Identity of the polymorphisms for esterase D and S-formylglutathione hydrolase in red blood cells

Summary The S-formylglutathione hydrolase (FGH) polymorphism of human red blood cells was studied in unrelated individuals, both by isoelectric focusing and starch gel electrophoresis, and with the substrates S-acetylglutathione and 4-methylumbelliferyl-acetate (the standard substrate for esterase D (ESD)). With both separation techniques the two substrates consistently gave similar and identically located zymograms. Thus, FGH (E.C.3.1.2.12) appears to be identical to ESD (E.C.3.1.1.1).     

Polymorphism of the red cell acid phosphatase in the Swiss population

Summary The distribution of the red cell acid phosphatase groups was studied on 1365 blood samples of Swiss individuals. The distribution is in agreement with the Hardy-Weinberg equilibrium. Gene frequencies similar to those observed in other Caucasian populations were obtained (P^A=0.345, P^B=0.607, P^C=0.049). In 331 mother/child-combinations, we found no theoretically impossible combinations.     

Polymorphism of red cell phosphoglucomutase, adenylate kinase and adenosine deaminase in a Polish population.

Genetic variants of PGM1, AK and ADA were studied in a sample of unrelated individuals from the Polish population. The gene frequencies observed are: PGM1/1: 0.715, AK1: 0.962 AND ADA1: 0.940.     

Polymorphism of red cell glyoxalase 1

Summary Glyoxalase polymorphism has been studied in 7296 persons from populations in South Asia, Southeast Asia, Oceania, Iran, and Colombia. The GLO¹ frequencies are very low in most of Oceania, including Australia, somewhat higher in Southeast Asia, and intermediate though variable in India. In Iran the GLO¹ frequency is similar to that in Europe. The value for the single amerindian group in Colombia is nearly 30%.     

Unique regulation of the active site of the serine esterase S-formylglutathione hydrolase

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.     

Polymorphism of red cell acid phosphatase in dogs

In fresh red cell haemolysates from Labrador retriever dogs three acid phosphatase (Pac) phenotypes were found by starch gel electrophoresis. Family data were consistent with the theory that the Pac phenotypes are controlled by two codominant, autosomal alleles, designated Pac ^F and Pac ^S.     

Cloning and characterisation of S-formylglutathione hydrolase from Arabidopsis thaliana: a pathway for formaldehyde detoxification

The toxic accumulation of formaldehyde in plant cells can occur from a number of sources: atmospheric pollution, endogenous P450 enzymes de-methylating herbicides and cell wall expansion. Any accumulated formaldehyde is rapidly bound to tetrahydrofolate or coupled to nucleophiles such as glutathione (GSH) resulting in S-hydroxymethylglutathione which is subsequently utilised by a glutathione-NAD-dependent formaldehyde dehydrogenase to form S-formylglutathione. The aim of this study was to examine the little understood pathway of formaldehyde detoxification in Arabidopsis thaliana, by cloning and characterising the key esterase S-formylglutathione hydrolase (FGH). The activity of FGH crucially recycles glutathione and renders formate available to the C1 pathway. Previously identified in bacteria, yeast and humans FGH from A. thaliana was cloned by RT-PCR, with a translated open reading frame that consisted of 284 amino acids and showed appreciable similarity with human esterase D. Over-expression using a pET vector system in combination with Escherichia coli resulted in a protein 30 kDa in size as determined by SDS-PAGE. Soluble A. thaliana FGH extracted from E. coli demonstrated a clear affinity for S-formylglutathione (K_m 0.318 mM) and was inhibited, 48% and 59%, respectively, by the addition of 1 μm 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) and p-hydroxy-mercuribenzoic acid (PHMB) indicating that an SH group may be essential for hydrolytic activity. The activity of S-formylglutathione hydrolase in the leaves of A. thaliana demonstrated that this enzyme might be part of a universal detoxification pathway shared by a variety of organisms.     

Polymorphism of S-adenosylhomocysteine hydrolase in Italy

S-adenosylhomocysteine hydrolase (SAHH) polymorphism has been investigated in the Italian population. Three common alleles, SAHH*1, SAHH*2 and SAHH*3, have been observed and the estimated gene frequencies are 0.968, 0.023 and 0.009, respectively. SAHH activity has been assayed in 50 healthy individuals and the mean activity was 0.043 +/- 0.017 mumol uric acid/min/g Hb at 37 degrees C. Five heterozygotes for adenosine deaminase deficiency and three heterozygotes for purine nucleoside phosphorylase deficiency showed SAHH within the range of the normal distribution. The effects of some thiol reagents on red blood cell SAHH electrophoretic pattern have been investigated.     

Polymorphism of red cell glyoxalase I in Serbia, Yugoslavia

Red cell glyoxalase I (GLO) phenotypes were determined in 258 unrelated adults from the population of Serbia (Yugoslavia). The GLO1 gene frequency was estimated to be 0.384.     

Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.     

Fatty acids of plasma and red blood cell lipids in a normal population

A detailed study of the fatty acid composition of each of the lipids present in plasma and red blood cells of 62 healthy subjects of our area (Barcelona and surrounding counties), by coupling thin-layer and gas chromatography techniques has been made. The results are presented as normative data for comparison with those found in pathological situations. No significant sex differences were found. With increasing age, there was a tendency for the proportion of linoleic acid to decrease. Correlation analyses between the fatty acid composition of different lipids suggested that the interchange of fatty acids between plasma and cells mainly affects the phosphatidylcholine of the latter.     

Polymorphism of human red cell glyoxalase I in six ethnic groups of China

Summary A total of 1242 individuals from six Chinese ethnic groups were studied with respect to the glyoxalase I polymorphism using agarose gel electrophoresis. The GLO1^*1 gene frequency and the number of subjects tested in each population are as follows: Uygur 0.2466 (219), Hui 0.1621 (219), Dong 0.1866 (201), Bai 0.1921 (203), Tujia 0.1075 (200), and Maio 0.1600 (200). The differences in the GLO1 gene frequencies between some of these populations are significant.