Analysis of catalytic subunit microheterogeneity in ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum.

Urea isoelectric focusing of dissociated, carboxymethylated Nicotiana tabacum ribulose-1,5-bisphosphate carboxylase/oxygenase reveals catalytic subunit microheterogeneity. Aggregated or nonaggregated sucrose gradient-purified preparations and the crystalline protein displayed essentially identical large subunit multiple polypeptide patterns. Various pretreatments which fully dissociate the holoenzyme did not alter catalytic subunit microheterogeneity. Direct comparison of the carboxymethylated and noncarboxymethylated crystalline and sucrose gradient-purified proteins demonstrated that the large subunit multiple polypeptide pattern was not an artifact of carboxymethylation. The inclusion of the seryl protease inhibitor phenylmethylsulfonyl fluoride during purification of the holoenzyme did not affect the large subunit multiplicity. However, the addition of leupeptin, a potent thiol proteinase inhibitor, to all solutions during purification of the native protein markedly reduced large subunit polypeptide L3 and increased the staining of polypeptide L2, suggesting that L3 is a leupeptin-sensitive proteinase degradation product of L2. Polypeptide L1 also appeared to be a purification-related artifact, but derived from a modification of L2 other than that which yielded L3. We conclude that polypeptide L2 is the single, native isoelectric form of the catalytic subunit of tobacco ribulosebisphosphate carboxylase/oxygenase.