Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate.

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

On the physiological role(s) of the paraoxonases.

In recent years several lines of evidence have indicated that serum paraoxonase (PON1), and perhaps other mammalian paraoxonases, act as important guardians against cellular damage from toxic agents, such as organophosphates, oxidized lipids in the plasma low density lipoproteins (LDL), and against bacterial endotoxins. For some of these protective activities but not all, PON1 requires calcium ion. The catalyzed chemical reactions generally seem to be hydrolytic, but for some types of protection this may not be so. Several other metals have very high affinity for PON1 and may displace calcium. Replacement or substitution of calcium by other metals could extend the range of catalytic properties and the substrate specificity of the paraoxonases, as it does for the mammalian DFPases. Although this Third International Meeting on Esterases Reacting with Organophosphorus Compounds focuses on the organophosphatase activities of paraoxonase and related enzymes, it is important to also briefly review some of the current directions in several laboratories searching for additional functions of the paraoxonases to extend our understanding of the properties of this family of enzymes which now seem to have both physiological and toxicological importance.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

Characterization of an unstable variant (BChE115D) of human butyrylcholinesterase.

An unstable variant of human butyrylcholinesterase (BChE) is described in four apparently unrelated individuals sensitive to succinylcholine. Sequencing of genomic DNA revealed a single nucleotide substitution which results in the replacement of amino acid residue Gly115 by Asp. This variant can be recognized by its increased instability under extremes of temperature such as heating and also freezing and thawing, both in homozygous and heterozygous states. When in heterozygous combination with the Atypical variant, it produces dibucaine and fluoride numbers which are intermediary between those of Atypical homozygotes and heterozygotes. After repeated freezing and thawing, however, these values approach those of homozygous Atypical plasma. Measurement of activity and immunoreactive BChE protein in plasma of individuals representing different combinations of this allele indicated that the presence of the Usual or Atypical enzymes seems to partially protect this variant from denaturation in vivo. Phenotyping fresh serum or plasma samples, before they are frozen, is critical for the identification of this, and possibly some other, unstable variants.

The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.

A physiological role for paraoxonase (PON1) is still uncertain, but it catalyzes the hydrolysis of toxic organophosphates. Evidence that the human genome contains two PON1-like genes, designated PON2 and PON3, is presented here. Human PON1 and PON2 each have nine exons, and the exon/intron junctions occur at equivalent positions. PON1 and PON2 genes are both on chromosome 7 in human and on chromosome 6 in the mouse. Turkey and chicken, like most birds, lack paraoxonase activity and are very susceptible to organophosphates. However, they have a PON-like gene with approximately 70% identity with human PON1, PON2, and PON3. Another unexpected finding is that the deduced amino acid sequences of PON2 in human, mouse, dog, turkey, and chicken and of human PON3 are all missing the amino acid residue 105, which is lysine in human PON1. The expanded number of PON genes will have important implications for future experiments designed to discover the individual functions, catalytic properties, and physiological roles of the paraoxonases.

Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene.

The silent phenotype of human butyrylcholinesterase (BChE), present in most human populations in frequencies of approximately 1/100,000, is characterized by the complete absence of BChE activity or by activity <10% of the average levels of the usual phenotype. Heterogeneity in this phenotype has been well established at the phenotypic level, but only a few silent BCHE alleles have been characterized at the DNA level. Twelve silent alleles of the human butyrylcholinesterase gene (BCHE) have been identified in 17 apparently unrelated patients who were selected by their increased sensitivity to the muscle relaxant succinylcholine. All of these alleles are characterized by single nucleotide substitutions or deletions leading to distinct changes in the structure of the BChE enzyme molecule. Nine of the nucleotide substitutions result in the replacement of single amino acid residues. Three of these variants, BCHE*33C, BCHE*198G, and BCHE*201T, produce normal amounts of immunoreactive but enzymatically inactive BChE protein in the plasma. The other six amino acid substitutions, encoded by BCHE*37S, BCHE*125F, BCHE*170E, BCHE*471R, and BCHE*518L, seem to cause reduced expression of BChE protein, and their role in determining the silent phenotype was confirmed by expression in cell culture. The other four silent alleles, BCHE*271STOP, BCHE*500STOP, BCHE*FS6, and BCHE*I2E3-8G, encode BChES truncated at their C-terminus because of premature stop codons caused by nucleotide substitutions, a frame shift, or altered splicing. The large number of different silent BCHE alleles found within a relatively small number of patients shows that the heterogeneity of the silent BChE phenotype is high. The characterization of silent BChE variants will be useful in the study of the structure/function relationship for this and other closely related enzymes.

The genetic mapping and gene structure of mouse paraoxonase/arylesterase.

The physiological role of mammalian paraoxonase/arylesterase is unknown. However, paraoxonase is an HDL-associated protein, and recent studies indicate that it may have anti-atherogenic functions. We describe the chromosomal localization and structure of the mouse paraoxonase gene (Pon1) to establish Pon1 as a candidate gene for genetically determined traits or pathological states in the mouse. The coding portion of Pon1 extends over approximately 25-26 kb and consists of nine exons and eight introns. We also present nucleotide sequences from the 5'-flanking region of Pon1 containing numerous consensus sequences for DNA binding proteins. Haplotype analysis of 94 N2 progeny from an interspecific cross indicates that Pon1 is localized on proximal mouse chromosome 6 near D6Mit86. This assignment excludes Pon1 as a candidate for the atherosclerosis susceptibility genes Ath1, Ath2, and Ath3. However, Pon1 is a promising candidate for the remaining unmapped Ath genes.

Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

An improved method for butyrylcholinesterase phenotyping.

An improved method for the identification of butyrylcholinesterase phenotypes is proposed. It is based on modifications of a method that uses alpha-naphthyl acetate as substrate and DL-propranolol and Ro2-0683 as inhibitors. The proposed modifications make the method more rapid and increase the accuracy of the determinations of the phenotypes tested (BCHE U, BCHE UF, BCHE UA, BCHE AK, BCHE AF, and BCHE A). These modifications make the method even more adequate for population studies and clinical routine.

[Identification of two different genetic mutation associated with silent phenotypes for human serum cholinesterase in Japanese].

Two different gene mutations associated with the silent phenotype for human serum cholinesterase were demonstrated. DNA from five individuals with silent gene phenotype of three unrelated Japanese families was amplified by the polymerase chain reaction (PCR) and analyzed by direct sequencing. The first instance demonstrated a G----C transversion at codon 365 from GGA (Gly) to CGA (Arg), which was seen in three individuals of the two families. This mutation was resulted to create a new Taq 1 restriction site (TCGA). The second mutation was shown by a double heterozygous condition with two different silent gene mutations in two members of remaining one family. These mutations were as follows: 1) one type was a frameshift mutation, in which an extra A was inserted in codon 315 (ACC----AACC) to create a new stop codon at position 322 and 2) the other was the same point mutation at codon 365 as seen in the first instance. These results indicated that many silent variants can be distinguished by direct sequence analyses of genomic DNA.

The C5 isozyme of serum cholinesterase and adult weight.

The relationship between the CHE2 locus of serum cholinesterase (BChE) and adult human weight was studied in a sample of 225 CHE2 C5+ individuals and 225 CHE2 C5- controls matched for sex, height, age and race. With respect to the intensity of the C5 band staining (scored 1-6), 113 individuals had faint C5 bands (scores 1-3) and 112 intense C5 bands (scores 4-6). The individuals with intense CHE2 C5+ phenotype showed a significantly lower mean adult weight (64.66 +/- 0.73 kg) when compared to their controls (70.59 +/- 0.97 kg) and a significant reduction in weight variance (59.81 and 105.18, respectively). Individuals with faint C5 bands, although showing a negative correlation between weight and C5 band intensity, did not differ from their controls in mean weight.

Serum cholinesterases polymorphism (CHE1 and CHE2 Loci) in indians from the Amazon Region of Brazil: Urubu-Kaapor and Assurini tribes

Freqüencias das variantes atípica e C5+ da colinesterase do soro foram estimadas em 210 índios Urubu-Kaapor e 162 índios Assurini, da Amazônia brasileira. O alelo CHE1*A näo foi detectado nas duas tribos, e o fenótipo C5+ foi encontrado apenas no grupo Urubu-Kaapor, com a elevada freqüência de 26,2%

Studies on serum cholinesterase (CHE1 and CHE2) in Brazilian Indian and admixed populations

Onze amostras brasileiras - sete tribos indígenas dos Estados do Amazonas (3), Rondônia (1), Goiás (1) e Paraná (2), e quatro comunidades rurais tri-híbridas do Amazonas (Branco/Indio/Negro) - foram estudadas quanto aos dois locos da colinesterase do soro (CHE1 e CHE2). Os índios näo apresentaram a variante CHE1*A e mostraram freqüências de CHE2*C5+ variando de 0 a 13%. Duas populaçöes tri-híbridas (Tapuá e Canutama) apresentaram prevalências de CHE1*A diferentes de zero e diferentes entre si (0,3% e 2,4%). As freqüências de CHE2*C5+ variaram de 4% a 8%, nos grupos tri-híbridos, num gradiente norte-sul. O alelo CHE2*C5+ aumentou a atividade da colinesterase do soro em 51,2% e regressöes múltiplas escalonadas mostraram que a atividade enzimática diminuiu com a idade em duas tribos indígenas (Sateré-Mawé e Pacaás Novos). Além disso, os três grupos indígenas, estudados quanto à atividade enzimática, revelaram níveis médios mais baixos do que os esperados

Frequencies of atypical serum cholinesterase among Caucasians and Negroes from southern Brazil.

Frequencies of the CHE1*A allele were estimated on the basis of a sample of 999 Caucasians (1.5%) and 1,015 Negroids (0.84%) from Curitiba, Brazil. The frequency found in the Negroid subsample allows an estimate of 50 +/- 15% of Caucasoid admixture and an average gene flow in the white-black direction of the order of 5.6% per generation.