forms of enkephalinase designated A1 and A2. Both enkephalinase A1 and A2 are bound to immobilized lentil lectin while chromatography on immobilized wheat germ lectin resolved each of the principle forms into two subforms, A1, 1, A1, 2, A2, 1, and A2, 2. All four enkephalinase forms have similar, if not identical kinetic properties. The possible implications of multiple molecular forms of enkephalinases are discussed.

A simplified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase.

A simple procedure has been developed for the large scale purification of fetal bovine serum acetylcholinesterase (AChE) (EC 3.1.1.7). The procedure involves two steps: batch adsorption of the AChE from 250 L of serum onto a procainamide affinity Sepharose 4B gel; and analytical procainamide affinity chromatography of the step-1 product. Over 100 mg of AChE was purified in 10 days to apparent homogeneity with this procedure.

Physical and chemical characterization of a horse serum carboxylesterase.

The serine carboxylesterase from horse serum was characterized by amino acid composition, peptide mapping, molecular and subunit weights, and sequencing of the amino acids around the essential serine residue at the active site. A protocol was developed for using reversed-phase high-performance liquid chromatography as the final step to obtain homogeneous preparations of horse serum carboxylesterase. Amounts sufficient for determining the amino acid composition and for peptide maps were obtained from a partially purified starting material which contained approximately 55% carboxylesterase. The amino acid composition, like the subunit weight (70,800 +/- 1400), was similar to the corresponding values reported for other serine carboxylesterases. However, the amino acid sequence of the tryptic digest fragment containing the essential nucleophilic seryl residue differed significantly from the corresponding sequences of other mammalian serine carboxylesterases.

Peptide capillary zone electrophoresis mass spectrometry of recombinant human erythropoietin: an evaluation of the analytical method.

An evaluation of capillary zone electrophoresis-mass spectrometry (CZE-MS) as an analytical methodology for the separation and characterization of complex glycopeptides and nonglycopeptide structures has been performed. The evaluation employed endoproteinase V8 digested recombinant human erythropoietin (rHuEPO) that was further fractionated by reverse phase chromatography. The peptides were subjected to sequence analysis and evaluated by capillary electrophoresis, with or without mass detection, for peptide purity. The peptide mass determined from the sequence was then compared to the mass obtained from CZE-MS. Glycosylation sites and carbohydrate branch patterns were easily determined, site specific microheterogeneity (either O-acetylation of N-acetylneuraminic acids or lactosamine extensions of the carbohydrate chain length) was assessed directly, glycosylation site occupancy was evaluated qualitatively, and nonglycopeptides were resolved and analyzed on-line with ease. Incomplete peptide digestion products were detected and identified by CZE-MS. Protein sequence coverage by CZE-MS was 98.2 percent complete from a single map. Off-line evaluation of peptide purity by CZE greatly aided the interpretation of multiple sequence analysis and, in validating that, the CZE-MS was detecting all peptides present. All off-line CZE and on-line CZE-MS experiments employed a capillary that was dynamically coated with Polybrene in the presence of polyethylene glycol; separations were conducted in 0.67 M formic acid.

Influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis.

The influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis (HPCE) is presented. The major effect of temperature is to shorten the analysis time by decreasing the viscosity, but specific temperature effects on the protein migration behavior were also observed. Myoglobin, under high field (350 V/cm), was essentially temperature stable from 20 to 45 degrees C, but at constant current, a second form of myoglobin could be detected at both 214 and 410 nm. The initial form appeared to correspond to the Fe3+ and the second to the Fe2+ oxidation state of the heme iron. The rate of conversion from Fe3+ to the reduced Fe2+ in myoglobin, under given electrophoretic conditions, followed first-order kinetics with a rate constant at 30 degrees C of 304 s-1. A second protein, alpha-lactalbumin type III, demonstrated a conformational transition that resulted in asymmetric peaks and sigmodial mobility plots versus temperature in the transition region.

Multiple peptide fraction collection by capillary electrophoresis with reinjection analysis.

This papers addresses many of the optimization parameters necessary to convert from high resolution capillary electrophoresis (CE) analytical separation parameters to automated, micropreparative multiple fraction collection using software-controlled, interrupted applied voltage. Optimization of two parameters are crucial: 1) preparative sample loading and 2) the determination of peak collection windows. Factors affecting sample loading volume are discussed, such as capillary inner diameters, sample temperatures and sample injection times. Peak collection windows have been determined experimentally and offer an advantage to windows calculated using a linear mobility relationship, especially for long run times, high current levels, and multiple voltage ramping required for multiple fraction collection. Reinjection analysis of both non-glycopeptides and glycopeptides are examined, and clearly indicate peak mobility can be employed for identifying the collected peptides. Difficulties associated with quantitation of the collected peaks by CE are described and appear to be predominantly associated with sample matrix effects.

Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis.

Multiple sequential fraction collection of peptides and glycopeptides by high-performance capillary electrophoresis (HPCE) under applied voltage has been demonstrated from complex tryptic peptide maps. The collection methodology was adapted from a high-resolution glycopeptide mapping procedure and, as such, requires active temperature control of the sample, electrophoresis vials, and collections vials because the electrophoresis buffer system is higher conductive. Resolution was compromised in the preparative HPCE separation due to heavy sample loading and to reduced voltage. The latter was a requirement for this buffer system in order to control Joule heating at the current levels employed; collections were routinely performed at approximately 1.5 W/m. The collection buffer was optimized by the addition of 12% methanol (v/v), thereby improving collection yields. Tryptic non-glycopeptides were group collected; secondary analysis of the HPCE collections agreed with analytical separations with respect to the number of peptides contained in a given fraction. Sequentially collected peptide fractions were analyzed by Edman sequencing and MALDI mass spectrometry to verify peptide identity and sequence. Consistent peptide sequence or mass measurements were obtained for repeat collections. The isolation of the single pure glycopeptide indicates that unique glycopeptide structures can be collected by HPCE and then analyzed by other methods.