Biochemical characterization of CD39L4.
Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components 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 recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated 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 disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.
Mulero
J J
JJ
Functional Genomics Department, Immunology Group, Hyseq Inc., 670 Almanor Avenue, Sunnyvale, California 94086, USA.
Yeung
G
G
Nelken
S T
ST
Bright
J M
JM
McGowan
D W
DW
Ford
J E
JE
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001081:Apyrase / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
D004220:Disulfides; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase