Human red cell acid phosphatase: purification and properties of the A, B and C isozymes

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

Association of acid phosphatase locus 1*C allele with the risk of cardiovascular events in rheumatoid arthritis patients

Introduction

Acid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in a number of different biological functions in the cell. The aim of this study was to determine the contribution of ACP1 polymorphisms to susceptibility to rheumatoid arthritis (RA), as well as the potential contribution of these polymorphisms to the increased risk of cardiovascular disease (CV) observed in RA patients.

Methods

A set of 1,603 Spanish RA patients and 1,877 healthy controls were included in the study. Information related to the presence/absence of CV events was obtained from 1,284 of these participants. All individuals were genotyped for four ACP1 single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247, and rs3828329, using a predesigned TaqMan SNP genotyping assay. Classical ACP1 alleles (*A, *B and *C) were imputed with SNP data.

Results

No association between ACP1 gene polymorphisms and susceptibility to RA was observed. However, when RA patients were stratified according to the presence or absence of CV events, an association between rs11553742*T and CV events was found (P = 0.012, odds ratio (OR) = 2.62 (1.24 to 5.53)). Likewise, the ACP1*C allele showed evidence of association with CV events in patients with RA (P = 0.024, OR = 2.43).

Conclusions

Our data show that the ACP1*C allele influences the risk of CV events in patients with RA.     

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.     

Chinook salmon NADP+-dependent cytosolic isocitrate dehydrogenase: Electrophoretic and genetic dissection of a complex isozyme system and geographic patterns of variation

Species in the genus Oncorhynchus express complicated isocitrate dehydrogenase (IDHP) isozyme patterns in many tissues. Subcellular localization experiments show that the electrophoretically distinct isozymes of low anodal mobility expressed predominantly in skeletal and heart muscle are mitochondrial forms (mIDHP), while the more anodal, complex isolocus isozyme system predominant in liver and eye is cytosolic (sIDHP). The two loci encoding sIDHP isozymes are considered isoloci because the most common allele at one of these loci cannot be separated electrophoretically from the most common allele of the other. Over 12 electrophoretically detectable alleles are segregating at the two sIDHP* loci in chinook salmon. Careful electrophoretic comparisons of the sIDHP isozyme patterns of muscle, eye, and liver extracts of heterozygotes reveal marked differences between the tissues with regard to both relative isozyme staining and the expression of several common alleles. Presumed single-dose heterozygotes at the sIDHP isolocus isozyme system exhibit approximate 9:6:1 ratios of staining intensity in liver and eye, while they exhibit approximate 1:2:1 ratios in skeletal muscle. The former proportions are consistent with the equal expression of two loci (isolocus expression), while the latter are consistent with the expression of a single locus. Screening of over 10,000 fish from spawning populations and mixed-stock fishery samples revealed that certain variant alleles (*127, *50) are detectable only in liver and eye, while other alleles (*129, *94, and *74) are strongly expressed in muscle, eye, and liver. The simplest explanation for these observations is that the "isolocus" sIDHP system of chinook salmon (and that of steelhead and rainbow trout) results from the expression of two distinct loci (sIDHP-1* and sIDHP-2*) that have the same common allele (as defined by electrophoretic mobility). IDHP expression in skeletal muscle is due to the nearly exclusive expression of the sIDHP-1* locus, while IDHP expression in eye and liver tissues is due to high levels of expression of both sIDHP-1* and sIDHP-2*--giving rise to the isolocus situation in these latter tissues. Direct inheritance studies confirm this model of two genetically independent (disomic) loci encoding sIDHP in chinook salmon. Extensive geographic surveys of chinook salmon populations from California to British Columbia reveal marked differences in allele frequencies at both sIDHP-1* and sIDHP-2* and considerably more interpopulation differentiation than was recognized previously when sIDHP was treated as an isolocus system with only five recognized alleles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Comparative studies on the properties of the different isozymes

The properties of the isozymes of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) found in unfertilized frog egg have been compared to those found in adult tissues of Rana pipiens. Chromatographic, kinetic, and electrophoretic data indicate that, of the five electrophoretic forms found in egg, the isozyme with the least anodic mobility (isozyme I) is the same molecular species as the only isozyme found in heart, and the egg isozyme with the greatest anodic mobility (isozyme V) is identical to the major isozyme found in liver.The activity of egg isozyme I was markedly inhibited by the antibody to the skeletal muscle enzyme, which has been shown previously to cross-react with the cardiac enzyme, but was unaffected by the antibody to liver isozyme V; the opposite effects were observed with egg isozyme V. The antibody to the skeletal muscle enzyme inhibited egg isozymes II > III > IV whereas the antibody to the liver enzyme gave the reverse inhibitory pattern, e.g., isozyme IV > III > II.In vitro dissociation-reassociation of mixtures of isozyme I and V led to the formation of the other three isozymes. Similar experiments performed individually with either egg isozyme III or IV resulted in the production of predominantly isozymes III, II, and I due to the instability of isozyme V during the hybridization procedure.The above results indicate that isozymes I and V are tetramers of the respective parental subunits and that isozymes II, III, and IV are hybrid molecules with subunit assignments of (I₃V₁), I₂V₂), and (I₁V₃), respectively.     

Erythrocyte acid phosphatase: species specificity in activity modulation by purine analogs

The previous studies of the interaction of purine analogs and human erythrocyte acid phosphatase isozymes were extended to include erythrocyte acid phosphatase from seven other species. Consistent responses, similar to the observations with the several genotypically different human isozymes, were observed. The isozyme from chimpanzee erythrocytes was similar to the human B-type isozyme while the baboon and cow isozymes were at the other extreme in responsiveness and were more divergent from the B-isozyme than was the human A-type isozyme. The ACP from rabbit, dog, sheep and rhesus erythrocytes exhibited intermediate levels of responsiveness but did differ from the human A-type isozyme. Additional studies indicated some differences between the responsiveness of the partially purified erythrocyte enzyme and the low molecular weight ACP from liver.     

A new variant glyoxalase I allele that is readily detectable in stimulated lymphocytes and lymphoblastoid cell lines but not in circulating lymphocytes or erythrocytes

We describe an allele of the human glyoxalase GLO locus that encodes an enzymatically inactive form of the protein, which would not have been detected if only circulating erythrocytes and lymphocytes had been studied. The new allele is named GLO*3 and its protein product, GLO 3. Circulating blood cells of GLO*2/GLO*3 heterozygotes have just one electrophoretic band that migrates as the normal 2-2 dimer. Lymphoblastoid cell lines and phytohemagglutinin-stimulated lymphocytes from the same individuals have two electrophoretic bands, one with the mobility of the 2-2 dimer and one with the mobility of the 2-1 dimer that is present in GLO*2/GLO*1 heterozygotes, but a band with the mobility of the 1-1 dimer is not present. Therefore, the GLO*3 allele encodes a monomer that has the electrophoretic mobility of GLO 1 but is enzymatically inactive unless it is combined with normal monomers in 2-3 and 1-3 heterodimers. The failure to detect the GLO 3 protein in red cells and unstimulated lymphocytes is attributed to a relatively great instability or small rate of production in those cells. Consistent with this interpretation is the reduction of GLO activity in red cells of GLO*2/GLO*3 and GLO*1/GLO*3 heterozygotes to 65% or less of that in normal homozygotes and heterozygotes, while the activity of GLO*3 heterozygous lymphoblastoid cells is about 80% of normal. In contrast, the GLO activity of lymphoblastoid cells that had one copy of the GLO locus deleted by γ-irradiation was 50%–60% of normal. Our observations indicate that certain kinds of mutant alleles of the GLO locus, and perhaps other loci, may not be detected in electrophoretic surveys on circulating blood cells only. The segregation of alleles that are not expressed in circulating red and white blood cells could confuse attempts to determine parentage, as they might have in the family described here. The observations also demonstrate the feasibility of mapping human genes by using ionizing radiation to create partial chromosome deletions in cultured cells.     

Discrimination of walleye, Stizostedion vitreum vitreum (Mitchill), stocks by isozyme analysis with cellulose acetate

Summary. Five walleye, Stizosfedion vitreum vitreum (Mitchill), populations from within the state of Minnesota were examined for isozyme gene frequencies. Of 26 loci identified and scored, nine polymorphic loci were found. The ADH*, MDH-3* and PROT-4* loci were found to be the most informative for distinguishing the populations. A north-south cline in the frequency of PROT-4* alleles was found, with a higher frequency of the fast allele being found in the southern population. This is probably a reflection of genetic isolation caused by glaciation. Cellulose acetate was used to resolve the isozymes. The advantages of cellulose acetate over starch in terms of decreased run time, smaller sample and stain volumes and superior resolution advocate the use of cellulose acetate for fish isozyme analyses. Comparisons between starch and cellulose acetate separations showed similar results with some exceptions. Cellulose acetate resolved FH* and PROT-2* polymorphisms that were not resolved on starch, but failed to resolve the MDH-1 locus. These differences did not affect the results.     

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.     

Genetics of sunflower alcohol dehydrogenase: Adh 2 ,nonlinkage to Adh 1 and Adh 1 early alleles

Alcohol dehydrogenase (ADH) isozymes in annual sunflowers (Helianthus annuus) are dimers whose subunits are produced by two genes, Adh ₁ and Adh ₂ .The codominant F and S alleles of Adh ₁ produce the slower-migrating set of three isozymes. The faster-migrating set of three isozymes is controlled by Adh ₂ , which also has at least two alleles, F and S. Hybridization experiments indicated that the Adh ₂ alleles segregate in expected Mendelian fashion and that Adh ₁ and Adh ₂ are not linked. A third common 1-locus allele is designated early (E) because when homozygous it results in a blank at the 1FF isozyme position in mature seeds, but in developing seeds produces a normal-appearing band at the 1FF position. Hybridization studies showed that the early alleles segregated normally. Correlation between genotype and presence or absence of isozymes electrophoretically intermediate between those of Adh ₁ and Adh ₂ suggests that four intergenic isozymes may be formed as a result of dimerization of the four basic subunits. Studies of zymograms of developing seeds suggest that the remaining but inconstant zymogram bands are mature seed isozymes which have altered charges during early morphogenesis and thus are developmental artifacts.     

Enzyme polymorphism and clinical variability of diseases: study of acid phosphatase locus 1 (ACP1) in obese subjects

The ACP1*A allele of erythrocyte acid phosphatase (ACP1) has a lower enzymatic activity when compared to other ACP1 alleles and is associated with maximal rate of body growth during intrauterine life. In three different samples of obese subjects (total number = 218). ACP1*A was associated with extreme body mass deviations. No difference in ACP1 allele distribution was observed between obese and nonobese subjects. These data suggest that a genetically determined variability of ACP1 influences the degree of obesity, but only when obesity itself has been triggered by some other factors.     

Genetic Polymorphism of the Blood Groups and Erythrocytic Enzymes in Three Ethnic Territorial Groups from the North of European Russia

Using the data on five red cell markers (AB0, PGM1, ACP1, GLO1, and ESD) polymorphisms, the population genetic structure of three ethnic territorial groups from the north of European Russia (Continental Nentsy, Kola Saami, and Russian Coast-dwellers) was described. In general, the groups studied a Caucasoid pattern of the frequency distribution of erythrocytic marker alleles. However, a substantial contribution of a Mongoloid component to the Nenets gene pool, expressed as a high frequency of the PGM1*1allele along with a low frequency of the GLO1*1allele, was observed. Three ethnic territorial groups examined were close to one another with respect to the distribution of classical biochemical markers. The interpopulation diversity was low (the mean F _ST= 0.015). The differences observed were for the most part caused by the genetic characteristics of Nentsy. The maximum interpopulation diversity was observed for the GLO1*locus (F _ST= 0.056).     

Occurrence of the ACP 1 0 allele in Czechoslovakia

Summary During a paternity test an unexpected type of red cell acid phosphatase isozyme (ACP₁) was found in one family. The mother was of type A and type B was diagnosed in the son. The whole family was then subjected to ACP₁ phenotyping and to the enzyme assay. Five members of the family were found to have unexpected types of ACP₁ isozymes. The average activity was approx. 50% of normal values. It is presumed that a silent ACP ₁ ⁰ allele was found in the family investigated and that the grandfather was its first carrier.     

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.     

Alcohol dehydrogenase isozymes inGossypium hirsutum and its putative diploid progenitors: The biochemical consequences of enzyme multiplicity

Electrophoretic variation in alcohol dehydrogenase (ADH) was examined in tetraploidGossypium hirsutum and its putative diploid progenitorsG. ramondii, G. herbaceum, and a close relative,G. arboreum. All the diploids had three isozymes, while strains of the tetraploidG. hirsutum had either 4 or 6. Each isozyme was eluted from starch gels and significant differences in activity were noted between several of the isozymes relative to pH, substrate, temperature and salinity. This suggests that an increase in enzyme heterozygosity can result in higher levels of developmental homeostasis, but it depends on the isozyme alleles involved. Michigan Agricultural Experiment Station Journal Article No. 10379.     

Isozyme gene markers in Vicia faba L.

Summary This study was conducted to assess the genetic basis of the variability observed for the glutamate oxaloacetate transaminase (GOT), Superoxide dismutase (SOD), esterase (EST), and malate dehydrogenase (MDH) isozyme systems in different open-pollinated Vicia faba varieties. Individual plants showing contrasting zymogram patterns were simultaneously selfed and cross-combined. Crossing was unsuccessful in producing progeny, and only selfed progenies were suitable for genetical analysis of isozyme variability. Three zones of GOT activity were made visible. The isozyme of GOT-2 and GOT-3 zones were dimeric and under the control of three alleles at the Got-2 locus and two alleles at the Got-3 locus, respectively. The isozymes of the GOT-1 zone did not show any variability. Three zones of SOD isozyme activity were made visible. The isozymes occurring in the SOD-1 (chloroplastic isozyme form) and SOD-2 (cytosol isozyme form) zones were dimeric and under the control of two alleles at the Sod-1 and Sod-2 loci. The isozyme visualized in the SOD-3 zone (mitochondrial isozyme form) were tetrameric and under the control of two alleles at the Sod-3 locus. Apparently the isozymes made visible in the most anodal esterase zones EST-1, EST-2, and EST-3 were monomeric, and the occurrence of two alleles at each of two different loci explained the variability observed in the EST-2 and EST-3 zones. For MDH, only two five-banded zymogram pattern types were found, and every selfed progeny showed only one of the two zymogram type, indicating that each individual possessed fixed alleles at the loci controlling MDH isozyme. Got-2, Got-3, Sod-1, Sod-2, and Sod-3 appear to be five new isozyme gene markers that can be useful in Vicia faba breeding for linkage study, varietal fingerprinting, outcrossing rate estimate, and indirect selection for quantitative characters.     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.This research was supported by the CNPq