Identification of the plasma membrane Ca2+-ATPase and of its autoinhibitory domain

• Published in: Plant physiology (Bethesda) Vol. 108; no. 1; pp. 105 - 113
• Main Authors: Rasi-Caldogno, F. (Universita di Milano, Milano, Italy.), Carnelli, A, De Michelis, M.I
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01-05-1995
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Adenosine triphosphatases; Agronomy. Soil science and plant productions; ATPASA; ATPASE; Biological and medical sciences; CALCIO; CALCIUM; CALMODULINA; CALMODULINE; Cell Biology and Signal Transduction; Cell membranes; COLORANT; COLORANTES; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Economic plant physiology; Enzymes; ESTRUCTURA CELULAR; Fundamental and applied biological sciences. Psychology; Gels; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; Lanthanum; Metabolism; Microsomes; Nutrition. Photosynthesis. Respiration. Metabolism; Phosphorylation; Plant physiology and development; Plants; PLANTULAS; PLANTULE; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PROTEINAS; PROTEINE; PROTEOLISIS; PROTEOLYSE; PURIFICACION; PURIFICATION; Radishes; RAPHANUS SATIVUS; STRUCTURE CELLULAIRE; THIOCYANATE; TIOCIANATOS; TRIPSINA; TRYPSINE; Enzyme; Enzyme inhibitor; Binding site; Raphanus sativus; Molecular weight; transporting ATPase; Vegetable crop; Cruciferae; Proteolysis; Dicotyledones; Angiospermae; Plasma membrane; Hydrolases; Spermatophyta; Ca; Calmodulin
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.108.1.105

The effect of controlled proteolysis on the plasma membrane (PM) Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125I-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase comigrates with the phosphorylated intermediate of the enzyme-- labeled by incubation with [gamma-32P]GTP in the presence of calcium--on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme