Hans Lüdi

Separation of the tryptic fragments of sarcoplasmic reticulum ATPase with high performance liquid chromatography: Identification of the calcium binding site

The four tryptic fragments of the Ca2+‐ and Mg2+‐dependent ATPase from sarcoplasmic reticulum were separated on a TSK‐G 4000 SW preparative high performance liquid chromatography column in the presence of sodium dodecyl sulfate. Using atomic absorption spectroscopy, the analysis of the protein peaks demonstrated that the calcium binding sites with high affinities are located in the smallest fragment (A2; 25 kDa).

State of aggregation of detergent-solubilized sarcoplasmic reticulum adenosine triphosphatase investigated by high-performance liquid chromatography

The state of aggregation of purified sarcoplasmic reticulum adenosine triphosphatase (ATPase) was investigated by high-performance liquid chromatography (LKB TSK-G 4000 SW column) in the presence of various detergents: sodium dodecylsulphate, dodecyl octaethylene glycol monoether (C12E8), sodium deoxycholate, Triton X-100 and myristoylglycerophosphocholine. When the protein (5 mg ml-1) was solubilized with detergent (2 mg per mg protein) and the column was equilibrated with 1 mg ml-1 of the respective detergent, a molecular weight for the monomeric ATPase protein ranging from 100,000 to 200,000 was obtained. In addition to the monomeric form, significant amounts (more than 20%) of aggregated ATPase protein were observed when C12E8 or deoxycholate was used. These results are in agreement with the observation of a great tendency for self-aggregation of the ATPase protein in conventional gel filtration chromatography and ultracentrifugation experiments. The dimeric form of the ATPase protein was detected only when deoxycholate and, probably, when C12E8 was used.

Tryptophan fluorescence of sarcoplasmic reticulum ATPase. A fluorescence quench study

The calcium-dependent change in the tryptophan fluorescence intensity of the sarcoplasmic reticulum Ca2+- and Mg2+-ATPase was investigated using different quenching reagents. It is demonstrated that only those compounds which are bound to the enzyme (i.e., 1-(9,10-dibromomyristoyl)-sn-2-glycerophosphorylcholine and 1-(9,10-dibromostearoyl)-sn-glycero-3-phosphorylcholine) are able to decrease the amplitude of the fluorescence decrement observed after removal of calcium ions. From the position of the bromine atom within the lysophosphatidylcholines, it is concluded that the tryptophan residues involved are located in the hydrophobic part of the ATPase molecule and are in contact with the hydrocarbon chains of the phospholipids.

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

The influence of sodium trichloroacetate on the tryptophan fluorescence of sarcoplasmic reticulum ATPase.

The chaotropic anion trichloroacetate quenches the tryptophan fluorescence of the sarcoplasmic reticulum calcium transport ATPase. Half-maximum quenching was observed at 50 mM trichloroacetate. In contrast to native preparations, in trichloroacetate-treated sarcoplasmic reticulum vesicles a decrease of the tryptophan fluorescence is observed on addition of millimolar concentrations of calcium. It is concluded that trichloroacetate renders the tryptophan fluorescence of the ATPase sensitive to the occupancy of its low-affinity sites.

The influence of detergents on the Ca2+- and Mg2+-dependent adenosine triphosphatase of the sarcoplasmic reticulum.

Abstract During the stepwise solubilization of sarcoplasmic reticulum vesicles with detergents, the following changes in the structural and enzymatic properties of the preparation are observed: 1. The viscosity of the vesicular suspension initially rises. This change is accompanied by the formation of elongated tubules. Subsequently the membranes are completely desintegrated, resulting in a considerable reduction of the viscosity. 2. A decrease in the activity of the Ca2+-dependent ATPase, which is restored after complete solubilization. 3. A decrease in the change of intrinsic tryptophan-fluorescence on removal of calcium ions, which is also restored after complete solubilization. 4. A decrease of the calcium affinity of the ATPase. 5. A decrease in the amount of phosphorylated protein formed by the incorporation of inorganic phosphate. On the other hand, the amount of phosphoprotein formed from ATP is not affected during solubilization. 6. The dependence of the initial rates of phosphoprotein formation from inorganic phosphate on either magnesium or inorganic phosphate at low concentrations of the respective ligand changes from an S-shape profile to a normal hyperbolic profile after solubilization.

Inactivation of the Sarcoplasmic Reticulum Calcium -Transport-ATPase by Lasolocid in Combination with Triton X-100

Abstract The calcium-transport-ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzymes activity. In the presence of Lasolocid the enzyme is most susceptable to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, ~ 0.2 mg · ml-1. Lasolocid becomes effective at a concentration of 10 μᴍ and produces rapid inactivation at 100 μᴍ . The enzyme is more rapidly inactivated in the active than in the inactive state.

Fluorescence Studies on N−(3−Pyrene)Maleinimide−Labeled Sarcoplasmic Reticulum ATPase in Native and Solubilized Membranes

Abstract Fluorescence polarization and formation of excimers were studied in N-(3-pyrene)maleinimide-labeled sarcoplasmic reticulum vesicles. 1. The polarization of pyrenemaleinimide labeled vesicles does not change with temperature and shows a pronounced decrease at labeling concentrations larger than 1 mol pyrenemaleinimide per 10 mol ATPase. 2. Solubilization of the membrane with myristoylglycerophosphocholine renders the polarization temperature dependent, but does not affect the concentration dependent depolarization observed in native vesicles. 3. The polarization of labeled vesicles is much smaller than to be expected from the temperature independent polarization indicating that the pyrenemaleinimide polarization did not monitor the rotation of the entire ATPase. Thus segmental motion occurs. 4. Pyrene excimers are observed at label concentrations larger than 1 mol label per 2.5 mol ATPase. 5. The amount of excimers was critically dependent on added detergents. From the fact that non-solubilizing amounts of myristoylglycerophosphocholine strongly reduced the amount of pyrene excimers it is concluded that in the native sarcoplasmic reticulum vesicles at least two ATPase molecules must be in close contact.