Stabilization of hepatic colchicine-binding activity by organic acids

Previous work has shown that the total hepatic tubulin pool and the hepatic microtubule-derived tubulin pool do not have identical [3H]colchicine binding properties. Rapid loss of colchicine-binding activity was noted in the microtubule-derived fractions of liver tubulin. Furthermore, quantitative determination of the total and polymerized tubulin in the liver by the [3H]colchicine-binding assay was hampered by rapid and unequal loss of binding sites under assay conditions. The organic acids, glutamate and glucose 1-phosphate, have been shown to stabilize calf brain tubulin against loss of colchicine-binding sites. Therefore, these compounds were tested as possible protecting agents against loss of colchicine binding activity of liver tubulin. It was found that these agents stabilized liver tubulin under [3H]colchicine-binding conditions. Additional experiments showed that these agents also prevented the rapid loss of colchicine-binding activity that occurred when purified brain tubulin was exposed to liver supernates. These results suggest that the inclusion of the organic acids, glutamate and glucose 1-phosphate, may modify the time decay properties of liver tubulin in solution. Further, these data suggest that these protecting agents may be of analytical value in [3H]colchicine-binding assay systems for liver tubulin.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.