Characterization, crystallization and preliminary X-ray crystallographic analysis of the complex between barley alpha-amylase and the bifunctional alpha-amylase/subtilisin inhibitor from barley seeds.
The complex between a member of the barley malt alpha-amylase isozyme 2 family (AMY2-2), and the endogenous bifunctional alpha-amylase/subtilisin inhibitor, BASI, has been crystallized by the hanging drop vapour diffusion technique at a AMY2-2: BASI molar ratio of 1:1. Crystals have been grown within 4 days from solutions containing polyethylene glycol and calcium chloride. Analysis of single crystals by gel electrophoresis showed the presence of both proteins in the crystal lattice. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 74.5 A, b = 96.9 A, c = 171.3 A and they diffract to 2.0 A resolution. The presence of two molecules of the 1:1 complex in the asymmetric unit gives a solvent content of 45% by volume. The 1:1 stoichiometry of the complex was confirmed by the molecular replacement method, using as a search model the recently determined three-dimensional structure of the barley alpha-amylase.
Vallée
F
F
Laboratoire de Cristallographie et Cristallisation des Macromolécules Biologiques, CNRS URA 1296, Université d'Aix-Marseille II, Marseille, France.
Kadziola
A
A
Bourne
Y
Y
Abe
J
J
Svensson
B
B
Haser
R
R
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D012639:Seeds; D013381:Subtilisins / Q000037:antagonists & inhibitors; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; C048195:PKI-3 protein, Triticum aestivum; D010940:Plant Proteins; D000516:alpha-Amylases; D013381:Subtilisins