PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was
prepared from Sulfolobus solfataricus, from which PP1-Arch was purified
over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G-
100), and Mono Q FPLC chromatography. PP1-Arch was identified from
the fmal purified sample by renaturation on an SDS-polyacrylamide gel. The
molecular size of PP1-Arch was determined by both gel filtration
chromatography and SDS-PAGE as 28 kDa and 33 kDa, respectively, which
suggests that PP1-Arch is a monomer. PP1-Arch was found stable at
temperatures as high as 90°C. Activation constants for the divalent metal
ions Mn2+ and Ni2+, and the Km for phosphocasein were determined. Myosin
light chain was found to be a substrate for PP1-Arch in vitro. EDTA, Cu2+,
Zn2+, Pi' and PPi were shown to be inhibitors of PP1-Arch, while many compounds known to affect eukaryotic protein phosphatase activities were
found to be without noticeable effect.
N-terminal and an internal peptide sequence of the enzyme were
obtained. The gene for PP1-Arch was cloned by a combination of
"touchdown" PCR and conventional cloning techniques. The PP1-Arch gene
was sequenced on both strands, and the sequence was compared with ones
from eukaryotes and bacteriophage λ. The sequence homology between
PP1-Arch and PP1/PP2A/PP2B suggests that they belongs to the same
genetic family.
A recombinant plasmid which was derived from pT7-7 was constructed
for expression of PP1-Arch. The PP1-Arch gene was expressed in E. coli and the activity of the expressed enzyme was tested and shown to be divalent
metal ion-dependent. Formation of inclusion bodies of expressed PP1-Arch
was demonstrated.
next to an author's name indicates that all files or directories associated with their ETD are accessible from the Virginia Tech campus network only.
![[VT]](http://scholar.lib.vt.edu/images/ETD-db/restricted.gif)
