Abstract
A phosphoinositide-specific phospholipase C (PLC) was solubilized from the isolated nuclei of rat ascites hepatoma AH7974 cells by ultrasonication in 2 M KCl. The extract was then subjected to five steps of column chromatographies in the order of Sephacryl S-300, phosphocellulose, Mono Q, Mono S, and Superose 6. Four forms of PLC (tentatively designated as N1, N2, N3, and N4) were purified 440-1400-fold. N1, N2, N3, and N4 showed apparent molecular masses of 85, 83, 80, and 88 kDa, respectively, on SDS-polyacrylamide gel electrophoresis. N1 cross-reacted with the antibody against the delta 1 isoform, while the other three forms did not cross-react with any of the antibodies against PLC-delta 1, -gamma 1, -gamma 2, and -beta 1. They hydrolyzed phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) but did not show any activities against phosphatidylcholine and phosphatidylethanolamine. They showed the same optimal pH:pH 6.5 for PI hydrolysis and pH 7.0 for both PIP and PIP2 hydrolyses. They absolutely required Ca2+ for activity, with optimal concentrations of 10(-3)-10(-5) M for PIP and 10(-4)-10(-5) M for PIP2. For PI hydrolysis, N1, N2, and N3 required a Ca2+ concentration higher than 10(-2) M whereas N4 revealed significant activity even at 10(-5) M Ca2+ concentrations. Two forms of plasma membrane PLC and three forms of cytosolic PLC were purified from AH7974 cells by the same procedure as for nuclear PLC. Comparative study with these three groups revealed that all of the purified PLC isoforms shared similar enzymological properties except N4, which showed an exceptionally high affinity to Mono S column and was active at low concentrations of Ca2+ for PI as substrate. Furthermore, when PLC isoforms of nuclei from adult resting rat liver were compared with those from regenerating rat liver after partial hepatectomy, a PLC isoform corresponding to N4 of AH7974 cells was found only in regenerating liver nuclei. From these results, it was suggested that the nuclei of growing liver cells possessed a unique form of PLC (N4).
Highlights
The extract was subjected to five steps of column chromatographies in the ordeofr Sephacryl S-300, phosphocellulose, Mono Q, Mono S, and Superose 6
We attempted to purify nuclear phospholipase C (PLC) in order to identify the PLC isoforms in nuclei using rat ascites hepatoma cells (AH7974).We have identified four formsof PLC in nuclei, designated as N1, N2, N3, and N4
For PI hydrolysis, N1, N2, cytosol of the samecells, one of the nuclearPLC isoforms, N4, and N3 required a Ca2+concentration higher than lo-' M was shown to be unique with respect to thebinding affinity to whereas N4 revealed significant activity even at
Summary
Calculated from the optical densityat 280 nm usinga SMART system. isolatednucleiwasestimated to bemore than 90% by measuring marker enzyme activities and by electron microscopy [23, 27]. Calculated from the optical densityat 280 nm usinga SMART system. Isolatednucleiwasestimated to bemore than 90% by measuring marker enzyme activities and by electron microscopy [23, 27]. Especially,the contaminationof plasma membrane to nuclei was shown to be negligible by thedetermination of 5'-nucleotidase activity. Plasma membrane andcytosol were preparedby methods described previously [27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
