Divalent cations stabilize the conformation of plasma cell membrane glycoprotein PC-1 (alkaline phosphodiesterase I).
The plasma cell-membrane glycoprotein PC-1 is an ectoenzyme with alkaline phosphodiesterase I/5'-nucleotide phosphodiesterase (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It contains sequence motifs which closely match the consensus EF-hand (helix-loop-helix) Ca(2+)-binding regions of parvalbumin, troponin-C and calmodulin, and its enzymic activity is increased in the presence of divalent cations and decreased in the presence of chelating agents. We have undertaken experiments to determine whether divalent cations affect the conformation of the PC-1 protein, as assessed by their effect on thermal stability, resistance to proteolysis and binding of polyclonal antibodies to the whole native protein and monoclonal antibodies to a putative Ca(2+)-binding region. Divalent cations were found to protect solubilized PC-1 against thermal denaturation and proteolysis. They also stabilized PC-1 on intact cells; this form was much more resistant to proteolysis than Triton X-100 solubilized PC-1. Ca2+, Mg2+ and Zn2+ ions were equally effective. Monoclonal antibodies to the bacterially expressed C-terminal EF-hand homology region only bound to mammalian PC-1 in the absence of Ca2+. In contrast, the great majority of polyclonal antibodies to native PC-1 bound regardless of whether Ca2+ was present or not, but with increased binding when Ca2+ was present. These results provide evidence that divalent cations bind to PC-1 and stabilize its conformation.
Belli
S I
SI
Department of Pathology and Immunology, Monash Medical School, Victoria, Australia.
Sali
A
A
Goding
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry; D002413:Cations, Divalent / Q000494:pharmacology*; D002460:Cell Line; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects*; D011755:Pyrophosphatases
D002352:Carrier Proteins; D002413:Cations, Divalent; D008562:Membrane Glycoproteins; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D002118:Calcium