The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A.
Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.
Malki
N
N
Laboratoire de Biochimie, Faculté de Pharmacie, Lille.
Balduyck
M
M
Maes
P
P
Capon
C
C
Mizon
C
C
Han
K K
KK
Tartar
A
A
Fournet
B
B
Mizon
J
J
D000510:Alpha-Globulins / Q000737:chemistry*; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D017421:Sequence Analysis; D014361:Trypsin Inhibitors / Q000737:chemistry*
D000510:Alpha-Globulins; D002241:Carbohydrates; D011134:Polysaccharides; D014361:Trypsin Inhibitors; D003208:Concanavalin A; C011877:inter-alpha-inhibitor