Identification and biochemical characterization of an 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei.
The primary aim of these studies was to identify and biochemically characterize GTP-binding proteins in the nucleus. We found that an 80 kDa protein was responsible for the majority of the GTP-binding activity detected in rabbit liver nuclear preparations as assayed by photoaffinity labeling with [alpha-32P]GTP. The GTP-binding activity was partially extracted only after treatment of nuclear envelope preparations with 0.5 M NaCl and 1% Triton-X 100, which suggested that this GTP-binding protein was a component of the nuclear pore/lamina fraction. The Triton-X-100/NaCl-solubilized 80 kDa protein was purified by a series of steps that included DEAE-Sephacel, Mono-Q, and Ultrogel AcA34 chromatographies. Microsequence analysis of two peptides generated by trypsin digestion of the 80 kDa protein indicates that it shares sequence similarity with the tissue transglutaminases. Purified preparations of the 80 kDa protein show a Ca(2+)-stimulated transglutaminase activity, as assayed by the incorporation of [3H]putrescine into caesin, which is strongly inhibited by GTP but not by GDP. A 36 kDa GTP-binding protein copurified with the 80 kDa GTP-binding protein through all of the chromatography steps and sequence analysis suggests that the 36 kDa protein represents a proteolytic fragment of the amino-terminal half of the 80 kDa protein and thus serves to mark the GTP-binding domain within the 80 kDa protein. The 36 kDa fragment has a significantly higher efficiency of [alpha-32P]GTP incorporation compared to the 80 kDa protein, suggesting that the carboxyl-terminal half of the GTP-binding protein/transglutaminase imparts a negative constraint on GTP-binding activity or on the subsequent incorporation of radiolabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002467:Cell Nucleus / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011817:Rabbits; D011503:Transglutaminases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011503:Transglutaminases; D019204:GTP-Binding Proteins