Enzyme polymorphism and clinical variability of diseases: a study of diabetes mellitus

We investigated possible relations among four common neonatal manifestations of diabetic pregnancy (macrosomia, hypoglycemia, hypocalcemia, jaundice) and four enzyme polymorphisms (PGM1, ADA, AK1, ACP1 in a sample of infants born of diabetic mothers. The pattern of associations observed between the two sets of variables is consistent with known differences in enzymatic activity within phenotypes of each system, suggesting that low enzymatic activity may have unfavorable effects on fetal development and on adaptability of the neonate to the extrauterine environment, Some of the polymorphic enzymes studied influence fetal growth in normal pregnancy as well. Analysis of relations between genetic polymorphisms and the clinical pattern of common diseases may provide a better understanding of the genetic basis of the clinical variability of diseases within and between human populations.     

Erythrocyte acid phosphatase (ACP1) activity

Summary Erythrocyte acid phosphatase (ACP₁) activity was determined in the absence of modulators and in the presence of either adenosine or inosine as modulators in 154 samples of red blood cells collected from adult donors. Adenosine and inosine showed modulating effects (activation), that were genotype dependent in the allele order p^bac; the activation by inosine was much higher than by adenosine. The modulating effect was dependent on adenosine deaminase (ADA) genotype: In carriers of ADA² allele the activation with ACP₁ phenotype A was lower and that with phenotypes CA and CB was higher than in ADA¹/ADA¹ subjects. In addition, the basic ACP₁ activity (i.e., without modulators) also appeared to be dependent on ADA genotype: The lowest ACP₁ activity was observed in A and BA subjects carrying the ADA² allele. Since the deamination of adenosine to inosine associated with ADA2-1 phenotype is slower than that associated with ADA1, the interaction of ADA on ACP₁ activity may in fact be explained by a lower intracellular concentration of inosine in ADA² carriers and, therefore, by a lower modulating effect of this on acid phosphatase activity.     

Duplication of 2p25: confirmation of the assignment of soluble acid phosphatase (ACP1) locus to 2p25

Summary The regional localization of the gene coding for soluble acid phosphatase (ACP₁) has been under debate in the two different chromosome regions, 2p23 or 2p25. Gene dosage studies in a case with a karyotype of 46,XX,dir dup(2) (p25.1→p25.3) showed that the ACP₁ activity was increased to 1.4 times the mean value of normal individuals with the same ACP₁ phenotype, while the level of soluble malate dehydrogenase (MDH₁) was normal. These gene dosage effects indicated that the ACP₁ gene locus can be mapped to 2p25.     

Three new phenotypes of human red cell acid phosphatase: ACP1FA,ACP1GA,and ACP1GB

Summary Three new phenotypes of human erythrocyte acid phosphatase (ACP₁) have been detected and found to be unique by direct comparison with previously identified ACP₁ variants. One of these new electrophoretic variants, labeled as ACP₁FA, has been detected in the Hispanic population of California. The electrophoretic variants identified as ACP₁GA and ACP₁GB have been detected in a black family in North Carolina. A family study has shown that ACP ₁ ^G is transmitted as an allele of ACP₁.     

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.     

Human red-cell acid phosphatase (ACP1): kinetic and thermodynamic characterization of the KUK variant.

The Km constant and the sensitivity to inhibitors were determined for the rare variant KUK of red cell acid phosphatase. At the same time, the thermodynamic energies of activation and of inactivation were measured. The kinetic parameters were not very different from those of the usual ACP1 C alloenzymes. However, they differed in their thermodynamic energies: the conformational structure of the ACP1 KUK protein being less stable.     

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     

Association between ACP(1) genetic polymorphism and favism

An association between favism (a hemolytic reaction to consumption of fava beans), glucose-6-phosphate dehydrogenase deficiency (G6PD(-)) and acid phosphatase locus 1 (ACP(1)) phenotypes has been reported; the frequency of carriers of the p(a) and p(c) ACP(1) alleles was found to be significantly higher in G6PD(-) individuals showing favism than in the general population. Here, we investigated the hypothesis that favism is caused by toxic Vicia faba substances, which in some ACP(1) phenotypes cause increased phosphorylation and consequently increased glycolysis, with strong reduction in reduced glutathione production, resulting in hemolysis. It has been demonstrated that ACP(1) f isoforms have physiological functions different from those of s isoforms and are responsible for most of the phosphatase activity, in addition to being less stable in the presence of oxidizing molecules. Thus, the C, CA and A phenotypes, characterized by lower concentrations of f isoforms, could be more susceptible to damage by oxidative events compared to the other phenotypes. To test this hypothesis, the (f+s) enzymatic activity of different ACP(1) phenotypes with and without added V. faba extract was analyzed. Enzymatic activities of ACP(1) A, -CA, -C groups (low activity) and -B, -BA, -CB groups (high activity) were significantly different after addition of V. faba extract. Phenotypes A, CA and C had extremely low enzymatic activity levels, which would lead to low levels of reduced glutathione and bring about erythrocyte lysis.     

Human red cell acid phosphatase (ACP1): Evidence for differences in the primary structure of the two isozymes encoded by the ACP1*B allele

Molecular properties of the two isozymes expressed by the B allele at the red cell acid phosphatase locus (ACP1) have been studied to distinguish between possible mechanisms for their production. The difference in electric charge exhibited by the native isozymes was retained under denaturing conditions; the unfolded peptide chains renatured without conversion of one form to the other. Chromatographic analysis [thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC)] of tryptic digests showed 12 peptides common to both isozymes but also revealed 5 peptides unique to one isozyme and 3 (possibly 4) peptides unique to the other. These findings argue against both conformational isomerization and simple posttranslational modification as the mechanism of generation of the two isozymes. We suggest that the two isozymes are synthesized as discrete molecular entities.     

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.     

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.     

Genetic variation of an acid phosphatase (Acp-2) in the laboratory rat: Possible homology with mouse AP-1 and human ACP2

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in the rat (Rattus norvegicus) is described. The locus, designed Acp-2, is not expressed in erythrocytes but is expressed in all other tissues studied. The product of Acp-2 hydrolyzes a wide variety of phosphate monoesters and is inhibited by l(+)-tartaric acid. Inbred rat strains have fixed either allele Acp-2^a or allele Acp-2^b. Codominant expression is observed in the respective F₁ hybrids. Backcross progenies revealed the expected 1:1 segregation ratio. Possible loose linkage was found between the Acp-2 and the Pep-3 gene loci at a recombination frequency of 0.36±0.06.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/15) and by a grant from the Alexander-von-Humboldt-Stiftung (VB2-FLF).     

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.     

Neuromedin beta: P73T polymorphism in overweight and obese subjects

Neuromedin beta (NMB) is a member of the bombesin-like peptide family expressed in brain, gastrointestinal tract, pancreas, adrenals and adipose tissue. The aim of our study was to compare the frequency of P73T polymorphism in overweight and obese patients (37 men: age 50.6+/-11.7 years, BMI 41.1+/-7.8 kg/m(2); 255 women: age 49.0+/-11.9 years, BMI 37.9+/-6.8 kg/m(2)) with that of healthy normal weight subjects (51 men: age 28.2+/-7.1 years, BMI 22.3+/-2.0 kg/m(2); 104 women: age 29.1+/-9.1 years, BMI 21.5+/-1.9 kg/m(2)) and to investigate the polymorphism's influence on anthropometric, nutritional and psychobehavioral parameters in overweight/obese patients both at the baseline examination and at a control visit carried out 2.5 years later, regardless of the patient s compliance with the weight reduction program. No significant differences in the genotype distribution were demonstrated between normal weight and overweight/obese subjects. Male T allele non-carriers compared to T allele carriers had higher energy (p=0.009), protein (p=0.018) and fat (p=0.002) intakes and hunger score (p=0.015) at the beginning of treatment. Male T allele non-carriers had a more favorable response to weight management at the follow-up, as they exhibited a significant reduction in waist circumference, energy intake and depression score as well as a significant increase in dietary restraint. No significant differences between carriers and non-carriers were demonstrated in women at the baseline examination. Both female T allele carriers and non-carriers demonstrated similar significant changes in nutritional parameters and in restraint score at the follow-up. Nevertheless, only female non-carriers showed a significant decrease in the hunger score.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

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.