Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

Abstract

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.

Lanthanide Phosphate Nanoparticle-Based One-Step Optical Discrimination of Alkaline Phosphatase Activity

“…As shown in Figure 4a, the ratio of F t /F decreased with the delay of reaction time during the initial reaction stage; the slopes of different PPi substrate concentrations vs time can be calculated by linear fitting of the F t /F data within 12 min. The slopes of these plots increase with substrate concentrations as expected, because the initial substrate concentrations are far below the K m values reported in the previous literature; 71,72 then, the Michaelis−Menten curves are obtained (Figure 4b). From Lineweaver−Burk plots of the Michaelis−Menten equation (V = V max [S]/(K m + [S]), 73 the Michaelis−Menten constant (K m ) and maximum initial velocity (V max ) are calculated to be 9.84 × 10 −3 nM and 7.63 × 10 −2 nM•S −1 , respectively, which are smaller than those obtained by Thilagaraj et al using a pNPP sensor; 74,75 it is probably bound up with the sensitivity of the P 2 O 7 :Ce,Tb probe.…”

MC1R variation and melanoma risk in the Swedish population in relation to clinical and pathological parameters

“…The subjects in the control group were blood donors recruited on a voluntary basis (total n = 520) and newborns (n = 202, placental tissue was used for DNA preparation) (Beckman et al., 2001; Wennberg et al., 2002). Among the blood donors, males were overrepresented (Table 1).…”

4. Alkaline phosphatase

“…Thus, the tissues with the highest ALP concentrations (greatest to least) are the placenta, small intestine, bone, kidney, and liver. PLAP has many allozymes, which vary in their biochemical properties [19]. There are four different genes that encode for the different ALP isoenzymes: one gene on chromosome 1 and three genes on chromosome 2.…”