Improved purification of pyruvate decarboxylase from wheat germ. Its partial characterisation and comparison with the yeast enzyme.
An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The high-molecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of Mr = 33,000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mr alpha = 63,000-65,000 and Mr beta = 61,000-62,000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: alpha, -(Val)-(Ser)-(Ala)-Leu; beta, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (alpha beta)2 structure, the apoenzyme alpha beta. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.
Zehender
H
H
Trescher
D
D
Ullrich
J
J
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011767:Pyruvate Decarboxylase / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014908:Triticum
D000596:Amino Acids; D046911:Macromolecular Substances; D002262:Carboxy-Lyases; D011767:Pyruvate Decarboxylase