The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.

The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.

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We have studied the effect of temperature on transcription initiation in vitro at two related promoters ga/Pcon and gaIP1, which have the same nucleotide sequence around the -10 region and transcription start site, but differ in upstream sequences. One of the promoters, gaIPcon, carries the consensus -35 hexamer, 5'TTGACA 3', whilst gaIP1 contains a block of 'distortable' upstream sequences that allow promoter function in the absence of a -35 region consensus sequence. RNA polymerase can form complexes with both promoters at a range of temperatures. However, the thermal energy requirement for open complex formation differs: open complexes can form at gaIP1 at low temperatures, whereas gaIPcon requires higher temperatures. The thermal energy requirement for transcription from preformed open complexes is the same for both promoters.

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