Identification of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity associated with the mouse plasma cell differentiation antigen PC-1.
The protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was purified from MOPC 315 plasmacytoma cells. A single SDS/PAGE-purified 115-kDa protein band was used to produce a rabbit polyclonal antiserum. This antibody preparation precipitated alkaline phosphodiesterase I activity, indicating that the SDS/PAGE-purified protein was nucleotide pyrophosphatase/alkaline phosphodiesterase I. When used for Western blot analysis, the antiserum detected a 115-kDa protein as well as a 220-kDa protein band. Multiple overlapping cDNA clones were isolated from a cDNA expression library screened with this anti-nucleotide pyrophosphatase/alkaline phosphodiesterase I antiserum. Sequence analysis indicated that the isolated cDNA clones encoded PC-1, a murine plasma cell differentiation antigen. To confirm the suspected enzymatic identity of PC-1, a recombinant PC-1 fusion protein was expressed in bacteria, purified, and used to produce another rabbit polyclonal antiserum. This antiserum likewise immunoprecipitated alkaline phosphodiesterase I activity and recognized the 115-kDa and 220-kDa proteins in Western blot analyses of cell extracts. Furthermore, expression of nucleotide pyrophosphatase/alkaline phosphodiesterase I corresponded directly with mRNA and protein levels of PC-1 in cells known to express different levels of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity. Finally, steroid induction of enzymatic activity was mirrored by levels of PC-1 mRNA and protein expression. Together, these data indicate that the plasma cell differentiation antigen PC-1 is a membrane-bound enzyme, nucleotide pyrophosphatase/alkaline phosphodiesterase I.
Rebbe
N F
NF
Washington University Medical Service, Veterans Affairs Medical Center, John Cochran Division, St. Louis, MO 63106.
Tong
B D
BD
Finley
E M
EM
Hickman
S
S
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011233:Precipitin Tests; D011755:Pyrophosphatases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics
D000944:Antigens, Differentiation, B-Lymphocyte; D012333:RNA, Messenger; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase