Characterization of over-expressed alkaline phosphodiesterase I in tumour-derived fibroblasts from patients with neurofibromatosis.
Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6.0. The enzyme required Zn2+ for its activity, was heat labile, and nicked superhelical covalently closed circular phi X174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430,000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.
Maruyama
E
E
Division of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Tokyo, Japan.
Takashima
S
S
D000912:Antibodies, Neoplasm; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D008970:Molecular Weight; D017253:Neurofibromatoses / Q000201:enzymology*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I