Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay.

Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Research Abstract Details 

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Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Abstract Text:

W Nishii,T Muramatsu,Y Kuchino,S Yokoyama,K Takahashi,

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 K(m) value of 1.0 microM 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.

Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Publishing Authors By Initials

W Nishii,T Muramatsu,Y Kuchino,S Yokoyama,K Takahashi,

For similar investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research abstracts see: investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research

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Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 122

Page Numbers: 402-8

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Aug

YEAR: 1997

Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Keywords Mesh Terms:

KEYWORDS: Spectrometry, Fluorescence

MESH TERMS: methods

Chemical & Substance for Abstract: Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay. Information

Substance Name: Cysteine Endopeptidases

Registry Number: EC 3.4.22.-

Grant and Affiliation Information for Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay.

AFFILIATION: School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji.

Country: JAPAN

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MEDLINETA: J Biochem

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