Genetic polymorphism of erythrocyte esterase-D in pigs

The erythrocyte esterase-D (Es-D) of four European-American pig breeds, two Taiwan native pigs (Taoyuan and Short ear breeds), Philippine native pig and wild boar populations (Sus scrofa riukiuanus) in Japan was investigated by using starch gel electrophoresis. Analysis of parentage records of the pigs revealed that the phenotypic variation of erythrocyte esterase-D was controlled by two codomi-nant alleles Es-D^A and Es-D^B. The allele Es-D^b was mostly found in three European -American pig breeds, Landrace (0.100), Hampshire (0.135) and Duroc (0.102). All of the wild boar populations were monomorphic for allele Es-D^A.     

Serum alkaline phosphatase polymorphism in pigs

Genetic variants of serum alkaline phosphatase were studied by the method of starch gel electrophoresis in the Z?otnicka Pstra breed of pigs. Two regions of alkaline phosphatase migration were observed. A single fraction in region I and four different phenotypes: AB, B, BC and BD in region II, were found. For AB, B and BC phenotypes the genetic control by three alleles AkpA, AkpB and AkpC is suggested. The observed segregation ratios in some cases deviated significantly from the expected ones.     

Developmental modification and hybridization of allelic acid phosphatase isozymes in homo- and heterozygotes for the Acp-1 locus in rice

A number of alleles each specified a set of three major and three minor bands of acid phosphatase (E.C. 3.1.3.2) in wild and cultivated rice strains. Relative intensity of the major bands was found to differ significantly according to the developmental stages of the leaves, suggesting the presence of protein modification genes. In heterozygotes, six parental and three hybrid major bands were clearly observed in most of the heterozygotes, but the intensities of the hybrid bands were found to be generally lower than those theoretically expected due to random association of enzyme subunits. The cause of this phenomenon is discussed.Contribution No. 1342 from the National Institute of Genetics.     

Canine plasma alkaline phosphatase polymorphism and its relationship with the canine Tr blood group system

A canine blood group antigen, QN, which was detected by a naturally occurring alloantibody in the antiglobulin test, was shown to be antigenically related to the human A and cattle J antigens by absorption experiments. Family studies supported a dominant mode of inheritance with the gene controlling the production of QN being dominant to the gene responsible for its absence. Frequencies of the QN and Tr antigens and serological data strongly suggested that the two antigens are identical. Two canine plasma alkaline phosphatase variants, F and S, were detected by starch gel electrophoresis, pH 8.65. Assuming genetic control of two codominant alleles, ALPF and ALPS, the distributions of types in families differed significantly from expectation. A relationship between the ALP and Tr(QN) systems was demonstrated with Tr-positive animals having a significant deficiency of S alkaline phosphatase types.     

Catalytic properties and stability of three common variants of placental alkaline phosphatase

Three placental alkaline phosphatases purified to homogeneity, i.e., the F, I, and S variants, were investigated for catalytic and stability properties. All three forms of the enzyme were found to have almost identical pH optima (10.7–10.8), similar sensitivity to the uncompetitive inhibitors L-phenylalanine (70%) and L-leucine (30%), and identical K_m values against p-nitrophenylphosphate, -glycerophosphate, and -naphthylphosphate. Significant differences among the three types were observed in thermal stability. The F variant was found to be most stable and the I variant most labile at 79 C. At 70 C all three forms were stable.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 4217 and 03X-2725), from the Medical Faculty, University of Umeå, and Jubileumsklinikens i Umeå forskningsfond.     

Polymorphism of acid phosphatase in chicken leucocytes

Three individual variants of acid phosphatase in chicken leucocytes were found by means of starch gel electrophoresis. The phenotype in leucocytes showed the same appearance as polymorphic forms of liver acid phosphatase in the same bird. The study of the Hardy-Weinberg distribution of the phenotypes of acid phosphatase in leucocytes also indicated that they are controlled by the same pair of codominant autosomal alleles as the phenotypes in the liver.
Acid phosphatase is polymorphic in all six strains of chickens studied.     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

Intestinal leucine aminopeptidase and alkaline phosphatase: Genetic regulation and development in mice

Intestinal and serum leucine aminopeptidase (LAP) and alkaline phosphatase (AKP) were characterized by electrophoresis for eight inbred strains of laboratory mice. Intestinal LAP and AKP of adult mice were expressed concordantly within strains, as banded or diffuse, and concordantly for rate of migration within strains that had diffuse isozymes. All strains, except DD/S, had a single band of serum LAP and a single, diffuse zone of serum AKP. DD/S had a double band of serum LAP as well as isozymes of intestinal LAP and AKP unlike those of other strains. All strains displayed similar, neuraminidase-sensitive isozymes of intestinal LAP and of AKP prior to weaning, but after weaning there was marked sensitivity to neuraminidase only in DD/S. In interstrain crosses, banded/diffuse, migration rate, and neuraminidase sensitivity were inherited as independent autosomal traits, with indications of variable penetrance and genetic interaction. Support was provided by NIH Grant RR08117.     

Integration of alkaline phosphatase in the intestinal brush border membrane

Alkaline phosphatase has been solubilized from porcine intestinal mucosa by two different methods: treatment of the mucosa by Emulphogen BC 720 and papain hydrolysis of enterocyte brush border membrane vesicles. Two different enzyme forms have been obtained by these methods.The two enzyme forms (‘detergent form’ and ‘papain form’) have been purified to homogeneity by similar techniques and exhibit closely related molecular characteristics. However, the detergent form displays a hydrophobic behaviour and aggregates in media free of detergent. The two forms can be differentiated by their electrophoretic mobility on polyacrylamide gel in the absence of sodium dodecyl sulphate.By electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate, it has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively. The difference between these molecular weights has been attributed to the existence of a hydrophobic region in the detergent form which is present on each subunit.     

Purification and partial characterization of the I variant of placental alkaline phosphatase

The I variant of placental alkaline phosphatase was purified to homogeneity by means of DEAE-cellulose chromatography, isoelectric focusing, and gel filtration on AcA-34. The specific activity of the I variant was found to be 3.33 kat/mg. The enzyme is a dimer with an isoelectric point of 4.6 and a molecular weight of 120,000 as determined by sodium dodecylsulfate electrophoresis. The amino acid composition and other physicochemical properties of the I variant were compared with those of the more common F and S variants. The low activity associated with the I variant is apparently not due to a low specific activity, but to decreased molecular stability. The behavior in the ultracentrifuge and other observations suggest that the I variant differs from the F and S variants in surface charge distribution.This investigation was supported by grants from the Swedish Medical Research Council (projects No. 4217 and No. 03X-2725) and from the Medical Faculty, University of Umeå.     

Structural studies of human placental alkaline phosphatase in complex with functional ligands

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.     

Serum bone alkaline phosphatase in assessing illness severity of infected neonates in the neonatal intensive care unit

Background: Infections can influence bone metabolism of neonates, which may lead to changes in some bone metabolism biomarkers. The purpose of this study was to determine whether serum bone alkaline phosphatase (BALP), osteocalcin (OC) and beta carboxy-terminal peptide of type I collagen (CTX), as specific biomarkers of bone metabolism, can be used to assess the severity of neonatal infections.Methods: Sixty-three neonates in the NICU were enrolled in this study. The neonates were divided into infected group (n=33) and non-infected group (n=30). The scores for neonatal acute physiology-perinatal extension II (SNAPPE-II) were calculated and interleukin-6 (IL-6), procalcitonin (PCT), BALP, OC and CTX were measured among the neonates with or without infections, and among the infected neonates before and after treatment.Results: The serum BALP levels were lower in the infected group than that in the non-infected group (p<0.01). The serum BALP levels increased markedly in the infected neonates after treatment (p<0.01). The serum BALP levels were also inversely correlated with SNAPPE-II of infected neonates before and after treatment (r=-0.56, p<0.05; r=-0.37, p<0.05, respectively). In infected neonates, the differences between serum BALP levels before and after treatment were inversely correlated with those of IL-6 levels (p<0.05). There were no significant changes in the OC, CTX and PCT levels in the infected or non-infected group before and after treatment.Conclusion: Our data suggest that serum BALP level might be used as a marker for assessing the severity of illness in infected neonates.     

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates

Increases of atmospheric CO₂ cause ocean acidification (OA) and global warming, the latter of which can stratify the water column and impede nutrient supply from deep water. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although the function of AP is known to require pH > 7, how OA affects AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examined the effects of pH conditions (5.5–11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH > 8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, and Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum and K. mikimotoi had the broadest pH niche for AP (7–10) and F. kawagutii the second (8–10), Am. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of Am. carterae AP to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These results in concert suggest OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected more acidified and nutrient-limited future ocean.     

Prolein polymorphism in a randombred chicken population

Polymorphism in plasma amylase, plasma alkaline phosphatase, non-specific esterase and red cell esterase-D of the Athens-Canadian randombred (ACRB) population of chickens was determined by polyacrylamide and starch gel electrophoresis. Amylase alleles Amy-1^A and Amy-1^B were segregating in the ACRB population with frequencies of 0.45 and 0.55 respectively. For the plasma alkaline phosphatase the F and S bands, the B band and a new isozyme migrating at a faster rate than the previously reported F band were detected. A genetic nomenclature for plasma alkaline phosphatase is suggested which considers the difference between the F and S bands as the presence or absence of sialic acid attached to a primary protein.
Plasma esterase activity was observed in all four of the regions previously reported, but there was no polymorphism found in any of the loci. All birds in this population showed the same red-cell esterase-D phenotype which consisted of a main band with sub-bands on each side.     

Blood protein polymorphism in the one-humped camel (Camelus dromedarius) in the Sudan

The blood protein polymorphism of serum albumin, haptoglobin, transferrin, ceruloplasmin and haemoglobin have been studied in 135 samples from one-humped Arabian camel (Camelus dromedarius) of the Sudan by starch gel electrophoresis. Only the serum albumin and haptoglobin systems exhibited polymorphism with the estimated frequencies of 0.0222, 0.2227 and 0.7773 for Alb^v, Hp¹Hp⁰respectively. The frequency of Hp was 0.0325. No electrophoretic variant was observed at transferrin, ceruloplasmin and haemoglobin loci in the camel. The activity of the ceruloplasmin of the camel sera was weak.     

Low activity of alkaline phosphatase in two eutrophic reservoirs

We studied recycling of phosphate by enzymatic hydrolysis in two temperate very eutrophic reservoirs. To assess the potential importance of phosphate regeneration by alkaline phosphatase, we determined the activity of this enzyme in lake water concomitantly with the determinations of the concentrations of phosphomonoesters, soluble reactive phosphate, total soluble phosphate and total phosphate. Contrary to our expectations for such productive waters where algal blooms are frequent, during the study period this process of phosphate regeneration was not significant, probably because the product of hydrolysis (contained in the soluble reactive phosphate fraction) was always abundant. We conclude that, in spite of what has been observed repeatedly in natural lakes with similar trophic characteristics, the readily available fraction of phosphate in these reservoirs is large and for that reason alkaline phosphatase production is low. Therefore hydrolysis by this enzyme is not significant for growth. What seems intriguing is the small amount of phosphomonoesters found in the water; with no phosphatase activity this phosphate fraction should always be high, unless hydrolysis takes place either during phosphomonoester release or later due to their instability.     

Serum albumin polymorphism and its relationship to economic traits in crossbred cattle

The serum albumin genotypes of 65 Jersey × Hariana (F₁), 75 Holstein Friesian × Hariana (F₁) and 47 Brown Swiss × Hariana (F₁) crossbred cows were determined by starch gel electrophoresis. Two albumin alleles Alb^F and Alb^s , but only either as Alb^F homozygotes or Alb^Fs heterozygotes, were observed amongst these animals. There were no Alb^S homozygotes or other genotypes. Highly significant relationships between albumin genotypes and both birth weights and first-lactation milk yields of these cows were observed. The Alb^F allele was associated with increased milk yield and greater birth weights.     

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.     

Polymorphism of the serum proteins of wild pigs

One hunderd and ninety five wild pigs from two different regions of Poland were investigated for transferrin, amylase and ceruloplasmin polymorphism. A new transferrin phenotype Tf PB was detected. This phenotype differed from Tf AB in the electrophoretic mobility of the more anodal transferrin. Tf P is assumed to be the product of a new allele Tf P at the Tf locus. Two amylase phenotypes Am 1–2 and Am 2 were observed. The Am 1 allele was absent from the pigs in the Poznan region. Only one ceruloplasmin phenotype, Cp B, was found.