There is evidence that sarcoplasmic reticulum isolated from muscle that has undergone a rapid post-mortem decline in pH has impaired calcium-accumulating ability when compared with that from normal muscle which has a slow rate of pH decline (Greaseret al., 1969a). The enzyme responsible for translocating the cations, namely the ATPase protein, was less affected. The implication is that ATP hydrolysis has been uncoupled from vectorial transfer of calcium through the reticular membrane. This study establishes the quantitative differences in calcium transport and ATPase activity of fragmented sarcoplasmic reticulum from the two muscle sources and has attempted to determine the nature of the diminished efficiency of sarcoplasmic reticular vesicles from muscles of diseased animals. In view of the membrane-bound nature of the pump protein, the influence of the lipid environment on its functional activity was considered and a detailed analysis of the lipids of the sarcoplasmic reticular membrane was carried out. The lipid studies included on analysis of whole muscle since a general derangement of muscle cell membranes is inferred in this disease from the diffusion of proteins and ions out of the muscle cell into the extracellular fluid. In addition, the nature of the lipid-ATPase interaction was examined by observing the effect of temperature on the functional activities of the sarcoplasmic reticulum.

In the first part of this study, the kinetics of decay of TNP-nucleotide superfluorescence was investigated with a view to understanding the role of nucleotides and Lys492 in later steps in the catalytic cycle of the skeletal muscle Ca²⁺ATPase. It has been found previously, and verified here, that tethering TNP-8N₃-AMP to the Ca²⁺ATPase via Lys492 retarded the Ca²⁺ initiated decay of Pᵢ-induced superfluorescence 10-fold compared with untethered nucleotide. The rapidity of the decay upon addition of EDTA suggested that the E₂ ↔ E₁ → E₁Ca₂ steps were being monitored rather than dephosphorylation per se. Tethered diand triphospho species did not accelerate the decay. While monophasic kinetics was observed with untethered TNP-AMP and TNP-8N₃-AMP, complex kinetics were observed with the di- and triphospho TNP-nucleotides. This was shown to be due to the utilization of TNP-ADP and -ATP, and the azido derivatives, as coupled substrates of the Ca²⁺ATPase in the forward direction of catalysis in the presence of Ca²⁺. The hydrolysis rates of TNP-ADP, TNP-ATP, TNP-8N₃ -ADP, and TNP-8N₃ -ATP were 10, 5, 15 and 10 nomoles/min/mg of protein, respectively, at room temperature and pH 5.5. Ca²⁺ transport was supported by all four nucleotides. This is the first time that a diphosphonucleotide has been shown to support Ca²⁺ transport. A new nonhydrolysable triphospho TNPnucleotide, TNP-AMP-PCP was synthesized and shown to interact with the Ca²⁺ATPase in a similar way, in terms of superfluorescence, as the other TNP-nucleotides. It did not show the complex kinetics on inhibition of the Pcinduced superfluorescence by Ca²⁺, but neither did it accelerate the kinetics. It was concluded that TNP-nucleotides do not accelerate the E₂ ↔ E₁ transition under these conditions, possibly because of the presence of glycerol in the medium. In the second part of the study, it was shown that addition of small amounts of chelators EGTA, EDTA, BAPTA, DTPA, HEDTA and NTA to a Ca²⁺ transport assay in which the free Ca²⁺ concentration is monitored by Fluo-3 causes the Ca²⁺ATPase to pump to apparently lower levels as seen in the [Ca²⁺] lim fluorescence. Addition of chelator retards pump function in the sense that it takes longer for 50 nmols Ca²⁺ to be accumulated. Increased thermodynamic efficiency of the pump and contaminating heavy metal ions were considered as possible mechanisms. To some extend Zn²⁺ and Cd²⁺, but not Fe²⁺ and Cu²⁺, appeared to reverse the partial inhibition. While interpretation of the results is difficult, it is suggested that heavy metal ions interact with luminal loops of the Ca²⁺ATPase and enhance Ca²⁺ release under conditions of high luminal Ca²⁺ concentrations.

Properties of the nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. The study centred around interaction of the high affinity ATP analog, 2'-3'-0-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, (TNP-ATP), with the Ca²⁺-ATPase. Defined fractions of the sarcoplasmic reticulum (SR), corresponding to the terminal cisternae (TC) and light SR (LSR), were isolated. The TC were shown to have distinctive morphological characteristics that differ from the LSR. The TC vesicles contained electron dense intravesicular material representative of Ca²⁺ binding proteins, and visible membranous "feet" structures, which are reported to interconnect with the transverse tubule. Functional characterisation of the isolated fractions provided evidence for the predominant localisation of Ca²⁺ release channels in TC, and concentration of Ca²⁺-ATPase molecules in LSR. These conclusions were based on the following observations: (a) decreased Ca²⁺ transport of TC versus LSR; ruthenium red, a Ca²⁺ channel blocker, enhanced Ca²⁺ transport and pumping efficiency in TC, (b) higher Ca²⁺-ATPase activity for LSR in the presence and absence of ionophore, (c) rapid Ca²⁺ efflux from TC which is inhibited by ruthenium red. Of special interest was the characterisation of the TC and LSR with respect to turnover-dependent TNP-ATP fluorescence. Fluorescence observed for TC was approximately 65% of that for LSR. This phenomenon may be attributable to either the decreased Ca²⁺ ATPase content of the TC vesicles or open Ca²⁺ release channels. Hence the TNP-ATP fluorescence characteristics appear to reflect the morphological and functional subspecialisation of the defined SR fractions.