Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Said A. Goueli, Alan T. Davis, Khalil Ahmed

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11 Scopus citations

Abstract

1. 1. Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. 2. 2. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nucleic, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. 3. 3. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. 4. 4. Both enzymes were active towards casein, phosvitin, dephosphosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. 5. 5. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. 6. 6. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kas 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.

Original languageEnglish (US)
Pages (from-to)861-873
Number of pages13
JournalInternational Journal of Biochemistry
Volume18
Issue number10
DOIs
StatePublished - 1986

Bibliographical note

Funding Information:
Acknowledgements--This work was supported in part by Research Grant CA-15062 from National Cancer Institute, D.H.H.S., P.H.S., and from the VA Medical Research Fund. Mr Gregory Buckley provided skilled technical assistance. Ms Camille L. Quaglio helped in the preparation of the manuscript.

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