Purification and characterization of two lipoxygenase isoenzymes from germinating barley.
Two lipoxygenase isoenzymes (linoleate: oxygen oxidoreductase, EC 1.13.11.12) present in the embryo of germinating barley seed have been purified to homogeneity and characterized. Both isoenzymes are monomeric proteins with a molecular mass of approx. 90 kDa and crossreact on Western blots with antibodies raised against pea lipoxygenase. They have an apparent Km of approx. 16 microM for linoleic acid. The isoenzymes differ in the product formed upon incubation with linoleic acid. One of the isoenzymes (lipoxygenase 1) solely forms the 9-HPOD as a product whereas the 13-HPOD is the major product formed by the other isoenzyme (lipoxygenase 2). Lipoxygenase 1 shows a pH-optimum of 6.5, is active in a broad pH range and has an isoelectric point of 5.2-5.3. Lipoxygenase 2 has the same pH optimum, but is active in a narrow pH range and has a significantly higher pI, namely 6.8-6.9. The occurrence of two isoenzymes was confirmed by peptide analysis of the proteins. Amino acid sequence data obtained from proteolytic fragments of lipoxygenase 1 show up to 50% identity with other plant lipoxygenases.
Doderer
A
A
Heineken Technisch Beheer, Zoeterwoude, Netherlands.
Kokkelink
I
I
van der Veen
S
S
Valk
B E
BE
Schram
A W
AW
Douma
A C
AC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012639:Seeds / Q000201:enzymology
D000596:Amino Acids; D007527:Isoenzymes; D008041:Linoleic Acids; D019787:Linoleic Acid; D008084:Lipoxygenase