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Human serum paraoxonase/arylesterase is an esterase with broad substrate specificity. It occurs in two genetically determined allozymic forms, which we have designated types A and B. These allozymes are presumed to be the products of two allelic genes located at the paraoxonase locus on chromosome 7, which is closely linked to the gene for cystic fibrosis. Paraoxonase activity of the B-type isozyme is considerably higher and stimulated more by 1 M NaCl than A-type paraoxonase. The ratio of paraoxonase activity/arylesterase activity of the B-isozyme is about 8, and that of the A-isozyme about 1. Purified isozymes A or B are free of nearly all other serum proteins, and the broad substrate specificity of the serum esterase is preserved after purification. A variety of substrates are hydrolyzed; these include: diisopropylfluorophosphate, soman, sarin, 4-nitro-phenylacetate, 2-nitro-phenylacetate, 2-naphthylacetate, and phenylthioacetate. The isozymic distinctions in kinetic properties and substrate specificity are preserved during purification. It is likely that the allozymes have very similar turnover numbers with phenylacetate (arylesterase activity), but differ considerably in their turnover numbers with paraoxon. Isozymes A and B have about the same minimal molecular weight of 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Further detailed studies on the individual isozymic proteins (or the DNA coding for their amino acid sequence) will be required to detect the exact structural differences in the isozymes.

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Evidence is presented that human serum contains a single enzyme with both paraoxonase and arylesterase activities. Throughout the steps of purification and after obtaining over 600-fold purification of the enzyme, the arylesterase activity (measured with phenylacetate as the substrate) co-eluted and retained the same ratio of activity to paraoxonase activity as it had in the initial plasma sample. Paraoxon and DFP (diisopropylfluorophosphate) both complete with phenylacetate as substrates; the inhibition is of mixed type with paraoxon and competitive with DFP. Paraoxonase and arylesterase activities require calcium, and both are inhibited to the same degree by EDTA. Purified arylesterase/paraoxonase is a glycoprotein with a minimal molecular weight of about 43,000. It has up to three sugar chains per molecule, and carbohydrate represents about 15.8% of the total weight. The enzyme has an isoelectric point of 5.1. Its amino acid composition shows nothing unusual, except for a relatively high content of leucine. We conclude that human serum arylesterase and paraoxonase activities are catalyzed by a single enzyme, capable of hydrolyzing a broad spectrum of organophosphate substrates and a number of aromatic carboxylic acid esters. Studies on the genetically determined polymorphism responsible for two allozymic forms (A and B) of the esterase are described in the following paper.

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Thrombolytic therapy has been shown to improve clinical outcome when administered early after the onset of symptoms of acute myocardial infarction; the mechanism of benefit is believed to be reestablishment and maintenance of coronary artery patency. Anistreplase is a second generation thrombolytic agent that is easily administered and has a long duration of action. To compare anistreplase (30 units/2-5 min) and therapy with the Food and Drug Administration-approved regimen of intravenous streptokinase (1.5 million units/60 min), a randomized, double-blind, multicenter patency trial was undertaken in 370 patients less than 76 years of age with electrocardiographic ST segment elevation who could be treated within 4 hours of symptom onset. Coronary patency was determined by reading, in a blinded fashion, angiograms obtained early (90-240 minutes; mean, 140 minutes) and later (18-48 hours; mean, 28 hours) after beginning therapy. Early total patency (defined as Thrombolysis in Myocardial Infarction grade 2 or 3 perfusion) was high after both anistreplase (132/183 = 72%) and streptokinase (129/176 = 73%) therapy, and overall patency patterns were similar, although patent arteries showed "complete" (grade 3) perfusion more often after anistreplase (83%) than streptokinase (72%) (p = 0.03). Similarly, residual coronary stenosis, determined quantitatively by a validated computer-assisted method, was slightly less in patent arteries early after anistreplase (mean stenosis diameter, 74.0%) than streptokinase (77.2%, p = 0.02). In patients with patent arteries without other early interventions, reocclusion risk within 1-2 days was defined angiographically and found to be very low (anistreplase = 1/96, streptokinase = 2/94). Average coronary perfusion grade was greater, and percent residual stenosis was less, at follow-up than on initial evaluation and did not differ between treatment groups. Enzymatic and electrocardiographic evolution was not significantly different in the two groups. Despite rapid injection, anistreplase was associated with only a small (4-5 mm Hg), transient (at 5-10 minutes) mean differential fall in blood pressure. In-hospital mortality rates were comparable for anistreplase and streptokinase (5.9%, 7.1%). Stroke occurred in one (0.5%) and three (1.6%) patients, respectively; one stroke was hemorrhagic. Other serious bleeding events and adverse experiences occurred uncommonly and with similar frequency in the two groups. Thus, for the end points of our study (patency, safety), anistreplase and streptokinase showed overall favorable and relatively comparable outcomes, with a few differences.(ABSTRACT TRUNCATED AT 400 WORDS)

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Human serum paraoxonase/arylesterase is a polymorphic enzyme, determined by two allelic genes at one autosomal locus. These two isozymes are called (A) and (B), and the three corresponding phenotypes A, AB and B. We measure paraoxon hydrolysis (paraoxonase activity), with, or without 1 M NaCl, and phenylacetate hydrolysis (arylesterase activity) with, or without 0.1 mM chlorpromazine. The resulting four measurements make it possible to determined essentially every individual's phenotype. The combination of qualitative tests, based on distinctive characteristics of the two isozymes, greatly improves our ability to determine individual phenotypes and detect other isozymic variants of the esterase. For several years, we have been interested in the possible correlation of the phenotypes of paraoxonase/arylesterase and sensitivity or resistance to organophosphate exposure [1]. Recent reports show that the paraoxonase gene may be closely linked to the gene for cystic fibrosis [2]. If this will be confirmed, it could be another very practical reason for typing people for this polymorphic marker. Being able to identify those heterozygous for the paraoxonase/arylesterase polymorphism, about 43% of the European population, should greatly enhance the value of phenotyping for this simple, convenient, polymorphic trait.

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Hydrolysis and covalent binding to nonessential esterases are two biochemical processes which can prevent paraoxon from reacting with the essential enzyme, acetylcholinesterase. Both processes have been proposed as the primary route of paraoxon detoxification in vivo. These experiments were designed to assess the relative contribution of each pathway to the disappearance of paraoxon in the rabbit. In vitro, paraoxon disappeared from whole rabbit blood with a t 1/2 of 17.7 sec. Hydrolysis by paraoxonase (EC 3.1.1.2) accounted entirely for this disappearance and covalent binding contributed essentially nothing. In vivo, following an iv injection of 0.15 mg/kg paraoxon, serum paraoxonase hydrolyzed as much as 41% of the injected dose within the first 30 sec. Pretreatment of rabbits with an ip injection of tri-o-tolyl phosphate eliminated more than 95% of the paraoxon binding sites. However, pretreatment with tri-o-tolyl phosphate had no significant effect on the t 1/2 or volume of distribution of paraoxon, indicating that covalent binding sites did not contribute significantly to the clearance of paraoxon from whole rabbits under these conditions. Hydrolysis of paraoxon by tissue paraoxonases, in addition to that catalyzed by paraoxonase in the blood, could account for its rapid metabolism. These findings demonstrate that paraoxonase has a major role in the disappearance of paraoxon in the rabbit. This suggests that susceptibility of people to chronic paraoxon poisoning may vary, according to their inherited level and type of serum paraoxonase.

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There is now considerable evidence that human serum contains an enzyme with both paraoxonase and arylesterase activities. The enzyme probably exists in two common isozymic forms with qualitatively distinctive properties. These isozymes differ particularly in their ratio of paraoxonase /arylesterase activities, one form having a ratio approximately seven times greater than the other. By measuring paraoxonase activity in the presence of 1 M NaCl, and arylesterase activity with phenylacetate under standard conditions, it is possible to classify individuals within one of the three phenotypes determined by a two-allele, single autosomal locus system at the ESA locus. These alleles are designated ESA*A and ESA*B. Pedigree analyses have also shown the anticipated Mendelian segregation of these traits within families. The frequencies of the ESA*A and ESA*B alleles in a sample population of unrelated Caucasian people in the United States were estimated to be 0.685 and 0.315, respectively, for the traits conferring relatively low and high paraoxonase activities. It still remains to be determined whether the distinctive phenotypes affect the degree of sensitivity or resistance to organophosphate agents, such as paraoxon. We do not know what additional functions these polymorphic enzymes may have in the metabolism of other compounds, particularly those of endogenous origin.

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The inhibition of cholinesterase of human serum by paraoxon can be predicted by a mathematical model which considers two competing reactions for paraoxon: one, the direct interaction with cholinesterase, and the other, enzymatic hydrolysis by paraoxonase. On the basis of the residual cholinesterase activity at various times during the incubation with paraoxon, it is possible to determine the rate constants for the reaction of paraoxon with cholinesterase (k1), and the reaction with paraoxonase (k2), the latter being directly proportional to paraoxonase activity. The percentage of initial activity remaining as residual cholinesterase depends primarily upon the paraoxonase level; it is influenced only slightly by variations in initial cholinesterase levels within the normal range. From these results, we conclude that the residual cholinesterase activity test is, in fact, an indirect measure of serum paraoxonase activity; it has the same limitations and is no more reliable a means of differentiating individual paraoxonase genotypes than measuring the level of serum paraoxonase activity directly. Our model suggests that there are conditions where paraoxonase genotype may alter the clearance of paraoxon and in turn the reaction of paraoxon with target sites. Whether similar results would be obtained in vivo is unknown. Since this model predicts the degradation of paraoxon well in vitro, it may be possible to extend the model and predict the effect of paraoxonase genotype on the clearance of paraoxon in vivo.

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The heterozygous human serum paraoxonase phenotype can be clearly distinguished from both homozygous phenotypes on the basis of its distinctive ratio of paraoxonase to arylesterase activities. A trimodal distribution of the ratio values was found with 348 individual serum samples, measuring the ratio of paraoxonase activity (with 1 M NaCl in the assay) to arylesterase activity, using phenylacetate. The three modes corresponded to the three paraoxonase phenotypes, A, AB, and B (individual genotypes), and the expected Mendelian segregation of the trait was observed within families. The paraoxonase/arylesterase activity ratio showed codominant inheritance. We have defined the genetic locus determining the aromatic esterase (arylesterase) responsible for the polymorphic paraoxonase activity as esterase-A (ESA) and have designated the two common alleles at this locus by the symbols ESA*A and ESA*B. The frequency of the ESA*A allele was estimated to be .685, and that of the ESA*B allele, 0.315, in a sample population of unrelated Caucasians from the United States. We postulate that a single serum enzyme, with both paraoxonase and arylesterase activity, exists in two different isozymic forms with qualitatively different properties, and that paraoxon is a "discriminating" substrate (having a polymorphic distribution of activity) and phenylacetate is a "nondiscriminating" substrate for the two isozymes. Biochemical evidence for this interpretation includes the cosegregation of the degree of stimulation of paraoxonase activity by salt and paraoxonase/arylesterase activity ratio characteristics; the very high correlation between both the basal (non-salt stimulated) and salt-stimulated paraoxonase activities with arylesterase activity; and the finding that phenylacetate is an inhibitor for paraoxonase activities in both A and B types of enzyme.

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A method for identifying two human serum paraoxonase phenotypes in vitro has been developed based upon the effect of NaCl upon paraoxonase activity. In a sample population of 336 individuals from the United States, 53.9% of the samples had serum paraoxonase that was highly stimulated (60%-257% above the control activity) by 1 M NaCl (salt-responsive), whereas the activity of the remaining samples was not salt-responsive (-23%-35%). The degree of stimulation was consistent and reproducible in frozen samples collected from an individual over a two-year period. Pedigree studies with 37 families indicate that the salt-responsive characteristic is inherited as a simple autosomal, Mendelian trait. Although the salt-responsive individuals on the average had a higher level of activity when assayed without added salt (basal activity) than did the non-salt-responsive individuals, there was considerable overlap in the basal paraoxonase activities. The quantitative polymorphism in serum paraoxonase activity observed in other laboratories is associated with a qualitative difference, quite possibly due to two distinct isozymic forms of the enzyme. A new designation for these alleles is proposed, and some preliminary studies on the molecular basis of the polymorphism are reported.

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Antiserum prepared against highly purified usual human serum cholinesterase (the most common phenotype) cross-reacted identically with the atypical serum cholinesterase. The level of circulating atypical enzyme protein, determined immunologically, was about 30% lower when the enzyme came from an atypical rather than a usual phenotype, and the level of enzyme activity measured enzymatically at Vmax with either o-nitrophenylbutyrate or benzoylcholine as substrate showed approximately the same degree of reduction. The average specific activity (activity at Vmax per microgram of enzyme protein) in sera from 28 usual and 20 atypical individuals did not differ significantly. These findings suggest that the atypical enzyme not only has altered catalytic properties (Km) but also might be synthesized more slowly, or cleared in vivo more rapidly, than the usual enzyme.

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The enzyme in human serum that rapidly hydrolyzes diacetylmorphine (heroin) to 6-acetylmorphine is identified in this report as serum cholinesterase (EC 3.1.1.8, acylcholine acylhydrolase; also called pseudocholinesterase or butyrylcholine esterase). The rate of heroin hydrolysis was measured spectrophotometrically at 245 nm using highly purified serum cholinesterase. The turnover number was 500 mumol of heroin hydrolyzed per min per mumol active site. The product was identified spectrophotometrically and by thin-layer chromatography to be 6-acetylmorphine. There appeared to be marked product inhibition of heroin hydrolysis, as 6-acetylmorphine (Ki = 0.015 mM) bound 7 times more tightly than heroin (Ki = 0.11 mM). Purified human serum arylesterase did not hydrolyze heroin. Purified serum cholinesterase accounted for all the observed heroin hydrolysis by whole serum. The genetic variants of human serum cholinesterase, silent and atypical cholinesterase, were also tested. Serum from a person identified as having silent cholinesterase did not hydrolyze heroin. Purified atypical cholinestearase hydrolyzed heroin, but the binding was less tight (Km = 0.45 mM) than with usual cholinesterase (Km = 0.11 mM). The possibility that heroin potency may be influenced by serum cholinesterase genotype or activity level remains to be investigated.

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Mating aggregates during conjugation directed by an F-like R factor in Escherichia coli were measured as the number of Lac+-Lac- sectored colonies present in a mating mixture. There is a high degree of correlation between the concentration of transconjugants produced in a mating mixture and the concentration of mating aggregates observed at several different concentrations of donor and recipient cells. The mating aggregates are sex pilus specific as demonstrated by the ability of donor-specific ribonucleic acid phage MS-2 to decrease both mating aggregates and transconjugants in a mating mixture. During entry exclusion by either a derepressed or a repressed F-like R factor, isogenic to the superinfecting R factor except for a resistance determinant, the number of transconjugants was markedly reduced, but the number of mating aggregates was not decreased. Entry exclusion by F-Gal toward the donor HfrH resembled that of the F-like R factor in that there was a reduction in the number of recombinants but no significant decrease in mating aggregates. These results suggest that entry exclusion inhibits conjugation at a stage after the formation of mating aggregates.

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