Properties of the Calcium‐Independent ATPase of the Membranes of the Sarcoplasmic Reticulum Delipidated by the Nonionic Detergent Triton X‐100

Abstract

1.When the sarcoplasmic reticulum membranes are solubilized in Triton X-100 their ATPases are modified : the calcium-dependent ATPase is activated whereas the basic ATPase is inhibited.2. Phospholipids are removed from the solubilized sarcoplasmic membranes completely by chromatography on a sepharose column equilibrated with Triton X-100. Thereby the activity of the calcium-dependent ATPase is abolished and cannot be restored by addition of phospholipids.3. The delipidated ATPase preparation, exhibits only low activity, which is not dependent on calcium but on magnesium alone. This calcium-independent ATPase is activated to the level of the calcium-dependent ATPase both by unsaturated and saturated fatty acids, by anionic phospholipids and various synthetic lipids.4. The enhancement of the ATPase activity produced by lipids is related to the binding of these compounds to the membrane protein in amounts comparable with those of bound Triton as demonstrated by dialysis experiments with dodecylsulfate. When the concentration of Triton in the medium is lowered, the dodecylsulfate/protein binding ratio increases, while Triton is detached from the protein.5. The activation of the calcium-independent ATPase by saturated fatty acids is dependent upon the chain-length of these aliphatic compounds. Only the long-chain fatty acids are strong activators. Moreover, the hydrophilic group of lipids affects maximal activities. No activation is observed with lecithin or dodecanol, while alkyl-sulfates or -phosphates activate most effectively.6. The calcium-independent ATPase differs from the calcium-dependent ATPase in that it is insensitive to N-ethylmaleimide, dinitrodithiobenzoic acid or salyrgan. The enzyme is strongly inhibited by prenylamine, sodium azide and silver ions. Similarly low alkali salt concentrations produce a strong reduction of the activity.7. The relative rates, with which the nucleoside triphosphates are split by the calcium-independent ATPase, differ considerably. As observed for the calcium-activated ATPase. The dependence on the substrate concentration suggests a positive cooperativity concerning Mg * ATP (Hill coefficient 1.46). 5001, of maximal activity is obtained a t 0.25 mM Mg * ATP. The enzyme is strongly inhibited by Mg * ADP.Kielley and Meyerhof have characterized a magnesium-activated ATPase found in aqueous muscle extracts, which was later identified as the ATPase of the membrane fragments of the sarcoplasmic reticulum [ 11. They demonstrated that treatment of the enzyme with phospholipase C caused inactiva- I n this paper a preparation is described from which essentially all phospholipids have been removed by use of the nonionic detergent TritonX-100 [8].

Correlation between Uncoupled ATP Hydrolysis and Heat Production by the Sarcoplasmic Reticulum Ca2+-ATPase

“…The sample containing the radioactive Ca 2ϩ was used to determine the incubation time when the vesicles are filled and the steady state 45 32 P]ATP not hydrolyzed during the reaction was extracted with activated charcoal as previously described (34). Two different ATPase activities can be distinguished in sarcoplasmic reticulum vesicles (7,35,36). The Mg 2ϩ -dependent activity requires only Mg 2ϩ for its activation and is measured in the presence of 2 mM EGTA to remove contaminant Ca 2ϩ from the medium.…”

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

“…At this concentration the ATPase protein which is present at only 0.05 mg • m l-1 is largely stripped of its native lipids, whereby mixed lipid Triton X-100 micelles are formed. The am ount of non-lipid bound Triton X-100 is presum ably sufficient to just cover the hydrophobic surface area of the protein [13,14]. In this state where the native lipids are removed and marginally replaced by Triton X-100 the enzyme becomes Lasolocid sensitive.…”

Participation of a non‐covalent phosphointermediate in ATP hydrolysis by the sarcoplasmic reticulum Ca2+‐transport ATPase

“…In the literature, support for both possibilities can be found. Walter and Hasselbach [22] have obtained a highly active sarcoplasmic reticulum ATPase, completely independent of Ca2+ after Triton X-100 treatment, delipidation, and reactivation with SDS. Scott and Shamoo [23] have demonstrated that occupation of one Ca2+-binding site suffices for stimulation of ATP hydrolysis; these authors used an enzyme preparation purified with detergents.…”