Diversity of puroindolines as revealed by two-dimensional electrophoresis.
Puroindolines are endosperm lipid binding proteins, which are separated by reversed phase-high-performance liquid chromatography or cation exchange chromatography into two isoforms, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Being very basic and close in molecular weight, PIN-a and PIN-b have never been separated using conventional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A two-dimensional electrophoresis method, linear immobiline pH gradient (IPGxSDS-PAGE), was developed, using 6-11 linear immobiline Dry Strips in the first dimension, which allowed the puroindolines to be focused between isoelectric point 10.5 and 11. Immunoblotting revealed that both PIN-a and PIN-b were each composed of several spots. Two-dimensional patterns from unrelated wheat varieties revealed that several spots can be highlighted among varieties. Matrix-assisted laser desorption/ionization-time of flight spectrometry allowed the majority of the spots revealed in the puroindoline zone to be identified. The two-dimensional IPGxSDS-PAGE of these very basic wheat endosperm proteins, puroindolines and related grain softness proteins should facilitate the identification of the proteins associated with wheat endosperm texture that have a strong effect on milling, dough properties and end-uses of wheats.
Branlard
Gérard
G
UMR INRA ASP-UBP, Clermont Ferrand, France. branlard@clermont.inra.fr
Amiour
Nardjis
N
Igrejas
Gilberto
G
Gaborit
Thérèse
T
Herbette
Stephane
S
Dardevet
Mireille
M
Marion
Didier
D
D015153:Blotting, Western; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000737:chemistry*; D011110:Polymorphism, Genetic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000378:metabolism
D002412:Cations; D010940:Plant Proteins; C082942:puroindoline protein, Triticum aestivum