The spinach plasma membrane Ca²⁺ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

Abstract

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 lackin a terminal fragment involved in calmodulin regulation. This was confirmed by 125I‐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 45Ca2+ in isolated inside‐out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca2+ and ATP. Calmodulin increased the Vmax. for Ca2+ pumping 3‐fold, and reduced Km for Ca2+ from 1.6 to 0.9 µM. The Km for ATP (11 µM) 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 Vmax. for Ca2+ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.

H+/Ca2+ exchange driven by the plasma membrane Ca2+‐ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin‐affinity purification

“…1B, lane 1) absent in the CaM‐regulated Ca 2+ ‐ATPases of plant endomembranes [4]. In agreement with previous reports [4,12,24], the 123 kDa band (Fig. 1C, lane 1) also cross‐reacted with an antiserum against the large cytosolic loop of At ‐ACA1p, a putative Ca 2+ ‐ATPase of the plastid envelope [27].…”

“…The EDTA-eluted fraction so obtained contained about 15% of the loaded CaM-stimulated Ca 2 -ATPase activity (Table 1). In the assay conditions used (10 WM free Ca 2 ), the percent stimulation of Ca 2 -ATPase activity by CaM ranged between 250 and 400%; this is likely an underestimation since CaM, beside increasing the V max of the reaction, also decreases the apparent K M of the enzyme for free Ca 2 [10,12,13], making the Ca 2 -ATPase more sensitive to inhibition by high Ca 2 concentrations (L. Luoni, unpublished results).…”

“…The PM of plant cells is endowed with a calmodulin (CaM)‐regulated Ca 2+ ‐ATPase belonging to the same subfamily (type IIB) of P‐type ATPases as its animal counterpart [4–7]. Like the PM Ca 2+ ‐ATPase of animal cells [8,9], the plant PM Ca 2+ ‐ATPase binds CaM at an autoinhibitory domain which can be selectively cleaved by controlled trypsin treatment [10–12]. However, the plant PM Ca 2+ ‐ATPase has some distinctive biochemical and structural features which clearly differentiate it from the PM Ca 2+ ‐ATPase of animal cells.…”

The inhibition of ammonium uptake in excised birch (Betula pendula) roots by batatasin‐III

“…Plasma membranes were isolated from expanding leaves of 4–5‐week‐old plants. The membranes were purified from a microsomal fraction of spinach leaves by partitioning in an aqueous polymer two‐phase system, as described in detail by Olbe and Sommarin (1998). The final membrane preparations were suspended in 0.25 M sucrose, 1 m M dithiothreitol and 10 m M HEPES‐BTP (pH 7.5), and stored as aliquots (ca 500 μl containing 2–5 mg of membrane protein) in liquid nitrogen until further use.…”

Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

“…Plasma membranes were purified from a microsomal fraction of spinach leaves by partitioning in an aqueous polymer two-phase system, and checked for purity as described in detail previously (Olbe and Sommarin, 1998). Plasma membranes were suspended (10-15 mg of membrane protein per mL) in 330 mm Suc, 10 mm HEPES-KOH, pH 7.5, 1 mm DTT, and 0.1 mm EDTA, and stored in liquid nitrogen until use.…”