Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 May;85(10):3314–3318. doi: 10.1073/pnas.85.10.3314

Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

K Maruyama 1, D H MacLennan 1
PMCID: PMC280199  PMID: 2966962

Abstract

Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis.

Full text

PDF
3314

Images in this article

3315Image
on p.3315
3315Image
on p.3315
3316Image
on p.3316
3317Image
on p.3317

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen G., Green N. M. A 31-residue tryptic peptide from the active site of the [Ca++]-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum. FEBS Lett. 1976 Mar 15;63(1):188–192. doi: 10.1016/0014-5793(76)80223-2. [DOI] [PubMed] [Google Scholar]
  2. Allen G., Trinnaman B. J., Green N. M. The primary structure of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum. Peptides derived from digestion with cyanogen bromide, and the sequences of three long extramembranous segments. Biochem J. 1980 Jun 1;187(3):591–616. doi: 10.1042/bj1870591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Brandl C. J., Green N. M., Korczak B., MacLennan D. H. Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986 Feb 28;44(4):597–607. doi: 10.1016/0092-8674(86)90269-2. [DOI] [PubMed] [Google Scholar]
  6. Brandl C. J., deLeon S., Martin D. R., MacLennan D. H. Adult forms of the Ca2+ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. J Biol Chem. 1987 Mar 15;262(8):3768–3774. [PubMed] [Google Scholar]
  7. Carrasco N., Antes L. M., Poonian M. S., Kaback H. R. lac permease of Escherichia coli: histidine-322 and glutamic acid-325 may be components of a charge-relay system. Biochemistry. 1986 Aug 12;25(16):4486–4488. doi: 10.1021/bi00364a004. [DOI] [PubMed] [Google Scholar]
  8. Ebashi S., Endo M., Otsuki I. Control of muscle contraction. Q Rev Biophys. 1969 Nov;2(4):351–384. doi: 10.1017/s0033583500001190. [DOI] [PubMed] [Google Scholar]
  9. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  10. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  11. Hackett N. R., Stern L. J., Chao B. H., Kronis K. A., Khorana H. G. Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F. J Biol Chem. 1987 Jul 5;262(19):9277–9284. [PubMed] [Google Scholar]
  12. Inesi G. Mechanism of calcium transport. Annu Rev Physiol. 1985;47:573–601. doi: 10.1146/annurev.ph.47.030185.003041. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. MacLennan D. H., Brandl C. J., Korczak B., Green N. M. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 1985 Aug 22;316(6030):696–700. doi: 10.1038/316696a0. [DOI] [PubMed] [Google Scholar]
  16. MacLennan D. H., de Leon S. Biosynthesis of sarcoplasmic reticulum proteins. Methods Enzymol. 1983;96:570–579. doi: 10.1016/s0076-6879(83)96049-4. [DOI] [PubMed] [Google Scholar]
  17. Mitchinson C., Wilderspin A. F., Trinnaman B. J., Green N. M. Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+ -dependent adenosine triphosphatase of sarcoplasmic reticulum. FEBS Lett. 1982 Sep 6;146(1):87–92. doi: 10.1016/0014-5793(82)80710-2. [DOI] [PubMed] [Google Scholar]
  18. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pick U., Bassilian S. Modification of the ATP binding site of the Ca2+ -ATPase from sarcoplasmic reticulum by fluorescein isothiocyanate. FEBS Lett. 1981 Jan 12;123(1):127–130. doi: 10.1016/0014-5793(81)80035-x. [DOI] [PubMed] [Google Scholar]
  20. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sarkadi B., Enyedi A., Földes-Papp Z., Gárdos G. Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. J Biol Chem. 1986 Jul 15;261(20):9552–9557. [PubMed] [Google Scholar]
  23. Sompayrac L. M., Danna K. J. Efficient infection of monkey cells with DNA of simian virus 40. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7575–7578. doi: 10.1073/pnas.78.12.7575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wong G. G., Witek J. S., Temple P. A., Wilkens K. M., Leary A. C., Luxenberg D. P., Jones S. S., Brown E. L., Kay R. M., Orr E. C. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science. 1985 May 17;228(4701):810–815. doi: 10.1126/science.3923623. [DOI] [PubMed] [Google Scholar]
  26. Yamada S., Ikemoto N. Reaction mechanism of calcium-ATPase of sarcoplasmic reticulum. Substrates for phosphorylation reaction and back reaction, and further resolution of phosphorylated intermediates. J Biol Chem. 1980 Apr 10;255(7):3108–3119. [PubMed] [Google Scholar]
  27. Zubrzycka-Gaarn E., MacDonald G., Phillips L., Jorgensen A. O., MacLennan D. H. Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site. J Bioenerg Biomembr. 1984 Dec;16(5-6):441–464. doi: 10.1007/BF00743238. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES