Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.
The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.
Lorand
L
L
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Velasco
P T
PT
Murthy
S N
SN
Wilson
J
J
Parameswaran
K N
KN
D000595:Amino Acid Sequence; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D003619:Dansyl Compounds; D007908:Lens, Crystalline / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D011503:Transglutaminases / Q000378:metabolism*
D003459:Crystallins; D003619:Dansyl Compounds; D009842:Oligopeptides; D011503:Transglutaminases; C008542:monodansylcadaverine; D008239:Lysine; D002103:Cadaverine