21
views
0
recommends
+1 Recommend
0 collections
    3
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1.

      Read this article at

      ScienceOpenPubMed
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Related collections

          Author and article information

          Journal
          J. Biol. Chem.
          The Journal of biological chemistry
          0021-9258
          0021-9258
          Jul 5 1988
          : 263
          : 19
          Affiliations
          [1 ] Cold Spring Harbor Laboratory, New York 11724.
          Article
          3288629
          6fb79cd9-736a-45c9-86c0-9428ef61859f
          History

          Comments

          Comment on this article

          Related Documents Log