ACP1GUA-1--a low-activity variant of human erythrocyte acid phosphatase: association with increased glutathione reductase activity.

ACP1GUA-1, a variant of human erythrocyte acid phosphatase, exists as a polymorphism (allele frequency of .132) in the Guaymi Indians of Central America. This variant has an electrophoretic mobility similar to the common B- and C-type variants, but individuals of the ACP1GUA-1 phenotype have a level of enzyme activity only 27% of the activity expected for the ACP1C variant. The GUA-1 variant is more thermostable than is the B variant, and the order of responsiveness to the modulation of activity by purine analogs and folate is always (B)-(A)-(GUA-1). Thus, the GUA-1 variant is a low-activity variant with C-like regulatory properties. Erythrocytes from individuals of the ACP1GUA-1 phenotype have increased basal levels of glutathione reductase, and a larger fraction of the glutathione reductase protein is present as the holoenzyme, indicating increased levels of flavin adenine dinucleotide in the erythrocytes of these individuals. This is consistent with the suggestion that ACP1 has a physiological function as a flavin mononucleotide phosphatase.     

ACP1 and human adaptability 1. Association with common diseases: a case-control study

Human red cell acid phosphatase (ACP1) is a polymorphic enzyme closely related to cytosolic low molecular weight acid phosphatases, a protein family broadly conserved among eukaryotes. Two different functions have been proposed for ACP1: flavin mononucleotide (FMN) phosphatase and phosphotyrosine phosphatase (PTPase). Given that genetic variants of ACP1 activity are common, the enzyme could have a role in regulating a large spectrum of cellular functions and, in turn, disease susceptibility. In the present paper we report a study of ACP1 genetic polymorphism in 1088 normal subjects and in 1267 subjects from the population of Rome admitted to hospital for a number of common diseases. All ACP1 parameters investigated show highly significant differences among samples, suggesting that the enzyme may have a significant role in some of the diseases considered. In particular, consistent associations of ACP1 with developmental disturbances and with hemolytic favism have been observed. In the majority of diseases showing association with ACP1, only one of the two ACP1 isoforms, f and s, is involved, supporting the hypothesis of a functional differentiation between the two enzymatic fractions.     

Phenotype dependence in the inhibition of red cell acid phosphatase (ACP) by folates.

Red cell acid phosphatase (ACP) is shown to be inhibited by folic acid and various folates. The degree of inhibition is phenotype dependent with a pattern of variation differing from that of the well recognized variation in red cell activity levels. The pattern of variation is ordered ACP1B less than ACP1A less than ACP1C in terms of the relative allelic contributions to the observed inhibition. This pattern correlates with previously observed patterns of risk for two hemolytic disorders and may thus provide a key to their understanding.     

Human red-cell acid phosphatase (ACP1): a new mutant (ACP1*KUK) detected by isoelectric focusing, kinetics of thermostability and substrate activity

A new rare mutant of the red-cell acid phosphatase (ACP1) is described using conventional gel electrophoresis and isoelectric focusing migration. According to the electrophoretic patterns obtained, the new mutant ACP1* KUK is different from the ACP* H and ACP1* A' variants already described. The enzyme activities and the thermostability curves definitively confirm the existence of a new variant. The transmission of this mutant was followed through a pedigree of three generations. The family originated from Czechoslovakia. The frequency of the variant is probably less than 0.001.     

Low birth weight and allergy: possible pleiotropic effect of ACP1

The well-known relationship between low birth weight and allergies prompted us to investigate a possible pleiotropic effect of ACP1 on these conditions. ACP1 is a polymorphic enzyme that affects signal transduction of insulin and other growth factors, T-cell receptor signaling, and the regulation of flavoenzyme activity. Our aim was to compare the relationship between ACP1 and allergy with the relationship between ACP1 and birth weight. We studied 299 subjects from the Caucasian population of England, 124 subjects from the Caucasian population of central Italy, and 302 healthy puerperae and their newborn babies from the same Caucasian populations. ACP1 phenotype was determined by starch gel electrophoresis on RBC hemolysate and by DNA analysis. Subjects with high ACP1 activity (ACP1 C,B phenotype) show a lower level of IgE compared to subjects with low ACP1 activity (p = 0.01). The proportion of infants with a birth weight below the first quartile is lower among infants born to mothers with high ACP1 activity than among infants born to mothers with medium-low activity (p = 0.01). The data suggest a protective effect of high-activity ACP1 C,B phenotype from low birth weight and from allergic manifestations after birth.     

Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn)

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.     

Comparison of acid phosphatase ACP1 variants by isoelectric focusing and conventional electrophoresis: identification of three new alleles, ACP1*N, ACP1*P and ACP1*S

Three new alleles of human red cell acid phosphatase (ACP1) have been identified by comparison with previously reported variants using three different electrophoretic techniques. Family data are available on all the variants and show genetic transmission of the rare alleles ACP1*N, ACP1*P and ACP1*S. Further evidence of a rare allele demonstrating reversed 'A' activity is also described. The report documents the need to use several electrophoretic techniques to characterize new or rare variants.     

Functional characterization of Ape1 variants identified in the human population

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5′ to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited ~40–60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit ~10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.     

Modeling single nucleotide polymorphisms in the human AKR1C1 and AKR1C2 genes: implications for functional and genotyping analyses

Enzymes encoded by the AKR1C1 and AKR1C2 genes are responsible for the metabolism of progesterone and 5α-dihydrotestosterone (DHT), respectively. The effect of amino acid substitutions, resulting from single nucleotide polymorphisms (SNPs) in the AKR1C2 gene, on the enzyme kinetics of the AKR1C2 gene product were determined experimentally by Takashi et al. In this paper, we used homology modeling to predict and analyze the structure of AKR1C1 and AKR1C2 genetic variants. The experimental reduction in enzyme activity in the AKR1C2 variants F46Y and L172Q, as determined by Takahashi et al., is predicted to be due to increased instability in cofactor binding, caused by disruptions to the hydrogen bonds between NADP and AKR1C2, resulting from the insertion of polar residues into largely non-polar environments near the site of cofactor binding. Other AKR1C2 variants were shown to involve either conservative substitutions or changes taking place on the surface of the molecule and distant from the active site, confirming the experimental finding of Takahashi et al. that these variants do not result in any statistically significant reduction in enzyme activity. The AKR1C1 R258C variant is predicted to have no effect on enzyme activity for similar reasons. Thus, we provide further insight into the molecular mechanism of the enzyme kinetics of these proteins. Our data also highlight previously reported difficulties with online databases.     

Human spleen histone H1. Isolation and amino acid sequences of three minor variants, H1a, H1c, and H1d

Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminal sequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21,233 for the alanine variant and 21,261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22,178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22,218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.     

Immunological and structural relatedness of isozymes and genetic variants of 3-phosphoglycerate kinase from the mouse

Isozymes (PGK-1 and PGK-2) and genetic variants (PGK-2A, PGK-2B, and PGK-2C) of 3-phosphoglycerate kinase were purified by affinity chromatography using an 8-(6-aminohexyl)-amino-ATP-Sepharose column as the key step. Antisera raised against purified PGK-1 and PGK-2A were tested for specificity and cross-reactivity by application of double immunodiffusion and enzyme immunoinactivation methods. By double immunodiffusion, no precipitin lines were observed between anti-PGK-2A and PGK-1, but a weak cross-reactivity between anti-PGK-1 and PGK-2A was detected. In addition to specific inhibition of PGK-1 and PGK-2A by their respective antisera, anti-PGK-1 was shown to inhibit PGK-2 activity at high antiserum concentrations, whereas no inhibition of PGK-1 activity by anti-PGK-2A was observed. The amino acid compositions of PGK-1 and PGK-2 revealed a certain degree of homology. However, tryptic peptide maps showed no obvious similarity in the peptide spots between these two 3-phosphoglycerate kinase isozymes. Three electrophoretic variants of PGK-2 were compared biochemically and immunologically. PGK-2C from C57L/J mice, a low activity variant, was shown to be the result of a structural gene mutation that affects the active site of the enzyme.     

Report and characterization of a new variant, EB, of human red cell acid phosphatase.

A new variant phenotype of human red cell acid phosphatase, designated EB, was discovered in a male during a survey of blood donors from Copenhagen, Denmark. Electrophoretically, the variant revealed the two isozymes corresponding to the B type as well as two fast moving anodic isozymes. The enzyme activity and thermostability were found to be higher than in any earlier reported type. Isoelectric focusing of the variant type indicated that the isoelectric point of the variant enzyme is lower than in the common types.     

Naturally occurring quantitative variants of acid phosphatase-1 in Drosophila melanogaster

We have examined 111 wild Drosophila melanogaster lines for cis-acting quantitative variants of the Acph-1 gene, which codes for acid phosphatase-1 (ACPH). Three variants with obvious, reproducible phenotypes were isolated. All variants acted equally on all tissues and developmental stages examined. No recombinants were detected between one quantitative variant and the site determining the electrophoretic mobility of Acph-1 among 3885 flies examined. Several enzymatic properties of the variant enzymes were tested, including the K _m values for two substrates, inhibition by three different inhibitors, and thermal stability; the variant enzymes behaved identically to the wild-type enzyme in all cases. Immunological titration experiments showed that the variant enzymes had the same enzyme activity per molecule of ACPH as the wild-type enzyme. These results suggest that the quantitative variants we have identified are altered in the regulatory portion of Acph-1 so as to produce altered numbers of normal ACPH molecules.This work was supported by NIH Grant 21548. MAJ was supported by NIH Predoctoral Training Grant GM07413.     

Immunochemical characterization of human red cell acid phosphatase isozymes

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A < ACP1*B < ACP1*C < ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1* B < ACP1*A < ACP1*, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.     

Phenotypic variation in the phosphotransferase activity of human red cell acid phosphatase (ACP1)

Summary Red cell acid phosphatase (ACP1) catalyses the transfer of phosphate from phosphate ester substrates to suitable acceptor alcohols such as methanol and glycerol. The rate of substrate turnover in the presence of acceptors is increased by the increment of the phosphotransferase reaction, thus allowing this activity to be measured. There is specificity with regard to acceptors: (a) polyols (e.g., glycerol) are better acceptors than the corresponding n-alcohols, and (b) polyol configuration and chain length determine acceptor activity. Ribitol was the most efficient acceptor found. Each of the three common ACP1 alleles is represented electrophoretically by two isozyme bands; the phosphotransferase activity of the anodal isozyme was found to be more than twice that of the cathodal isozyme. The extent of phosphotransferase activity is also genotype dependent. In the presence of 2M glycerol, the relative phosphotransferase efficiencies for the three homozygote types were: ACP1^*B=3.7, ACP1^*A=3.4, and ACP1^*C =2.5. This pattern of B>A>C is the same as found for the modulation of ACP1 by purines and folates.Publication no. 278 of the Forensic Science Group, School of Public Health, University of California     

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.     

Phosphoglucomutase-1 polymorphism among Chinese in Taiwan.

Phosphoglucomutase-1 (PGM1) phenotyping of 1,128 Chinese blood donors was performed by thin-layer isoelectric focusing on agarose. The PGM1 gene frequencies were: 1A, 0.6005; 1B, 0.1500; 2A, 0.1510; 2B, 0.0973, and rare variants, 0.0058. The rare variants found in this series were PGM1 W21, W2, W3, W6 and W9 (or W10) with PGM1 W21 being the most common variant among Chinese with a phenotype frequency of 0.8%.     

Identification of base substitutions in ten types of rare variants of phosphoglucomutase-1 (PGM1) encountered in Japanese.

In a previous starch-gel electrophoresis study of erythrocyte phosphoglucomutase-1 (PGM1) in 23,095 Japanese from Hiroshima and Nagasaki, we detected 14 types of rare variant alleles. To determine sequence differences in these rare alleles, cell lines were established from peripheral B-lymphocytes from 24 unrelated individuals in whom nine types of rare variants are presumed to exist on the basis of earlier electrophoresis studies. cDNAs reverse transcribed from mRNAs extracted from these cell lines were amplified by polymerase chain reaction and sequences determined. Amino acid substitution types were deduced from each cDNA sequence. Although two individuals were reported to have an identical electromorph (PGM1 4HR3), sequence analysis revealed that alleles encoding these electromorphs possessed different base substitutions, and one was renamed PGM1 4HR4. As the amino acid substitution of ten different variants could be deduced by cDNA sequence in this study, the effect of each amino acid substitution on enzyme activity could be precisely simulated. The secondary structure of each variant predicted by computer simulations revealed that very decreased activity observed on PGM1 4HR2 protein was caused by significant secondary structure change introduced by the amino acid substitution. On the basis of the crystal structure, the amino acid substitutions of the ten types of rare variants seem to be outside the active center of this enzyme.     

Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.     

Effect of ACP1*C on early life viability

Associations with past malarial morbidity, season of conception, and common diseases such as obesity, type 2 diabetes, and allergy argue against neutrality of the ACP1 genetic polymorphism. Comparison of ACP1 distribution in mothers and their newborns and analysis of the joint wife-husband ACP1 phenotype distribution in couples with repeated spontaneous abortion suggest a negative effect of the ACP1*C allele on early life viability. Analysis of the polymorphism of the ACP1 gene suggests that, unlike the ACP1*A and ACP1*B alleles, the ACP1*C allele is independent of sequences in the 5' flanking region, resulting in an inverted F/S isoform ratio.