Mass spectrometric identification of the amino donor and acceptor sites in a transglutaminase protein substrate secreted from rat seminal vesicles.
Four different transglutaminase-modified forms of a protein secreted by the rat seminal vesicles (SV-IV) were synthesized in vitro and characterized. FAB maps of both the native protein and its derivatives, produced by the purified guinea pig liver enzyme in the presence or absence of the polyamine spermidine, were obtained by mass spectrometric analysis after proteolytic digestions. Two differently derivatized SV-IV molecular forms, both possessing only one glutamine residue out of two (Gln-86) cross-linked to endogenous lysine residues, were produced when spermidine was omitted from the reaction mixture: (i) an insoluble homopolymer in which Lys-2, -4, -59, -78, -79, and -80 were involved in the linkage; (ii) a soluble form of the protein with an intramolecular epsilon-(gamma-glutamyl)lysine isopeptide bond between Gln-86 and Lys-59. Two species of SV-IV-spermidine adducts were obtained when the protein was treated with transglutaminase in the presence of high concentrations of the polyamine. The first one was characterized by one spermidine molecule covalently bound to Gln-86 and the second one by two spermidine molecules respectively bound to Gln-9 and Gln-86.
Porta
R
R
Istituto di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Palermo, Italy.
Esposito
C
C
Metafora
S
S
Malorni
A
A
Pucci
P
P
Siciliano
R
R
Marino
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D006168:Guinea Pigs; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D029584:Prostatic Secretory Proteins; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D029589:Seminal Plasma Proteins; D012669:Seminal Vesicles / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry*
D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein; D011503:Transglutaminases