| Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6. | |
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MedLine Citation:
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PMID: 6417656 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo. |
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Authors:
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C J Le Peuch; R Ballester; O M Rosen |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 80 ISSN: 0027-8424 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 1983 Nov |
Date Detail:
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Created Date: 1984-01-07 Completed Date: 1984-01-07 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 6858-62 Citation Subset: IM |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Brain / enzymology* Calcium / pharmacology Chromatography, Gel Egtazic Acid / pharmacology Kinetics Phosphatidylserines / pharmacology Phosphorylation Protein Kinases / isolation & purification, metabolism* Rats Ribosomal Protein S6 Ribosomal Proteins / metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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5R01 AM09038/AM/NIADDK NIH HHS; AM20541/AM/NIADDK NIH HHS; IF05 TW03236-01/TW/FIC NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Phosphatidylserines; 0/Ribosomal Protein S6; 0/Ribosomal Proteins; 67-42-5/Egtazic Acid; 7440-70-2/Calcium; EC 2.7.-/Protein Kinases |
| Comments/Corrections | |
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