| Autor: |
Stirtan, W.G., Poulter, C.D. |
| Zdroj: |
Archives of Biochemistry and Biophysics; August 1, 1995, Vol. 321 Issue: 1 p182-190, 9p |
| Abstrakt: |
Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in leucine by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). The Saccharomyces cerevisiae genes encoding the α (RAM2) and β (CDC43) subunits of PGGTase-I were translationally coupled by overlapping the RAM2-CDC43 stop-start codons and by locating a ribosome-binding site near the 3' end of RAM2. Recombinant PGGTase-I was overproduced in Escherichia coli to give ~8% of total cellular protein and purified 12-fold to >95% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The purified heterodimer contained α- and β-subunits with molecular masses of 34 and 42 kDa, respectively. A continuous fluorescence assay was developed to measure PGGTase-I activity. The recombinant enzyme showed maximal activity at pH 7.5 and required both Mg2+ and Zn2+. Michaelis constants for GGPP (1.0 µM) and dansyl-Gly-Cys-Ile-Ile-Leu (2.4 µM) were similar to those reported for yeast protein farnesyltransferase (PFTase) with farnesyl diphosphate and dansyl-Gly-Cys-Val-Ile-Ala; Vmax = 0.20 µmol min−1 mg−1 for recombinant yeast PGGTase-I was similar to that reported for yeast PFTase.Copyright 1995, 1999 Academic Press, Inc. |
| Databáze: |
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