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The spinach plasma membrane Ca 2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region
Author(s) -
Olbe Malin,
Sommarin Marianne
Publication year - 1998
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1998.1030105.x
Subject(s) - calmodulin , proteolysis , spinacia , trypsin , spinach , biochemistry , atpase , egta , binding protein , biology , chemistry , enzyme , calcium , chloroplast , organic chemistry , gene
The spinach ( Spinacia oleracea L.) leaf plasma membrane Ca 2+ ‐ATPase is regulated by calmodulin (3‐fold stimulation) and limited proteolysis (trypsin; 4‐fold stimulation). The plasma membrane Ca 2+ ‐ATPase was identified as a 120‐kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120‐kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109‐kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109‐kDa polypeptide represents a Ca 2+ ‐ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by 125 I‐calmodulin overlay studies where calmodulin labeled the 120‐kDa band in the presence of Ca 2+ , while the 109‐kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP‐dependent accumulation of 45 Ca 2+ in isolated inside‐out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca 2+ and ATP. Calmodulin increased the V max. for Ca 2+ pumping 3‐fold, and reduced K m for Ca 2+ from 1.6 to 0.9 µ M . The K m for ATP (11 µ M ) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca 2+ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V max. for Ca 2+ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.