Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes.
The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.
Psarra
A M
AM
Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.
Sotiroudis
T G
TG
D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007527:Isoenzymes / Q000276:immunology; D008566:Membranes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D010764:Phosphorylase Kinase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology*
D007527:Isoenzymes; D010764:Phosphorylase Kinase; D002118:Calcium