Calcium-Dependent ATPase: Analytic Techniques
A common eukaryotic enzyme, calcium-dependent ATPase has been extensively investigated. The ion-transport enzyme uses energy derived from the hydrolysis of adenosine triphosphate (ATP) to move Ca2+ against a concentration gradient. Innumerable techniques have been applied to Ca2+-ATPase analyses. These have included proteolytic, genetic, immunologic, and molecular approaches.
Calcium-dependent ATPase was first isolated in 1970 (3:696-700). This heterogenous family of enzymes can be broadly subdivided into two separate groups. The plasma membrane Ca2+-ATPase occurs in most eukaryotic tissues. This 140-kDa enzyme binds calmodulin and is stimulated by calcium ion (10:285-297). Although it may be derived from plants, yeasts, or, for example, pig stomach smooth muscle, perhaps the most thoroughly studied plasma membrane Ca2+-ATPase is found in erythrocytes (1:303). The sarcoplasmic reticulum (SR) is also a very good source of Ca2+-ATPase. The SR Ca2+-ATPase is derived from the SERCA gene family. Perhaps the most well-known of these enzymes comes from the SERCA 1 gene. The SERCA 2a Ca2+-ATPase isoform is found in skeletal muscle; whereas, the SERCA 2b isoform occurs in smooth muscle.
The Ca2+-ATPase molecule extends about 60 + above the surface of any given plasma bilayer. Further, the enzyme's total length is approximately 100 + (4:365-370). According to MacLennan and associates (1985), calcium-dependent ATPases generally contain three major domains. The first of these consists of a transmembrane domain. This region contains two high affinity Ca2+ binding sites and a Ca2+ pathway. Second, the enzyme possesses a large pear-shaped, cytosolic head (5:303-309). This extramembranal head accommodates both the ATP binding site and an aspartyl residue phosphorylation site (Asp-351). A "P-type" ionic pump, the Ca2+-ATPase forms a phosphorylated intermediate during its ...