Specific sodium-22 binding to a purified sodium + potassium adenosine triphosphatase. Inhibition by ouabain.

Analysis of sodium-22 binding to purified sodium + potassium ion-activated adenosine triphosphatase (Na+, K+)-ATPase reveals the presence of two classes of binding sites. The higher affinity site (Kd = 0.2 mM) binds 6 to 7 nmol of sodium per mg of protein. Pretreatment of (Na+, K+)-ATPase with ouabain blocks the binding of sodium to this higher affinity site. Neither heat-denatured enzyme nor phospholipids extracted from the (Na+, K+)-ATPase contain a ouabain-inhibitable, higher affinity sodium binding site. The ouabain enzyme complex therefore appears to contain altered binding sites for cations.

Association of gylcogenolysis with cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum fragments isolated from dog cardiac muscle possess a calcium-accumulating system associated with a series of enzymes linked to glycogenolysis. These enzymes include: adenylate cyclase, cyclic AMP-dependent protein kinase, phosphorylase b kinase, phosphorylase (b/a, 30/1),"debrancher" enzyme, and glycogen (0.3 to 0.7 mg/mg of protein). The sarcoplasmic reticulum preparation produced glucose 1-phosphate and glucose from either endogenous or exogenous glycogen. Both the calcium-accumulating and glycogenolytic enzymes sediment in a single peak at 33% sucrose on a linear continous sucrose density gradient, and the complex remains intact throughout repeated washing. Glycogen particles appear to be associated with the sarcoplasmic reticulum in situ as well as in the isolated microsomal fraction. The sarcoplasmic reticulum-glycogenolytic complex, monitored by a linked enzyme spectrophotometric assay, shows several features: (a) activation of phosphorylase activity to peak rate occurs over a very rapid time course which cannot be duplicated using combinations of purified enzymes; (b) activation is inhibited by protein kinase inhibitor; (c) phosphorylase b functions as in the purified form with respect to AMP (Km, 0.3 mM); (d) in the presence of limiting amounts of glycogen, optimal phosphorylase b activity in the sarcoplasmic reticulum requires the presence of debrancher, and the activity is sensitive to inhibitors of that enzyme such as Tris, which suggests the possiblity that the enzymes bear a specific structual relationship to the glycogen present. Phosphorylase b leads to a activation in the sarcoplasmic reticulum was completely resistant to ethylene glycol bis(beta-aminoethyl either)-N,N'-tetraacetic acid (EGTA). Inhibition of calcium accumulation by or release of bound calcium from sarcoplasmic reticulum by X537A (RO 2-2985) did not alter the EGTA resistance. These results suggest that cardiac sarcoplasmic reticulum is a complex organelle containing functions that may be related to excitation-contraction coupling and intermediary metabolism.