Cytosolic serine hydroxymethyltransferase. Deamidation of asparaginyl residues and degradation in Xenopus laevis oocytes.
Rabbit liver cytosolic serine hydroxymethyltransferase exists as a set of subforms which exhibit different isoelectric points. Previous studies have shown that deamidation of an asparagine residue at position 5 of the amino acid sequence accounted for some of the charge heterogeneity (Artigues, A., Birkett, A., and Schirch, V. (1990) J. Biol. Chem. 265, 4853-4858). The present study has also identified asparagine 220 as being partially deamidated. An estimated 25-30% of the purified enzyme contains an isoaspartyl residue at position 220. This suggests that deamidation of asparagine 220 occurs by the beta-aspartyl shift mechanism. Western blot analysis of purified cytosolic serine hydroxymethyltransferase, after isoelectric focusing under reducing and denaturing conditions, showed four subforms of the individual subunits with respect to isoelectric point. Extracts from 3-day- and 3.5-year-old rabbit livers showed the presence of these same four subunit subforms. Purified cytosolic serine hydroxymethyltransferase was found to be degraded in 24 h after mechanical injection into Xenopus laevis oocytes. However, when the first 14 amino acid residues are removed from the enzyme by digestion with chymotrypsin, leaving a fully catalytically active enzyme, the rate and extent of degradation of the truncated enzyme in oocytes were significantly reduced. One of the deamidated asparagine residues is at position 5, suggesting that this deamidation site may be a signal for degradation of the enzyme.
Artigues
A
A
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
Farrant
H
H
Schirch
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology; D005260:Female; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D014982:Xenopus laevis
D010446:Peptide Fragments; D001216:Asparagine; D012696:Glycine Hydroxymethyltransferase