Partial Purification and Characterization of a CAAX-Motif-Specific Protease from Bovine Brain Using a Novel Fluorometric Assay.

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概要

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• Proteolytic trimming of isoprenylated proteins, including Ras, at the C-terminal CAAX motifs is a key event in their activation. However, the protease responsible for the proteolysis has not been well characterized yet. In this study, we established a novel assay method for the enzyme using a fluorescent substrate, dansyl (Dns)-KSKTKC (S-farnesyl)-VIM, with which we can assess the proteolytic activity with high sensitivity and more easily than by the former assay methods using radio-labeled substrates. Using this assay method, we purified the protease 104-fold from bovine brain microsomal membranes by Sepharose CL-6B gel filtration and DE-52 chromatography. The partially purified enzyme was shown to be an endoprotease specific to the farnesylated peptide and to have a Km value of 1.0μM for Dns-KSKTKC (S-farnesyl) VIM. o-Phenanthroline and zinc chloride strongly inhibited the activity. Interestingly, however, m- and p-phenanthrolines were as effective as o-phenanthroline, indicating that the inhibition by o-phenanthroline is not simply due to its chelating action. The molecular mass of the protease was deduced to be 480 kDa by gel filtration. The enzymatic activity was lost during further attempts at chromatographic purification, but was partially recovered by mixing the chromatographic fractions which had apparently lost the activity. These results suggest that this protease consists of multiple subunits.

• 社団法人 日本生化学会の論文

著者

• YOKOYAMA Shigeyuki

  Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo

• MURAMATSU Tomonari

  Biophysics Division, National Cancer Center Research Institute

• NISHII Wataru

  School of Life Science, Tkyo University of Pharmacy and Life aascience

• Yokoyama Shigeyuki

  Department Of Biophysics And Biochemistry Faculty Of Science The University Of Tokyo

• Takahashi Kenji

  School Of Life Science Tokyo University Of Pharmacy And Life Science

• Kuchino Yoshiyuki

  Biophysics Division, National Cancer Center Research Institute

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