324
Biochimica et Biophysica Acta, 587 (1979) 324--332
© Elsevier/North-Holland Biomedical Press
BBA 29068 A CYCLIC GMP-DEPENDENT HISTONE KINASE BOUND TO LIVER NUCLEOLI
ANNIKKA LINNALA-KANKKUNEN and PEKKA H. MAENP~,A Department of Biochemistry, University of Kuopio, P.O. Box 138, SF-70101 Kuopio 10 (Finland)
(Received February 26th, 1979) Key words: Cyclic GMP; Histone kinase; (Liver nucleolus)
Summary A m e t h o d of steady-state electrophoresis in polyacrylamide gels was used to analyze the presence of cyclic nucleotide binding components in cell extracts. Multiple cyclic AMP and cyclic GMP binding c o m p o n e n t s were detected in soluble cytoplasmic and nuclear extracts derived from avian liver, but only a single cyclic GMP binding protein was found in the 0.3 M NaC1 extract of liver nucleoli. In the presence of cyclic GMP, this protein phosphorylated efficiently a calf t h y m u s histone mixture and an endogenous nucleolar protein, which migrated identically with histone H4 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the cyclic GMP-binding protein was 4.8. Addition of cyclic GMP did not influence the activity of the endogenous nucleolar R N A polymerase. Introduction Cyclic nucleotides have been implicated as intracellular mediators during processes associated with growth in tissues of higher animals and cells in culture. The intracellular distribution of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic G M P ) b i n d i n g proteins in cellular extracts has been examined with techniques of ion~exchange chromatography and i m m u n o c y t o c h e m i s t r y [1,2]. We have employed a new technique of steady-state electrophoresis in polyacrylamide gels to separate and identify cyclic nucleotide binding proteins in various cellular subfractions. The m e t h o d was initially .described in studies of steroid binding proteins [3], b u t with some modifications it can also be used in analysis of cyclic nucleotide binding components in cellular extracts. The method gives good separation and a quantitative estimate of the individual binding c o m p o n e n t s in the sample,
325 provided that all components reach steady-state binding conditions with the free radioactive ligand in the gel [3]. This communication will describe the method and a partial characterization and properties of a cyclic GMP
Biochimica et Biophysica Acta, 587 (1979) 324--332
© Elsevier/North-Holland Biomedical Press
BBA 29068 A CYCLIC GMP-DEPENDENT HISTONE KINASE BOUND TO LIVER NUCLEOLI
ANNIKKA LINNALA-KANKKUNEN and PEKKA H. MAENP~,A Department of Biochemistry, University of Kuopio, P.O. Box 138, SF-70101 Kuopio 10 (Finland)
(Received February 26th, 1979) Key words: Cyclic GMP; Histone kinase; (Liver nucleolus)
Summary A m e t h o d of steady-state electrophoresis in polyacrylamide gels was used to analyze the presence of cyclic nucleotide binding components in cell extracts. Multiple cyclic AMP and cyclic GMP binding c o m p o n e n t s were detected in soluble cytoplasmic and nuclear extracts derived from avian liver, but only a single cyclic GMP binding protein was found in the 0.3 M NaC1 extract of liver nucleoli. In the presence of cyclic GMP, this protein phosphorylated efficiently a calf t h y m u s histone mixture and an endogenous nucleolar protein, which migrated identically with histone H4 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the cyclic GMP-binding protein was 4.8. Addition of cyclic GMP did not influence the activity of the endogenous nucleolar R N A polymerase. Introduction Cyclic nucleotides have been implicated as intracellular mediators during processes associated with growth in tissues of higher animals and cells in culture. The intracellular distribution of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic G M P ) b i n d i n g proteins in cellular extracts has been examined with techniques of ion~exchange chromatography and i m m u n o c y t o c h e m i s t r y [1,2]. We have employed a new technique of steady-state electrophoresis in polyacrylamide gels to separate and identify cyclic nucleotide binding proteins in various cellular subfractions. The m e t h o d was initially .described in studies of steroid binding proteins [3], b u t with some modifications it can also be used in analysis of cyclic nucleotide binding components in cellular extracts. The method gives good separation and a quantitative estimate of the individual binding c o m p o n e n t s in the sample,
325 provided that all components reach steady-state binding conditions with the free radioactive ligand in the gel [3]. This communication will describe the method and a partial characterization and properties of a cyclic GMP
