Olbe M, Sommarin M
The spinach plasma membrane Ca2+ pump is a 120-kDa polypeptide regulated by calmodulin-binding to a terminal region
Physiologia Plantarum: 1998 103:35-44
The spinach (Spinacia oleracea L.) leaf plasma membrane Ca2+-ATPase is regulated by calmodulin (3-fold stimulation) and limited proteolysis (trypsin, 4-fold stimulation). The plasma membrane Ca2+-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 Ca2+-ATPase lacking a terminal fragment involved in calmodulin regulation. This was confirmed by I-125-calmodulin overlay studies where calmodulin labeled the 120-kDa band in the presence of Ca2+, while the 109-kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP-dependent accumulation of Ca-45(2+) in isolated inside-out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca2+ and ATP. Calmodulin increased the V-max for Ca2+ pumping 3-fold, and reduced K-m for Ca2+ from 1.6 to 0.9 mu M. The K-m for ATP (11 mu M) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca2+ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V-max for Ca2+ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.
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