RESUMEN
The bovine beta-crystallin Bp chain is organized into two very similar domains, with short extensions at both N- and C-termini, and two alternative models for the beta Bp dimer have been proposed (Wistow, G., Slingsby, C., Blundell, T., Driessen, H.P.C., De Jong, W.W. and Bloemendal, H. (1981) FEBS Lett. 133, 9-16). By limited proteolysis the C-terminal arms can be cleaved off rapidly from the beta Bp dimer, while the N-terminal arms are more difficult to remove. Trypsin divides the beta Bp chain into two fragments which approximately correspond to the two structural domains. Dissociation and reassociation of the different products of limited proteolysis indicated that: the C-terminal arm extends freely from the surface and is not involved in subunit-contact; at least one N-terminal arm seems required for dimer formation; the N-terminal domains have a greater tendency to associate than the C-terminal domains and, when mixed, the purified domains reassociate partially to a Mr 50 000 structure like native beta Bp. These findings support the more extended dimer model of beta Bp.
Asunto(s)
Cristalinas/metabolismo , Animales , Bovinos , Quimotripsina , Electroforesis en Gel de Poliacrilamida , Cinética , Sustancias Macromoleculares , Peso Molecular , Fragmentos de Péptidos/análisis , Conformación Proteica , Tripsina/metabolismoRESUMEN
Incubation of calf lens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different beta-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamylyltransferase, EC 2.3.2.13). One of these is beta Bp, the predominant subunit of beta-crystallin, of which the amino acid sequence is known. The site of amine-labeling in beta Bp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled beta-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled beta subunits.