Showing papers by "Pál Gergely published in 1995"

Endothall thioanhydride inhibits protein phosphatases-1 and -2A in vivo

Abstract: The objective of this study was to relate the toxicity of several cantharidin-derivative pesticides with their abilities to inhibit protein phosphatases-1 (PP1) and -2A (PP2A). Cantharidin (CA), endothall, and endothall thioanhydride (ETA) inhibited the activity of PP1 and PP2A, and the potency sequence was CA > endothall > ETA in vitro. We determined the inhibitory potency of these pesticides on hepatic protein phosphatases by administration of the toxins into the portal vein of rats. The potency sequence of ETA > CA > endothall was established for the inhibition of PP1 and PP2A in vivo and shows close correlation with the sequence of relative toxicity. ETA predominantly targets PP1 for inhibition in liver, as revealed by assays specific for PP1 or PP2A. Studies using 3T3 fibroblasts showed that only ETA, but not CA or endothall, induced marked morphological changes. These effects included cell rounding and detachment as well as extensive reorganization of actin filaments and are characteristic for the cell-permeable phosphatase-inhibitory toxins. It is suggested that the in vivo effectiveness is related to enhanced uptake of ETA, because this is permeable across the plasmalemma.

Changes of the 78 kDa glucose-regulated protein (grp78) in livers of diabetic rats.

Abstract: The 78 kDa glucose-regulated protein (grp78) is an abundant member of the 70 kDa molecular chaperone family in the lumen of the endoplasmic reticulum participating in the quality control of secretory proteins. In the present paper we have analysed the synthesis and level of grp78 in livers of control, streptozotocin-diabetic, and the spontaneously diabetic Zucker rats. The level of grp78 mRNA significantly decreased in streptozotocin-diabetic rats. The effect was reversed by insulin treatment. In case of Zucker rats we did not detect any significant change in grp78 mRNA, grp78 protein level showed opposite changes being essentially unchanged in streptozotocin-diabetes and significantly reduced in Zucker rats. Autoradiograms of Ca-dependent phosphorylation of postmitochondrial supernatants of control and streptozotocin-diabetic livers indicated no significant changes in the 70 kDa region. Decrease in the availability of grp78 may participate in the attenuation of hepatic protein secretion in diabetes.

Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa

Abstract: The catalytic subunit of protein phosphatase 2A (PP2A c ) was purified from Neurospora crassa extract by (NH 4 ) 2 SO 4 -ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/ g with a 2% yield. The apparent M r of PP2A c was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2A c was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the a-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2A c is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2A c did not cross-react with N. crassa PP2A o indicating sequence differences outside the catalytic core of the enzyme.