Biochemical characterization of CD39L4

Nucleotides are involved in regulating a number of important processes rang ing from inflammation to platelet aggregation. Enzymes that can modulate le vels of nucleotides in the blood therefore represent important regulatory c omponents in these physiological systems, CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the re combinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the mo lecular masses before and after glycosidase treatment. Activity measurement s of CD39L4 isolated from tunicamycin-treated, transiently transfected COS- 7 cells indicate that glycosylation is not required for full ADPase activit y. Recombinant human CD39L4 protein isolated from stable insect cells was g lycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfid e-linked dimer. We determined that the monomer is the most active form of C D39L4 by measuring the activity of sucrose density gradient fractions of mo nomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.