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Structure, genomic DNA typing and kinetic characterization of the D allozyme of placental alkaline phosphatase

Wennberg, Charlotte (author)
Kozlenkov, Alexey (author)
Umeå universitet,Institutionen för medicinsk biovetenskap
Mauro, Sonia Di (author)
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Fröhlander, Nils (author)
Beckman, Lars (author)
Hoylaerts, Marc F. (author)
Millán, José Luis (author)
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 (creator_code:org_t)
2002-02-13
2002
English.
In: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 19:3, s. 258-267
  • Journal article (peer-reviewed)
Abstract Subject headings
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  • 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 kcat and Km, 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.

Keyword

alkaline phosphatase
placental; PLAP; ALPP; ALPL; ALPPL2; ALPI; isozyme; negative selection; spontaneous abortion; gene therapy; genetic disease; placental function; SNuPE

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