Increase in negative charge of cytosolic aspartate aminotransferase in vitamin B6 deficiency and during incubation.
Several subforms of cytosolic aspartate aminotransferase (AspATc) in the crude extracts of rat liver and kidney were separated by isoelectric focusing using immunoblotting and staining of activity to detect the enzyme protein and activity, respectively. Vitamin B6-deficiency resulted in decrease in the subforms with higher isoelectric points and increase in those with lower ones both in the liver and in the kidney. When pyridoxal phosphate was added to those preparations from vitamin B6-deficient rats, the isoelectric focusing pattern of kidney was recovered to the similar one to that of the controls. However, the liver preparation was affected only partially by the addition of PLP. The pattern of subforms was altered during in vitro incubation at 37 degrees C for 24 h in both liver and kidney preparations, and their patterns were very similar to that of liver preparation from VB6-deficient rats. The enzyme activity also decreased during this incubation, especially in preparations of the enzyme from the liver of vitamin B6-deficient rats. This loss of enzyme activity was not affected by addition of PLP alone, but was almost completely prevented by addition of substrate. The inactivation was recovered by addition of substrate and pyridoxal phosphate simultaneously. This finding suggests that the inactivation may be related with a conformational change around the catalytic site of the AspATc molecule.
Okada
M
M
Faculty of Health and Living Sciences, Naruto University of Education, Tokushima, Japan.
Nagai
M
M
Harima
H
H
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004563:Electrochemistry; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D026681:Vitamin B 6 Deficiency / Q000201:enzymology*
D007527:Isoenzymes; D007656:Ketoglutaric Acids; D011732:Pyridoxal Phosphate; D008623:Mercaptoethanol; D001219:Aspartate Aminotransferases