Factors affecting the phosphorylation of a 41,000 dalton protein of hippocampal subcellular fractions.

The phosphorylation of the alpha-subunit of mitochondrial pyruvate dehydrogenase may be involved in the development of long-term potentiation in the hippocampus. Study of this hypothesis is hampered by variability in the incorporation of 32P into pyruvate dehydrogenase of hippocampal subcellular preparations, in vitro. 32P from [gamma-32P]ATP was incorporated into pyruvate dehydrogenase present in mitochondria and in a membrane-enriched synaptic particulate fraction from hippocampus. However, the presence of the synaptic fraction decreased isotopic labeling of the mitochondrial protein. This effect was not due to inhibition of the protein kinase or activation of a protein phosphatase, but the rate of ATP hydrolysis was found to be higher in the synaptic fraction than in the mitochondria (34 nmol/mg protein/min vs 14 nmol/mg protein/min). These data raise a variety of questions about the interpretation of the in vitro phosphorylation assay. It is concluded that variability in in vitro labeling can be minimized if the effect of ATP hydrolysis is diminished by use of a higher concentration of ATP. In addition, these data indicate that quantitative comparisons of the in vitro phosphorylation of diverse subcellular preparations must take into account differential rates of ATP hydrolysis.