title: Promotion of insulin aggregation by protein disulfide isomerase.
abstract: We examined the aggregation of insulin as a result of reduction of disulfide bonds catalyzed by protein disulfide isomerase (PDI) using various techniques. We demonstrated the kinetic correlation between PDI-catalyzed insulin reduction and the aggregate formation, the relationship between aggregation and amyloid formation, and the structural information on the secondary structure of the aggregates. The initial rate of PDI-catalyzed reduction of insulin, apparent rate constants of aggregate growth for sigmoidal features, and lag times were obtained by changing the PDI concentration, temperature, and insulin concentration. In situ kinetics were studied using the dyes; thioflavin T (ThT) and Congo red (CR) in addition to turbidimetry with the insulin reduction by PDI. The ThT and CR binding assay revealed sigmoidal kinetics, and the spectrum of binding CR showed a red shift against time, suggesting an orderly formation of insulin aggregates. The secondary structure of the PDI-promoted insulin aggregates showed antiparallel beta-sheet conformation by FT-IR measurement.
authors.affiliation: Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu, Fukuoka 802-0985, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Maeda
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ado
authors.forename: Naohiro
authors.initials: N
authors.lastname: Takeda
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D011232:Chemical Precipitation; D003224:Congo Red / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D004220:Disulfides; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D019704:Protein Disulfide-Isomerases