Abstract
A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.
Highlights
Human Pin1 belongs to the parvulin family of peptidyl-prolyl cis/trans isomerases (EC 5.2.1.8) [1]
Isolation of DlPar13—The chromatographic isolation of a phospho-specific parvulin from cells grown as proembryogenic masses (PEMs) of D. lanata Ehrh. strain VIII was carried out by applying a sensitive PPIase assay for detection of phosphorylation-specific enzyme activity in crude protein solutions
We detected in cell lysates of PEMs of D. lanata a 90-fold higher total PPIase activity toward the unphosphorylated substrate Ac-Ala-Ser-Pro-Tyr-NH-Np than toward the side chain phosphorylated peptide derivative (Ac-AlaSer(OPO3H2)-Pro-Tyr-NH-Np)
Summary
Enzyme Assay—For general screening purposes the protease-coupled PPIase microplate assay was performed [22]. The specificity constants kcat/Km were determined according to Fischer et al [23]. The sensitivity of the PPIase assay was calculated with the equation [lowest detectable PPIase concentration] ϭ 4*ko/kcat/KM), with ko as the uncatalyzed first-order rate constant. After an affinity chromatography step (Fractogel TSK AF-Blue, 80 ϫ 15 mm, Merck) the fractions containing the highest phospho-specific activity were further separated by reverse phase HPLC (Nucleosil 300 –5 C18, 125 ϫ 3 mm, Macherey-Nagel, Duren, Germany) applying a linear gradient of 30 –50% acetonitrile in 0.1% trifluoroacetic acid for 40 min at 40 °C with a flow rate of 0.5 ml minϪ1. The molecular masses of the obtained fragments were determined by matrix-assisted laser desorption time-of-flight mass spectrometry and sequenced using sequencer 476A (Applied Biosystems, Weiterstadt, Germany) according to the manufacturers instructions or as described by Pfeifer et al [30]. The resulting 128-base pair PCR fragment was cloned into a TOPO
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