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Reaction of four of the eight sulfhydryl groups per subunit of rat liver glycogen synthetase D through mixed disulfide formation with oxidized glutathione leads to inactivation of the enzyme and its dissociation from glycogen. The differential protection offered by glucose 6-phosphate against inactivation and dissociation permits determination of the number of sulfhydryl groups involved in each process. Modification of the first sulfhydryl group had no effect on enzyme activity or glycogen binding. Reaction of the second sulfhydryl group led to complete inactivation of the enzyme and was accompanied by an increase in the Ka for glucose-6-P. Modification of two more sulfhydryl groups per subunit resulted in release of the enzyme from glycogen. Gel filtration experiments revealed that disruption of the glycogen-glycogen synthetase D complex was a consequence of dissociation of the enzyme itself into a subunit (mol wt 86,000) which could not bind glycogen. The enzyme-glycogen complex could be reconstituted from glycogen and glycogen-free, inactive glycogen synthetase D by treatment with glucose-6-P. Reduction with dithiothreitol, which by itself failed to restore the complex, significantly lowered the concentration of glucose-6-P required to fully achieve reconstitution. Treatment with glucose-6-P, but not reduction with dithiothreitol, reassociated the inactive subunit into a high molecular weight species which is the form capable of binding glycogen. These results suggest that glucose-6-P plays an important role in maintaining the glycogen-glycogen synthetase D complex.

Meyer et al. [1 ] have described the preparation of a particulate suspension from rabbit muscle containing glycogen and a number of enzymes. They suggest that the glycogen binding enzymes are attached to the large glycogen molecules in the suspension and they term these aggregates 'glycogen particles'. The enzymes present include phosphorylase b, phosphorylase b kinase, phosphorylase phosphatase and other enzymes of glycogen metabolism, trace glycolytic enzymes and some of the enzymes concerned with nucleotide interconversions. Studies by the same group have shown that some of these enzymes have anomalous properties when bound to the glycogen particles and they postulate specific interactions either between the enzymes themselves or with the carbohydrate matrix to account for this. They also suggest that these protein-glycogen complexes are the glycogen granules seen in sections of muscle by electron microscopy and that the 'glycogen particle' is therefore a cellular oganelle [2 -5 ] . Transient production of phosphorylase a could be generated in this glycogen particle rich fraction by the addition of ATP, calcium and magnesium ions. This 'flash activation', was completely calcium dependent because of the calcium requirement of phosphorylase b kinase, and was terminated by the action of phosphorylase phosphatase on the phosphorylase a after the ATP had been used up [2]. Using spin-labelled phosphorylase b it has been possible to monitor the conformational changes which occur both when ligands bind to the enzyme and when it is activated by conversion to phosphorylase a [6,7]. This clearly offers a way of studying the behaviour of phosphorylase attached to the glycogen particle and in this paper we describe the use of spin labelled phosphorylase b to monitor flash activation and the interactions of ligands with phosphorylase during this process.