Zoller, M. J., Kuret, J., Cameron, S., Levin, L., Johnson, K. E. (July 1988) Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1. J Biol Chem, 263 (19). pp. 9142-8. ISSN 0021-9258
Abstract
In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Carrier Proteins/genetics/*isolation & purification/metabolism Chromatography, Affinity *Genes, Fungal *Genes, Structural *Intracellular Signaling Peptides and Proteins Kinetics Molecular Weight Protein Kinases/*isolation & purification Research Support, U.S. Gov't, P.H.S. Saccharomyces cerevisiae/*enzymology/genetics |
| Subjects: | biotechnology > chromatography organs, tissues, organelles, cell types and functions > organs types and functions > metabolism bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase > Protein kinase C |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Gail Sherman |
| Date: | 5 July 1988 |
| Date Deposited: | 14 Sep 2017 19:57 |
| Last Modified: | 14 Sep 2017 19:57 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/35234 |
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