ABSTRACT
1,1-Dichloroethylene (DCE, 50 mg/kg) rapidly and selectively injures the bile canalicular membrane of zone 3 hepatocytes. Thus, DCE is of value as a tool to assess the consequences of alterations in canalicular membrane integrity on bile formation. Our objective was to characterize the effects of DCE on the biliary secretion of proteins and phospholipids in freely moving rats. DCE treatment caused a rapid and sustained decrease in total biliary protein output. In contrast, canalicular membrane-localized enzyme activities more slowly increased to 8- to 15-fold in bile from DCE-treated rats. Biliary output of lysosomal enzymes was altered in a biphasic manner. Specifically, there was a transient fivefold increase within 30 min of DCE treatment and then a progressive decrease to approximately 10% basal levels by 4 h. Secretion of phospholipids into bile decreased rapidly in a striking and sustained manner, after DCE. Our findings of diminished lysosomal protein and phospholipid secretion following DCE treatment are consistent with an important role for canalicular membrane integrity in their entry into bile.
Subject(s)
Bile Canaliculi/drug effects , Dichloroethylenes/toxicity , Membrane Proteins/metabolism , Phospholipids/metabolism , Proteins/metabolism , Animals , Bile/metabolism , Bile Canaliculi/enzymology , Bile Canaliculi/metabolism , Cell Membrane/enzymology , Electrophoresis , L-Lactate Dehydrogenase/metabolism , Lysosomes/drug effects , Lysosomes/enzymology , Male , Rats , Rats, Sprague-DawleyABSTRACT
Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
