Molecular and Regulatory Properties of Leucoplast Pyruvate Kinase from Brassica napus (Rapeseed) Suspension Cells

Author： Plaxton W.C. Smith C.R. Knowles V.L.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.400, Iss.1, 2002-04, pp. : 54-62

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Abstract

Plastidic pyruvate kinase (PK_p) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 μmol phosphoenolpyruvate (PEP) utilized/min/mg protein. SDS–PAGE, immunoblot and gel filtration analyses indicated that this PK_p exists as a 380-kDa heterohexamer composed of equal proportions of 64- (α-subunit) and 58-kDa (β-subunit) polypeptides. The N-terminal sequence of the PK_p α- and β-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively. B. napus PK_p displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (K_m = 0.052 and 0.14 mM, respectively). 6-Phosphogluconate functioned as an activator (K_a = 0.12 mM) by increasing V_max by approximately 35% while decreasing the K_m(PEP) and K_m(ADP) values by 40 and 50%, respectively. 2-Oxoglutarate and oxalate were the most effective inhibitors (I₅₀ = 8.3 and 0.23 mM, respectively). A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B. napus leucoplasts. ©2002 Elsevier Science (USA).