Purification and characterization of cytosolic pyruvate <span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-bidi-font-family: HiddenHorzOCR">kinase from developing seeds <i>of Brassica campestris L</i> </span>

Abstract

Cytosolic pyruvate kinase (ATP: Pyruvate phosphotransferase, EC 2.7.1.40; PKc) was purified to apparent homogeneity with about 22% recovery from developing seeds of <i>Brassica campestris </i>using (NH₄)SO₄ fractionation,

DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive Blue Sepharose-CL-6B. The &nbsp;purified enzyme with molecular mass of about 214 kDa was a heterotetramer with subunit molecular mass of 55 and 57 kDa. The enzyme showed maximum activity at <i style="mso-bidi-font-style:normal">p</i>H 6.8 and absolute requirement for a divalent

(Mg²⁺) and a monovalent (K⁺) cation for activity. Typical Michaelis-Menten kinetics was obtained for both the substrates with <i>K_m </i>values of 0.10 and 0.11 <i>mM </i>for PEP and ADP, respectively. The enzyme could also use UDP or GDP as alternative nucleotides, but with lower V_maxand lesser affinities. The enzyme was inhibited by glutamate, glutamine, fumarate, citrate,

isocitrate, oxalate, 2-PGA. ATP, UTP and GTP and activated by glucose-6-phosphate, fructose-1,6-bisphosphate and Pi, suggesting its regulation mainly by TCA cycle intermediates and the cellular need for carbon skeletons for amino acid biosynthesis. ATP inhibition was of competitive type with respect to PEP and non-competitive with respect to ADP.

Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. Initial <span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-bidi-font-family:HiddenHorzOCR">velocity and product inhibition studies except for pyruvate inhibition were consistent for a compulsory-ordered tri-bi mechanism.

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