Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Platelet-derived endothelial cell growth factor (PD-ECGF) is known to stimulate angiogenesis in vivo.
• A significant amino acid sequence similarity exists between human PD-ECGF and Escherichia coli thymidine phosphorylase.

Purpose of the Study:

• To investigate the enzymatic activity of human PD-ECGF.
• To determine if PD-ECGF possesses thymidine phosphorylase activity.
• To explore the mechanism behind PD-ECGF's in vitro and in vivo effects.

Main Methods:

• Sequence alignment analysis to compare PD-ECGF with E. coli thymidine phosphorylase.
• Expression and purification of recombinant human PD-ECGF.
• Enzymatic assays to assess thymidine phosphorylase activity.
• Gel chromatography to determine the molecular weight and oligomeric state of PD-ECGF.
• Analysis of PD-ECGF's effect on [3H]thymidine incorporation assays.

Main Results:

• Human PD-ECGF shares 39.2% amino acid sequence similarity with E. coli thymidine phosphorylase over a 439 amino acid region.
• Recombinant human PD-ECGF demonstrated significant thymidine phosphorylase activity.
• PD-ECGF exists as a 90 kDa homodimer in solution, consistent with other thymidine phosphorylases.
• PD-ECGF affected [3H]thymidine assays independently of cell proliferation, suggesting an indirect mechanism.

Conclusions:

• PD-ECGF possesses enzymatic activity as a thymidine phosphorylase.
• The observed in vitro and in vivo effects of PD-ECGF are likely mediated through its enzymatic function.
• This finding provides a novel mechanistic insight into the biological roles of PD-ECGF beyond direct cell proliferation stimulation.