title: Non-enzymatic glycation of antithrombin III in vitro.
abstract: Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.
authors.affiliation: Laboratory of the Howard Hughes Medical Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
authors.forename: T
authors.initials: T
authors.lastname: Sakurai
authors.forename: J P
authors.initials: JP
authors.lastname: Boissel
authors.forename: H F
authors.initials: HF
authors.lastname: Bunn
mesh_terms: D000595:Amino Acid Sequence; D000990:Antithrombin III; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005947:Glucose; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D000990:Antithrombin III; D005947:Glucose