Solubilization of liver alkaline phosphatase isoenzyme during cholestasis in dogs.
OBJECTIVE
To determine the mechanism by which liver alkaline phosphatase (LALP) isoenzyme is converted from a membrane-bound enzyme to the soluble enzyme during cholestasis.


METHODS
Serum and tissues from 2 dogs.


METHODS
The LALP was purified by use of affinity chromatography in samples of serum from dogs with complete bile duct obstruction. Gas chromatography/mass spectrometry was used to detect myo-inositol residues that would be evident when serum LALP had been membrane-attached and released by phospholipase activity. Exclusion chromatography, gel electrophoresis, and octyl-sepharose phase separation of the serum isolate were used to confirm cleavage of the hydrophobic membrane anchor. Western immunoblot analysis was used to distinguish release by glycosylphosphatidylinositol phospholipase D (GPI-PLD) from that by glycosylphosphatidylinositol phospholipase C (GPI-PLC). Intact hepatocytes were incubated with canine serum GPI-PLD to test sensitivity of LALP to release by GPI-PLD. Hepatocyte membrane fragments were treated with serum GPI-PLD and mixtures of taurocholate and taurodeoxycholate to test effects of bile acids on LALP release.


RESULTS
Amounts of myo-inositol per mole of serum LALP isolate were equal to amounts detected with LALP isolated from hepatic tissue. Evaluation of results of western immunoblot analysis and electrophoretic mobility suggested release by GPI-PLD rather than by GPI-PLC. Membrane-bound LALP was resistant to serum GPI-PLD activity in the absence of bile acids; however, incubation in the presence of bile acids caused release of LALP.


CONCLUSIONS
Solubilization of LALP during cholestasis involves cleavage of its membrane anchor by endogenous GPI-PLD activity. Action of GPI-PLD is likely enhanced by increased concentrations of hepatic bile acids during cholestasis.
Solter
P F
PF
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61802, USA.
Hoffmann
W E
WE
D000469:Alkaline Phosphatase / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western / Q000662:veterinary; D002779:Cholestasis / Q000201:enzymology / Q000503:physiopathology / Q000662:veterinary*; D002846:Chromatography, Affinity / Q000662:veterinary; D002847:Chromatography, Agarose / Q000662:veterinary; D004283:Dog Diseases / Q000201:enzymology* / Q000503:physiopathology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D008401:Gas Chromatography-Mass Spectrometry / Q000662:veterinary; D017261:Glycosylphosphatidylinositols / Q000502:physiology; D007202:Indicators and Reagents / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D007294:Inositol / Q000032:analysis; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D012995:Solubility; D013656:Taurocholic Acid / Q000502:physiology
D000911:Antibodies, Monoclonal; D017261:Glycosylphosphatidylinositols; D007202:Indicators and Reagents; D007211:Indoles; D007527:Isoenzymes; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D007294:Inositol; D013656:Taurocholic Acid; D000469:Alkaline Phosphatase