Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules

Citation
Sa. Mccloud et al., Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules, PHYSL PLANT, 111(3), 2001, pp. 283-290
Citations number
49
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PHYSIOLOGIA PLANTARUM
ISSN journal
00319317 → ACNP
Volume
111
Issue
3
Year of publication
2001
Pages
283 - 290
Database
ISI
SICI code
0031-9317(200103)111:3<283:PPACOP>2.0.ZU;2-L
Abstract
Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cyt osolic fraction of N-2-fixing soybean (Glycine max \L\ Merr.) root nodules, The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major ph osphoenolpyruvate (PEP)-utilizing enzyme in legume root nodules, Latency ex periments with sonicated nodule extracts showed that Bradyrhizobium japonic um bacteroids do not express either PK or PEPC activity in symbiosis, In co ntrast, free-living B. japonicum bacteria expressed PK activity, but not PE PC activity. Antibodies specific for the cytosolic isoform of PK from casto r bean endosperm cross-reacted with a 52-kDa polypeptide in the partially p urified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extrac ts was 2.6 times the corresponding PK activity, This would tend to favour P EP metabolism by PEPC over PEP metabolism by PK, However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activ ity was unaffected. Thus, we propose that PK and PEPC activity in legume ro ot nodules may be coordinately regulated by fluctuations in malate concentr ation in the plant cytosolic fraction of the bacteroid-containing cells. Re duced uptake of malate by the bacteroids, as a result of reduced rates of N -2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.