Electron Microscopical Examination of Different Aggregation and Decomposition States of Phosphorylase Kinase. Identification and Computer Averaging of the αγδ Fragment

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Abstract
A sample of phosphorylase kinase aged by storage was subjected to chromatography on a size exclusion column. Samples from the four major peaks were analysed by electron microscopy. The first major peak consisted of oligomers of the enzyme monomers which showed no structural order. The second peak mainly consisted of dimers of phosphorylase kinase connected to each other by the outer tips of the "wings", thus identifying a binding locus through cross-labelling. The third peak showed the already known typical forms of phosphorylase kinase ("butterflies", "chalice form"). The last peak yielded structures identical to the .alpha..gamma..delta. fragments obtained by lithium bromide cleavage of the intact enzyme. These structures correspond to the curved outer parts of the cup subtructures of the chalice and butterfly forms. The result could be verified by computer averaging of the .alpha..gamma..delta. fragments. This finding confirms reports (Chan, K.-F.J. and Graves, D.J. (1984) Calcium Cell Funct. 5, 1-31) that the .beta.-subunits (missing in the distally situated fragment) are located in the central parts of the molecules. The good quality of the averages of the low molecular mass fragment (about 200 kDa) recommends computer averaging of electron micrographs of partial complexes as a suitable method for the study of the protein complexes.