Alan D. Wolfe

Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes.

Abstract Light scattering was used to determine the effects of the bacterial initiation factors on the 70 S ribosome subunit equilibrium: 70 S ⇌ k 2 k 1 30 S + 50 s. The equilibrium proportion of associated ribosomes was studied as a function of IF-3, IF-1 and IF-2 § concentrations, and the rate constants κ1 and κ2 were determined in the presence of IF-3 and IF-1. Our results strongly support the view that IF-3-induced dissociation of the 70 S couple results from its binding to the free 30 S subunit, thus shifting the equilibrium toward dissociation. IF-3 causes κ2 to decrease drastically, while κ1 is unaffected; moreover, equilibrium values obtained with A-type ribosomes (tight couples) are consistent with binding of IF-3 to the 30 S subunit (association constant K3 = 2·5 × 107 to 4·0 × 107 m −1) and with its exclusion from the 70 S ribosome. No significant variation of K3 was found, either with temperature (25 to 37 °C) or with Mg2+ concentration (2·2 to 5 m m ). IF-1 was found to aid IF-3-induced dissociation by increasing the rate constants κ1 and κ2. IF-1 alone showed a limited dissociating activity, interpreted as due to its stronger binding to the 30 S than to the 70 S particle. Accordingly, κ1 has a stronger dependence on IF-1 concentration than κ2. The affinity of IF-3 for the 30 S subunit was observed not to vary significantly on addition of IF-1, when both factors were used in nearly stoichiometric amounts (not more than threefold excess over the ribosomes). When, on the contrary, IF-1 and IF-3 were present in large excess, dissociation was more efficient than expected from simple one to one binding of the factors to the ribosome. Streptomycin slowed the rate of dissociation by IF-3 and IF-1, but did not entirely suppress the effect of the factors. IF-2 was found to cause association of the ribosomal subunits; the effect is stronger and more specific in the presence of GMPPCP or GTP than in their absence. In the latter case, increase of the light-scattering signal probably involves some aggregation. In. the presence of GMPPCP, data are consistent with the following binding constants of IF-2: K2 m −1 to the 30 S, and K2′ = 1 × 107 to 1·5 × 107 m −1 to the 70 S ribosomes.

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

Enzymes as pretreatment drugs for organophosphate toxicity

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

MODE OF ACTION OF CHLORAMPHENICOL. IX. EFFECTS OF CHLORAMPHENICOL UPON A RIBOSOMAL AMINO ACID POLYMERIZATION SYSTEM AND ITS BINDING TO BACTERIAL RIBOSOME.

Abstract Chloramphenicol at a constant concentration inhibited the polymerization of phenylalanine in a bacterial ribosome-polyuridylic acid system by a constant percentage when graded concentrations of ribosomes, polyuridylic acid, or transfer ribonucleic acid limited the activity of the system. [ 14 C]Cloramphenicol combined weakly but stereospecifically with 70-S ribosomes; the average number of chloramphenicol molecules binding to a ribosome was of the order of 1. The hypothesis is proposed that ribosome-bound chloramphenicol interferes with a function of messenger ribonucleic acid.

Use of cholinesterases as pretreatment drugs for the protection of rhesus monkeys against soman toxicity

Purified fetal bovine serum acetylcholinesterase (FBS AChE) and horse serum butyrylcholinesterase (BChE) were successfully used as single pretreatment drugs for the prevention of pinacolyl methylphosphonofluoridate (soman) toxicity in nonhuman primates. Eight rhesus monkeys, trained to perform Primate Equilibrium Platform (PEP) tasks, were pretreated with FBS AChE or BChE and challenged with a cumulative level of five median lethal doses (LD50) of soman. All ChE-pretreated monkeys survived the soman challenge and showed no symptoms of soman toxicity. A quantitative linear relation was observed between the soman dose and the neutralization of blood ChE. None of the four AChE-pretreated animals showed PEP task decrements, even though administration of soman irreversibly inhibited nearly all of the exogenously administered AChE. In two of four BChE-pretreated animals, a small transient PEP performance decrement occurred when the cumulative soman dose exceeded 4 LD50. Performance decrements observed under BChE protection were modest by the usual standards of organophosphorus compound toxicity. No residual or delayed performance decrements or other untoward effects were observed during 6 weeks of post-exposure testing with either ChE.

Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase

The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.

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. californica AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Erythromycin: Mode of Action

Erythromycin, a specific inhibitor of protein biosynthesis, inhibited the incorporation of phenylalanine by a cell-free ribosomal system prepared from Escherichia coli.

Amplification of the effectiveness of acetylcholinesterase for detoxification of organophosphorus compounds by bis-quaternary oximes☆

Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.

Cholinesterases as scavengers for organophosphorus compounds: Protection of primate performance against soman toxicity

The present treatment for poisoning by organophosphates consists of multiple drugs such as carbamates, antimuscarinics, and reactivators in pre- and post-exposure modalities. Recently an anticonvulsant, diazapam, has been included as a post-exposure drug to reduce convulsions and increase survival. Most regimens are effective in preventing lethality from organophosphate exposure but do not prevent toxic effects and incapacitation observed in animals and likely to occur in humans. Use of enzymes such as cholinesterases as pretreatment drugs for sequestration of highly toxic organophosphate anticholinesterases and alleviation of side effects and performance decrements was successful in animals, including non-human primates. Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase protected them against lethal effects of soman (up to 5 LD50) and prevented signs of OP toxicity. Monkeys pretreated with fetal bovine serum acetylcholinesterase were devoid of behavioral incapacitation after soman exposure, as measured by serial probe recognition or primate equilibrium platform performance tasks. Use of acetylcholinesterase as a single pretreatment drug provided greater protection against both lethal and behavioral effects of potent organophosphates than current multicomponent drug treatments that prevent neither signs of toxicity nor behavioral deficits. Although use of cholinesterases as single pretreatment drugs provided complete protection, its use for humans may be limited, since large quantities will be required, due to the approximately 1:1 stoichiometry between organophosphate and enzyme. Bisquaternary oximes, particularly HI-6, have been shown to reactivate organophosphate-inhibited acetylcholinesterase at a rapid rate. We explored the possibility that enzyme could be continually reactivated in animals pretreated with fetal bovine serum acetylcholinesterase, followed by an appropriate dose of reactivator, and challenged with repeated doses of sarin. In in vitro experiments, stoichiometry greater than 1:400 for enzyme:sarin was achieved; in vivo stoichiometry in mice was 1:65. Pretreatment of mice with fetal bovine serum acetylcholinesterase and HI-6 amplified the effectiveness of exogenous enzyme as a scavenger for organophosphate.