A detailed study of the interaction of glycolytic enzymes with the ultrastructure of mammalian skeletal muscle has been carried out and evidence of the dynamic nature of these interactions within whole muscle is presented. Moving boundary electrophoresis and ultracentrifugation have been used to estimate the binding parameters for these interactions, and kinetic investigations of enzyme bound to structural elements of muscle have revealed marked alterations in the catalytic properties of the enzyme compared with its soluble form . In addition, a specific interaction of at least three enzymes with muscle ultrastructure has been delineated using whole animals; furthermore, the binding of these enzymes has been shown to vary with the metabolic status of the muscle.
Aldolase has been used as a model for the in vitro studies, using purified components of the thin filaments of muscle. A significant salt-labile interaction-of this enzyme with the regulatory components, tropomyosin and troponin, or their complex indicated that these proteins, as well as actin, may be able to bind glycolytic enzymes within the muscle fibre. The presence of tropomyosin and troponin on actin filaments increased the capacity of these filaments to bind enzyme; it was revealed that this alteration was due chiefly to an increase in the number of binding sites per unit length of filament (from one enzyme molecule per fourteen actin monomers to a maximum of four) whilst the binding constant decreased slightly (2.0 to 0.5 x 10+5 M-1 at 0.0881). Filaments containing troponin were also shown to possess one calcium sensitive binding site, and, based on a repeat unit of fourteen actin monomers, such filaments bound one molecule of enzyme less when calcium was present than when this ion was absent.
Kinetic studies of aldolase bound to thin filaments are presented in chapter five. It was found that troponin-containing filaments increased the Vmax four-fold and the K about sixty fold compared with the soluble enzyme; additionally these catalytic modifications were shown to be calcium dependent, characterised by a reduction in the Vmax and Km, although these parameters were still markedly elevated above those of the soluble enzyme. Theoretical considerations indicated that flux through this enzyme in vivo may be regulated, by modulating the amount of enzyme adsorbed, over a much broader range of substrate concentration than would be expected from a study of the isolated enzyme.
The relevance of enzyme adsorption to the situation within the muscle fibre has been demonstrated by monitoring variations in the amounts of various glycolytic enzymes bound in mammalian muscle under different conditions. It was shown that electrical stimulation of a muscle prior to analysis caused an increase in adsorption of several enzymes; the effect was specific for phosphofructokinase, aldolase and glyceraldehyde-3-pnosphate dehydrogenase three enzymes which catalyse consecutive reactions in glycolysis. A survey of four different muscles of sheep using variable rates of stimulation revealed that each muscle had a characteristic response at different rates; a complementary study using rabbits indicated a possible role for different fibre types in these responses. The finding that the increases in enzyme binding were readily reversible, if the muscles were allowed to recover from the contractions before analysis, confirmed that the changes were a dynamic response to an altered physiological state.
Estimation of numerous intracellular metabolites provided evidence that the alterations in enzyme binding were associated with a change in the energy status of the tissue. In particular, a fall in the adenylate energy charge, a decrease in glycogen and an increase "in lactate were noted in muscles exhibiting increased levels of enzyme binding. The intimate role of glycogen and lactate changes in this phenomenon were confirmed by experiments where glycogen was eliminated using adrenalin, or the utilisation of glycogen and production of lactate were decreased by training, prior to the experiments. An indication that blood flow was also implicated in the muscle responses was provided by studies with ischemic preparations which showed maximum responses to stimuli.
The studies reported in those thesis have shown that F-actin-tropomyosin-troponin, a synthetic thin filament which most closely resembles the filaments of the l-band of muscle, is a better absorbent for aldolase than either F-actin or F-actin-tropomyosin, and that adsorption of enzyme to this filament markedly alters the enzymes kinetic parameters; both the adsorption and kinetic alterations are calcium sensitive. In addition, the present work has shown that alteration of enzyme binding is a dynamic phenomenon under physiological conditions and may be correlated with the metabolic status of the tissue.