Genomic and enzymatic characterization of the human low molecular weight red cell type acid phosphatase
Abstract
The human red cell acid phosphatase was purified to homogeneity from whole blood and characterized. Red cell acid phosphatase is encoded at the ACP1 locus of human chromosome 2 and the gene is expressed as two isoenzymes. The isoenzymes have 157 amino acids, sharing identical sequences, except for a short divergent segment in the protein, corresponding to residues 40-73 in the mature protein. The isoenzymes exhibit different catalytic and molecular properties, as demonstrated in kinetic studies with alkane diols, formaldehyde, and multiple substrates on recombinant enzyme. Immunoblotting studies with the tissue enzyme and the recombinant enzyme demonstrate that the red cell enzyme is identical to the placental enzyme. We now present results for the genomic sequence of the ACP1 locus. This genomic DNA sequence data supports the hypothesis that the two isoenzymes are generated by mutually exclusive alternative mRNA splicing. $\lambda$-FixII phage and P1 plasmid clones from a human genomic DNA library which encode the ACP1 gene have been isolated. This human cytosolic protein tyrosine phosphatase gene (HCPTP) is apparently comprised of 7 exons interrupted by 6 introns. DNA sequences of most introns, exons and 3$\sp\prime$-flanking regions have been determined. The alternatively spliced exons are 114 base pairs in length and encode amino acids 39-76, which, ultimately, is the region responsible for substrate recognition for the isoenzymes. The exons are interrupted by a short intron (41 bp). Southern analysis with both coding and non-coding regions used as probes revealed the presence of a single gene, further support for the hypothesis that these isoenzymes are generated via alternative splicing. The promoter region of the ACP1 gene is very GC rich and has no apparent TATA box or CCAAT box. This is typical of "housekeeping" gene promoters. The chromosomal assignment of the ACP1 gene was determined. Fluorescence in situ hybridization (FISH) results indicate that the ACP1 gene is localized to 2p25. This was an important finding due to some prior controversy over the location of ACP1 at either 2p23 or 2p25.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry|Molecular biology
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