Human Red Cell Acid Phosphatase Research Articles

Human red cell acid phosphatase (ACP1): the primary structure of the two pairs of isozymes encoded by the ACP1*A and ACP1*C alleles.

The Af, As, Cf and Cs isozymes encoded by the human red cell acid phosphatase ACP1*A and ACP1*C alleles, respectively, have been sequenced. All four isozymes consist of a single non-glycosylated peptide chain (157 residues), acetylated at the amino-terminal alanine residue. Each f isozyme differs from the corresponding s isozyme over the sequence segment 40-73, while the remaining four-fifth of the molecules are identical. These findings are consistent with results for the Bf and Bs isozymes encoded by the common ACP1*B allele and confirm that the presence of a specific f or s segment is a common property to ACP1 isozymes. This supports our hypothesis that f and s isozymes are generated by alternative splicing of exons in the primary RNA transcript. Cf and Cs are identical in sequence with Bf and Bs, respectively. Thus, the ACP1*B and ACP1*C alleles encode exactly the same pair of isozymes, the only difference at the protein level being the ratio of f and s isozyme. Af and As differ from the Bf and Bs isozymes by a single substitution at residue 105; Arg and Gln, respectively. These observations explain the electrophoretic identity of the B and C isozyme pairs and the higher P(i) of the A isozyme pair.

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.

Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele.

The pair of isozymes, Bf and Bs, encoded by the human red cell acid phosphatase ACP1*B allele has been sequenced. Similar but not identical primary structures were observed. Both isozymes consist of a single peptide chain of 157 amino acid residues, which is acetylated at the amino-terminal alanine residue. The Bf and Bs isozymes are not glycosylated, and the calculated molecular masses are 17,932 and 17,867 Da, respectively. They are identical except for the sequence segment 40-73, which is peculiar to the respective isozyme. This is consistent with our hypothesis that the two isozymes are generated as the result of alternative splicing of the primary RNA transcript. The finding of a signature sequence offers the basis for the characteristic differences in catalytic and molecular properties of the Bf and Bs isozymes. A high degree of homology was found between the Bs isozyme and the 18-kDa cytosolic acid phosphatase from bovine liver. No homology was observed with other sequenced proteins, and this establishes these low molecular weight acid phosphatases as products of a distinct gene family.

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.

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 voltage isoelectric focusing for the study of red cell acid phosphatase polymorphism: Improved phenotyping and better agreement with the Hardy Weinberg distribution

A low voltage isoelectric focusing (IEF) method for typing human red cell acid phosphatase (ACPl) polymorphism is reported. A clear differentiation of the ACPl*A, ACPl*B and ACPl*C allelic products is obtained by minimizing diffusion of the bands during electrophoresis and during enzyme visualization by using a hydrophilic cellophane film soaked with the substrate solution, heated to 50 degrees C, and by reading without incubation in a moist chamber. A population study of ACPl, carried out with this method, on a sample of 624 unrelated individuals of the population of Rome (gene frequencies: ACPl*A = 0.267; ACPl*B = 0.680; ACPl*C = 0.053), showed a good agreement between the observed and expected phenotype distribution (chi 2/2 = 1.35; 0.70 greater than p greater than 0.50).

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 less than ACP1*B less than ACP1*C less than 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 less than ACP1*A less than ACP1*C, 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.

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.

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

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 2 M 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 greater than A greater than C is the same as found for the modulation of ACP1 by purines and folates.

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.

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

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 ACP1G is transmitted as an allele of ACP1.

5.7. Human red cell acid phosphatase: Differences in concentration of isozyme protein as the cause of phenotypic differences in enzyme activity

5.7. Human red cell acid phosphatase: Differences in concentration of isozyme protein as the cause of phenotypic differences in enzyme activity

Quantitation and characterization of human red cell acid phosphatase by immunoelectrophoresis

Quantitation and characterization of human red cell acid phosphatase by immunoelectrophoresis

Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography

1. 1.|In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsinic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b 1 and b 2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. 2.|Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acid in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. 3.| p- Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. K i values of 4 · 10 −4 and 6 · 10 −4 M were found for the b 1 and b 2 forms, respectively. 4. 4.|Coupling of p- aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

Deletion mapping: Further evidence for the location of acid phosphatase (ACP1) within 2p23

The human red cell acid phosphatase (ACP1) locus was assigned to region 2p23 leads to 2pter by Ferguson-Smith et al [3], more specifically to 2p23 by Hamerton et al [5]. We describe two unrelated patients with deletion of chromosome 2, with similar breakpoints in the distal portion of band p23 (del(2) (p23)). ACP1 typing in both patients revealed heterozygous BA phenotypes. Thus, we assign the locus for ACP1 to the distal portion of 2p23.

The rare silent gene P o of human red cell acid phosphatase in a second family in Poland

Abstract The authors describe a Polish family (three generations) with a silent gene P o of human red cell acid phosphatase. The phosphatase levels of 4 family members were about half the normal value.

The rare silent gene P o of human red cell acid phosphatase in a second family in poland

The authors describe a Polish family (three generations) with a silent gene P o of human red cell acid phosphatase. The phosphatase levels of 4 family members were about half the normal value.

Quantitative evidence of a silent gene P° of human red cell acid phosphatase in south Polish population

The distribution of red cell acid phosphatase types in 3244 unrelated Polish adults is reported. Gene frequencies P a = 0.3594, P b = 0.5643 and P c = 0.0763 were obtained. In a forensic case of disputed paternity an apparent mother/child incompatibility respect to red cell acid phosphatase was found, the mother appearing as type B and the child as type A. Determination of acid phosphatase activity suggested the presence of a silent gene P°. The phosphatase levels were about half the values expected, as determined in 237 adults representing the different phenotypes.

VARIATION IN THE NUMBER OF GENES CODING FOR SALIVARY AMYLASE IN THE BANK VOLE, CLETHRIONOMYS GLAREOLA

A Danish population of bank voles is polymorphic for three electrophoretically different salivary amylases; A, H and S, of which A is the most common. Both single-, double- and triple banded phenotypes were observed, and in several crosses two electrophoretic forms cosegregated. In addition to the qualitative variation, some individuals show consistent quantitative variation in the relative activities of their amylase bands. This variation has been qualified by spectrophotometrical measurements of the relative amounts of amylase protein in the various bands. --Seventy wild chromosomes were analyzed by determining the amounts of amylase they produced when heterozygous with a laboratory stock chromosome known to carry two closely linked amylase genes, both coding for a fourth electrophoretic variant, B. The amount of A-protein divided by half the amount of B-protein was used as an estimate of the number of A-genes on the tested chromosomes. The wild chromosomes fell into three clearly distinguishable classes: 9 clustered around a gene number estimate of one, 45 chromosomes yielded estimates around two genes, and the gene number estimate of the remaining 16 was close to three. The integer values of the gene number estimates and the cosegregation of electrophoretically different salivary amylases are consistent with the model that the population is polymorphic for chromosomes with either one, two, or three closely linked amylase genes. It is suggested that such gene number variation may be more common than generally recognized, and some other reported cases of quantitative enzyme variation, for instance that of human red cell acid phosphatase, are interpreted in terms of variation in the number of genes involved.