Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum.
Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.
Lee
W K
WK
Center for Molecular Catalysis, Department of Chemistry, Seoul National University, Seoul 151-742, Korea.
Shin
S
S
Cho
S S
SS
Park
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D018870:Endoplasmic Reticulum, Rough / Q000201:enzymology*; D004795:Enzyme Stability; D005260:Female; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006358:Hot Temperature; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D005969:Glutamate Dehydrogenase