Kinetic, equilibrium and spectroscopic studies on cation association at the active center of acetylcholinesterase: topographic distinction between trimethyl and trimethylammonium sites.

This study examines the importance of electrostatic interactions on ligand association at the active center of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). The active-center serine was covalently modified with the dimensionally equivalent isosteric beta-(trimethylammonium)ethyl and 3,3-dimethylbutyl methylphosphonofluoridates. Reactivation of the 3,3-dimethylbutyl methylphosphono-conjugate by the bisquaternary mono-oxime HI-6, after accounting for the capacity for spontaneous reactivation, proceeded at a rate that was 20-fold greater than that for the cationic conjugate. Decidium, a fluorescent bisquaternary ligand that binds with its trimethylammonium moiety within the active center, exhibited affinity for the 3,3-dimethylbutyl conjugate that was within 2-fold that for the native enzyme, but 100-fold greater than for the cationic conjugate. Whereas association of n-alkyl mono- and bisquaternary ligands with the uncharged conjugate was virtually unaltered with respect to the native enzyme, the affinities of edrophonium, phenyltrimethylammonium and N-methylacridinium were reduced 100-fold for the uncharged conjugate relative to native enzyme. These results indicate that the orientations of the 3,3-dimethylbutyl and beta-(trimethylammonium)ethyl moieties with respect to the surface of the enzyme are not equivalent, that modification of the active center does not preclude cation association of active-center-selective ligands, and that aromatic cations associate at an anionic locus which is unique from that at which decidium and the n-alkyl mono- and bisquaternary cations associate. As such, the results point to the presence of a heterogeneity of cation binding sites within a circumscribed distance from the modified serine, and do not sustain the view proposed by Hasan et al. (J. Biol. Chem. 255 (1980) 3898-3904; 256, (1981) 7781-7785) that electrostatic interactions at the active center are subordinate to steric constraints imposed by a dimensionally restricted trimethyl site.

Behavior during hippocampal microinfusions: anticholinesterase-induced locomotor activation.

Awake, unrestrained rats received direct bilateral infusions of the cholinesterase inhibitor, echothiophate, into the dentate gyrus during a 40-min experimental session in a holeboard/activity chamber. Additional animals were similarly tested during intrahippocampal infusions of a novel cholinesterase inhibitor, NBD-AP-MPF. The computerized behavioral pattern monitor recorded the animals' locomotor activity, holepoking, and rearing in a manner that permitted the reconstruction and analysis of their sequential patterns of movement. Both echothiophate and NBD-AP-MPF infusions produced dose-dependent increases in locomotor activity that were accompanied by increased holepoking and rearing. The hyperactivity induced by anticholinesterase infusions was qualitatively similar to the atropine-sensitive effects of infusions of the cholinergic agonist, carbachol, as previously reported. Dye infusions revealed that spread of the infusate was restricted to the dentate gyrus of the anterodorsal hippocampal formation. The locomotor activation caused by the echothiophate infusions into the dentate gyrus were interpreted as indicative of a role for the cholinergic septo-hippocampal pathway in the release of motor responding.

The Tufts partnership for managed care education.

The authors describe the formation and the academic activities of the Tufts Managed Care Institute, a collaborative venture of Tufts University School of Medicine and Tufts Health Plan, an independent-practice-association (IPA)-model health maintenance organization (HMO). In 1994, the dean of the medical school and the CEO of the HMO recognized the need for collaboration to prepare students and practitioners for high-quality, cost-effective practice in a managed care environment. They established an advisory committee to oversee a six-month feasibility study to interview experts and opinion leaders and identify critical characteristics of the ideally prepared managed care practitioner. In 1995, with start-up funding from the HMO, the institute began its operations as a freestanding enterprise with board representation from the two sponsoring institutions. While many of the institute's programs have been developed for practicing physicians and other health care professionals, this article focuses on the academic activities. For medical students, the approach has been to blend managed care principles and practices into existing courses, problem-based learning cases, and clerkships, rather than creating separate managed care courses. For primary care residents, the institute has a grant to develop managed care curricula in conjunction with Tufts-affiliated residency training programs. Faculty development is accomplished through dedicated workshops and seminars, and through increased dialogue between traditional faculty and managed care professionals. In all of its programs and activities the institute has fostered greater contact and collaboration between colleagues from both sides of the health plan-academia "divide." Operationally, the institute structure, with dedicated full-time administrative staff, provides the singular focus necessary to establish managed care education as a top priority for the partnership. At the same time, sustaining this freestanding organization and infrastructure requires increased resources. Initial responses to the institute's programs and activities have been positive, both from the local Tufts community and from external parties. Yet the partnership must establish methods to evaluate the institute's long-term impact in its efforts to help practitioners succeed in a transforming landscape.

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Structural bases for the specificity of cholinesterase catalysis and inhibition.

The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.

The equity model: three commentaries.

In the current ferment of health care reform, advocates of most proposals agree that one of the goals to be achieved is maintaining an environment in which physicians and other caregivers can be comfortable. One such proposal, the equity model, is evaluated here from the physician's viewpoint by three directors of established health care plans. While all express concern over potential loss of physicians' autonomy and control, they see the threat as coming from different sources.

Fluorescence studies on the interactions of myelin basic protein in electrolyte solutions.

This paper examines the influence of electrolytes on fluorescence spectral properties of the single tryptophanyl residue, Trp-115, within the 18.5-kDa species of myelin basic protein from bovine brain. Steady-state fluorescence spectra and intensities and time-correlated fluorescence lifetimes increased in the presence of increasing concentrations of mono- and divalent electrolytes (Li+, Na+, K+, Mg2+, Ca2+, Cl-, ClO4-, SO4(2-), and PO4(3-)). In all cases, the increases closely paralleled the ionic strength of the bulk aqueous medium and resembled that observed upon immersion of the protein in solutions of urea. This behavior was therefore concluded to reflect changes in the solution conformation of myelin basic protein. Bimolecular quenching of Trp-115 by acrylamide was rapid (10(9) M-1 s-1), approaching the diffusion limitation, and markedly dependent on the viscosity of the bulk aqueous medium. Rotational depolarization of myelin basic protein was rapid (phi less than or equal to 1 ns), occurring at rates exceeding those predicted for a rigid particle of revolution, and markedly dependent on the viscosity of the surrounding medium. Whereas the bimolecular quenching constants were unaltered in the presence of electrolytes, rotational depolarization of myelin basic protein underwent substantial slowing as indicated by the appearance of an additional decay component characterized by a correlation time of 5-10 ns. These studies indicate that Trp-115 of myelin basic protein is readily accessible to the bulk aqueous medium and is associated with a highly mobile segment of the protein. The slowing of rotational depolarization upon immersion of myelin basic protein in electrolyte solutions is consistent with an electrolyte-induced self-association of myelin basic protein molecules and indicates a relationship between the lability of solution conformation on the one hand and the capacity for self-association on the other.

Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography.

The mechanism of dealkylation ("aging") of branched-alkyl organophosphonyl conjugates of acetylcholinesterase and the consequence of this reaction on enzyme conformation were examined by employing kinetic, equilibrium, and spectroscopic techniques. Aging of cycloheptyl methylphosphono-acetylcholinesterase proceeded as a unimolecular reaction in which the enzyme became refractory to oxime reactivation and was accelerated with increases in temperature and decreases in pH and ionic strength of the medium. While aging occurred in a manner invariant with the nature of the salt in buffers containing Na+, K+, Rb+, Cs+, Cl-, CH3COO-, SO2-(4), and PO3-(4), the influence of ionic strength on aging was opposite to that predicted for a mechanism requiring charge separation during formation of the polar transition state. Examination of the equilibrium enzyme conformation with decidium, a fluorescent active center-selective ligand, revealed marked alterations in ligand association and a greater ionic strength dependence for binding after aging. The explanation for this behavior focuses on the high net negative surface charge of the enzyme and proposes that acetylcholinesterase topography is governed by the strength of electrostatic interactions between charged, contiguous, mobile protein regions within the subunit. As such, these studies reveal a reciprocal relationship between acetylcholinesterase topography, surface charge, and ionic strength of the medium.

Yohimbine antagonism of the vasodepression elicited by organophosphates applied on ventral medulla oblongata.

The rostral ventral surface of medulla oblongata (RVMO) has been shown to constitute a selective target for organophosphate (op) cholinesterase inhibitors. The action of soman (S) as compared with (7-nitrobenz-2-oxa-1,3 diazole)aminopentyl methylphosphonofluoridate (NBD-AP-MPF), a fluorescent organophosphate has now been examined in anesthetized cats pretreated with atropine sulphate. Blood pressure (BP), electrocardiogram (ECG) and respiration (R) were recorded. In some animals a cannula was implanted into the right lateral ventricle. Chemicals were bilaterally applied on RVMO by means of a perspex cannula and removed after 5 min. The application of 2.5 micrograms S or 60 micrograms NBD-AP-MPF elicited severe fall of BP which recovered only after 2 h in the case of the former and up to 45 min in the latter. Smaller doses produced corresponding responses of lesser magnitude. Accompanying R changes consisted in most cases of increased rate and reduced amplitude whereas in others the opposite or mixed alterations occurred. Frequently, sigh-like movements intermingled at periodic intervals with regular R deflections. The sighs were interpreted as aiming to correct blood gases balance. After application of atropine on RVMO--but not by systemic administration--BP and R were restored whereas single repeated i.v. injection of 1 microgram/kg noradrenaline produced only transient reversals without influencing the course of long lasting vasodepression. In contrast, the intraventricular administration of 250-500 micrograms yohimbine considerably reduced both the magnitude and extent of the vasodepression elicited by topically applied organophosphates. It is postulated that central alpha 2-adrenoceptors in contrast to vascular sites are likely involved in the op-induced vasodepression. The present work provides an indication that effective antagonists might be developed considering blockade of these receptors.

Denervation-induced alterations of acetylcholinesterase in denervated and nondenervated muscle.

The influence of denervation on acetylcholinesterase (AchE) molecular forms in rat skeletal muscle for durations up to 30 days is examined in denervated anterior tibialis, the innervated contralateral muscle, and diaphragm. Denervated rats at a common age of 8.5 weeks are compared with age-matched, nondenervated animals. The results indicate that time-dependent losses of AchE in denervated muscle occur more rapidly than loss of muscle mass and are not uniform among the different molecular forms. Loss of the 4 S and 16 S forms is rapid and essentially complete within 3.5 days of denervation, while during this same period the 10.5 S form undergoes a transient twofold increase and its presence in denervated muscle is never abolished. Within 30 days of denervation, all forms of AchE including the 16 S species reappear. A salient finding of these studies is that the effects of denervation are evident also in anatomically remote, innervated muscle such as anterior tibialis of the contralateral limb and in diaphragm. These alterations appear as pronounced reductions in 4 S AchE and increases in 10.5 S AchE; the asymmetric collagen-tailed 16 S form is unaltered. Treatment of primary cultures of embryonic chick pectoral muscle with sera from denervated but not nondenervated rat causes reductions in AchE. These results indicate that the appearance and retention of AchE, in particular the 16 S form, occur in the absence of functional innervation. The effects of denervation on AchE metabolism in remote, innervated tissue are consistent with the action of a diffusible factor released from severed nerve or muscle, or both.

The influence of Pb2+ on expression of acetylcholinesterase and the acetylcholine receptor.

This paper examines the influence of inorganic lead (Pb2+) on the presence of acetylcholinesterase (AchE) molecular forms and the acetylcholine receptor (AchR) in two types of excitable tissue, primary cultures of skeletal muscle and neural retina from embryonic chick. Treatment of skeletal muscle with Pb2+ is observed to cause reductions in the 5/7S and 19S but not the 11.4S molecular forms of AchE. The reductions are dose-dependent, requiring submicromolar concentrations, slow in onset, requiring incubation times greater than 24 hr, and tissue specific, being pronounced in skeletal muscle but absent from neural retina. Significantly, the reductions in AchE occur without corresponding reductions in amounts of AchR and without reduction in activity of protein kinase C (PKC). These studies illustrate a tissue-specific action of inorganic lead that is not mediated through PKC.

Ligand exclusion on acetylcholinesterase.

This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.

Dihydropyridine receptor regulation of acetylcholinesterase biosynthesis.

The dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in primary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the 1-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a 2-fold reduction in the rate of secretion. The effects are markedly more pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187. These reductions are incompatible with accelerated protein degradation, alterations in posttranslational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis. Since AchE forms are thought to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and posttranscriptional processing of mRNA and ligand occupation of the dihydropyridine receptor.

Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates. Noncovalent determinants of enzyme chirality.

Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.

Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase.

This paper examines the chiral nature of the covalent conjugates formed upon reaction of acetylcholinesterase (AchE) with enantiomeric cycloheptyl, isopropyl, and 3,3-dimethylbutyl methylphosphonyl thiocholines. With the exception of the conjugate formed from reaction of AchE with RP-cycloheptyl methylphosphonyl thiocholine, all enantiomeric conjugates underwent oxime reactivation at rates that were within 2-3-fold of each other. Oxime reactivation was, therefore, independent of both initial configuration about phosphorus and the alkyl phosphonyl ester (-OR) moiety. Aging of the enantiomeric cyclopheptyl and isopropyl methylphosphonyl conjugates occurred exclusively for the conjugate formed from the SP-enantiomer and therefore displayed an absolute dependence on the initial configuration of the methylphosphonyl group. Equilibrium titrations with decidium, a fluorescent bisquaternary competitive inhibitor of AchE, provided an index of aging and enantiomeric configuration of the conjugates independent of enzyme activity. Decidium association with the enantiomeric conjugates (prior to aging) showed no marked dependence on the initial configuration about phosphorus but was measurably dependent on nature of the -OR moiety. These results are interpreted with respect to symmetry and nonrigidity of the organophosphonyl conjugates and are consistent with formation of final methylphosphonyl conjugates that are enantiomerically pure and of opposite configuration. These studies indicate that the active center of AchE comprises at least two kinetically distinct environments separate from the esteratic region but located within 5 A of the nucleophilic serine and differing in dipolar characteristics that promote charge separation and general acid catalysis.

Interaction of tetrahydroaminoacridine with acetylcholinesterase and butyrylcholinesterase.

This paper examines inhibition of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) by tetrahydroaminoacridine (THA), an acridine analog under consideration for palliative treatment of Alzheimer's dementia. THA causes linear mixed inhibition of AchE hydrolysis of acetylthiocholine, a cationic substrate (KI = 3.8 x 10(-9) M), and linear competitive inhibition of AchE hydrolysis of 7-acetoxy-4-methylcoumarin, an uncharged substrate (KI = 6.8 x 10(-9) M), and N-methyl-7-dimethylcarbamoxyquinolinium, a cationic carbamate (KI = 1.5 x 10(-8) M). Propidium association with AchE in the presence of saturating concentrations of THA is characterized by a dissociation constant of 7.7 +/- 0.7 x 10(-6) M, a value within 2-fold of the dissociation constant in the absence of THA. Association of THA with AchE is, therefore, not mutually exclusive with association of propidium at the peripheral anionic site. Moreover, THA causes dissociation of decidium complexes with AchE at concentrations compatible with a dissociation constant of 7.0 +/- 0.4 x 10(-9) M. Similar relationships were observed for THA inhibition of BuchE hydrolysis of butyrylthiocholine (KI = 2.5 x 10(-8) M) and dissociation of decidium complexes with BuchE (KD = 1.9 +/- 0.1 x 10(-8) M). These kinetic and equilibrium data uniformly indicate that THA associates with AchE and BuchE with high affinity and that the subsequent inhibition comes about through ligand association at the active center rather than at a peripheral site. The noncompetitive component of inhibition reflects association of THA with the acyl-enzyme intermediate, with subsequent effects on the rate of deacylation.

Specificity and orientation of trigonal carboxyl esters and tetrahedral alkylphosphonyl esters in cholinesterases.

We have examined the specificity of planar carboxyl and tetrahedral phosphonyl esters for mouse cholinesterases and have delineated the orientation of these ligands in the enzyme active center. The approach involved altering acyl pocket dimensions by site-specific mutagenesis of two phenylalanines and varying ligand size and enantiomer presentation. Substrate catalysis rates by wild type acetylcholinesterase (AChE) of acetyl-, butyryl-, and benzoylthiocholine diminished with increasing size of the acyl moiety. In contrast, substitution of the acyl pocket phenylalanines giving the mutants F295L and F297I of AChE yielded more efficient catalysis of the larger substrates and a specificity approaching that of butyrylcholinesterase. Extension from planar substrates to enantiomerically pure organophosphonates allowed for an analysis of enantiomeric selectivity. We found that AChE reactions are 200-fold faster with the Sp than the Rp enantiomer of of cycloheptyl methylphosphonyl thiocholine. Upon the acyl pocket size being enlarged, the Rp enantiomer became more reactive while reaction with the Sp enantiomer was slightly reduced. In fact, the F297I mutant displayed inverted stereospecificity. A visual correlation with the kinetic data has been developed by docking the ligands in the active site. Upon placement of the phosphonyl oxygen in the oxyanion hole and the leaving group being directed out of the gorge, the Rp, but not the Sp, enantiomer engendered steric hindrance between the alkoxyl group and the acyl pocket. Replacing F297 with Ile accommodated the bulky alkoxyl group of the Rp isomer in the acyl pocket, allowing similar orientations of the phosphonyl oxygen and the leaving group to the Sp isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum sulfonamide concentration after oral administration: comparison of results of chemical assay with two bioassay techniques.

Sulfonamide concentrations were studied in 210 serum samples from 10 volunteers after ingestion of sulfacytine, sulfisoxazole, and sulfadiazine. Results obtained by chemical assay were compared with bioassay values determined by two techniques: twofold broth dilution and agar diffusion. Although the correlation for individual samples was rather poor for the twofold broth-dilution method, the agar-diffusion bioassay correlated well with chemical determinations. The agar-diffusion assay tended to "underread" the chemical assay by an amount characteristic of each drug.