Galactokinase of Vicia faba seeds.
Galactokinase (EC 2.7.1.6) from the dormant seeds of Vicia faba was purified approximately 1300-fold with an 18% recovery through an eight-step procedure. The preparation showed the presence of only minor contaminations as judged by disc-gel electrophoresis. The native enzyme displayed a molecular weight of approximately 60 000 (determined by Sephadex G-100 gel-filtration) and the subunit value was 30 000. The isoelectric point of the enzyme was 5.3 and the amino acid analysis showed high percentage of acidic amino acids. The pH optimum of the enzyme was 7.3 at 25 degrees C. The relative activity for phosphorylating various monosaccharides followed the order, D-galactose greater than 2-deoxy-D-galactose greater than D-galactosamine; D-fucose, L-arabinose, L-galactose and D-glucose were not phosphorylated. Whereas ATP acted as an efficient phosphate donor, ADP, GTP and UTP were unable to act in this capacity. The Km and the V values of the substrates were determined. The metal ion requirement for the enzymic activity followed the order, Mg2+ greater than Co2+ greater than Mn2+ greater than Ni2+ greater than Ca2+. The enzymic reaction was inhibited by heavy metal ions and sulphydryl reagents indicating the participation of -SH group(s) in enzymic catalysis. Product inhibition was observed; galactose 1-phosphate and ADP were competitive and non-competitive inhibitors, respectively. Seed germination showed an increase in galactokinase level up to 24 h followed by a rapid decrease. The level of raffinose and stachyose decreased continually. The galactokinase level was found to be sufficiently high to phosphorylate the liberated galactose. No free galactose was observed at any stage of germination.
D000596:Amino Acids / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D007887:Fabaceae / Q000201:enzymology*; D005686:Galactokinase / Q000302:isolation & purification*; D007700:Kinetics; D010940:Plant Proteins / Q000302:isolation & purification*; D010946:Plants, Medicinal
D000596:Amino Acids; D010940:Plant Proteins; D005686:Galactokinase