Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

K. Nagata, H. Itoh, T. Katada, K. Takenaka, M. Ui, Y. Kaziro, Y. Nozawa

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46 Citations (Scopus)

Abstract

We have purified, characterized, and identified two GTP-binding proteins with M(r) of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the βγ subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, L., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low M(r) GTP-binding proteins already reported. c25KG bound about 1 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTPγS)/mol of protein, with a K(d) value of about 45 nM. [35S]GTPγS-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl β,γ-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate P(i) with the specific activity of 1.8 mmol of P(i)/mol of protein/min, which are different from the activities of the already purified low M(r) GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.

Original languageEnglish
Pages (from-to)17000-17005
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number29
Publication statusPublished - 1989
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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