Changes in the net charge and subunit properties of ribulose bisphosphate carboxylase--oxygenase during cold hardening of Puma rye.
Ribulose bisphosphate carboxylase--oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. The specific activity of the hardened form was twice that of the unhardened form. A difference in charge between the two forms of this  enzyme was proved by gel electrofocussing. The estimated isoelectric point (pI) values were 6.4 and 6.3 for the enzyme from the hardened and unhardened source respectively. The large subunit (55,000 molecular weight) of the enzyme from only the unhardened source formed at apparent dimer during sodium dodecyl sulfate (SDS) gel electrophoresis. At pH 6,8 it was also the source of an anomalous polypeptide with an apparent molecular weight of 47,000. This anomalous polypeptide appeared in both hardened and unhardened preparations after irreversible inactivation of RUBPCase activity by NaCl. It also appeared after preparation of the purified enzymes for SDS--PAGE in the absence of beta-mercaptoethanol, but this was reversible. The enzyme from the hardened source was less affected in the absence of reducing agent. Structural evidence was obtained for the previously reported cold hardening of the enzyme against freeze inactivation. A freeze-thaw cycle applied to the enzyme in vitro caused some polymerization of the large subunit and its anomalous polypeptide, in the absence of reducing agent, especially in the unhardened case. This increased with repeated cycles until the fifth cycle when the large subunit monomer and its satellite were abolished only in preparations from the unhardened source. These data indicate that the large subunit is a probable site of change that occurred in this enzyme during cold hardening.
D002262:Carboxy-Lyases / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D018521:Plant Physiological Phenomena; D010944:Plants / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D012434:Secale / Q000201:enzymology / Q000502:physiology
D046911:Macromolecular Substances; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase