THE SPINACH PLASMA-MEMBRANE CA2-KDA POLYPEPTIDE REGULATED BY CALMODULIN-BINDING TO A TERMINAL REGION( PUMP IS A 120)

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 ide ntified as a 120-kDa polypeptide on western immunoblots using two diff erent antibodies. During trypsin treatment the 120-kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109-kDa ban d correlated with the increase in enzyme activity following trypsin tr eatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109-kDa polypeptide represents a Ca2+-ATPase lack ing a terminal fragment involved in calmodulin regulation. This was co nfirmed by I-125-calmodulin overlay studies where calmodulin labeled t he 120-kDa band in the presence of Ca2+, while the 109-kDa band did no t bind calmodulin. The effects of calmodulin and limited proteolysis o n ATP-dependent accumulation of Ca-45(2+) in isolated inside-out plasm a membrane vesicles were studied, and kinetical analyses performed wit h respect to Ca2+ and ATP. Calmodulin increased the V-max for Ca2+ pum ping 3-fold, and reduced K-m for Ca2+ from 1.6 to 0.9 mu M. The K-m fo r 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 increa sed the V-max for Ca2+ pumping to a higher extent than calmodulin, ind icating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.