Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1988 Apr 15;251(2):527–540. doi: 10.1042/bj2510527

Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.

A P Jackson 1, C R Bagshaw 1
PMCID: PMC1149033  PMID: 2969726

Abstract

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.

Full text

PDF
537

Selected References

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

  1. Ashiba G., Asada T., Watanabe S. Calcium regulation in clam foot muscle. Calcium sensitivity of clam foot myosin. J Biochem. 1980 Sep;88(3):837–846. doi: 10.1093/oxfordjournals.jbchem.a133038. [DOI] [PubMed] [Google Scholar]
  2. Bagshaw C. R., Eccleston J. F., Eckstein F., Goody R. S., Gutfreund H., Trentham D. R. The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation. Biochem J. 1974 Aug;141(2):351–364. doi: 10.1042/bj1410351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bagshaw C. R., Trentham D. R. The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. Biochem J. 1974 Aug;141(2):331–349. doi: 10.1042/bj1410331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bagshaw C. R., Trentham D. R. The reversibility of adenosine triphosphate cleavage by myosin. Biochem J. 1973 Jun;133(2):323–328. doi: 10.1042/bj1330323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chalovich J. M., Chantler P. D., Szent-Gyorgyi A. G., Eisenberg E. Regulation of molluscan actomyosin ATPase activity. J Biol Chem. 1984 Feb 25;259(4):2617–2621. [PMC free article] [PubMed] [Google Scholar]
  6. Collins J. H., Jakes R., Kendrick-Jones J., Leszyk J., Barouch W., Theibert J. L., Spiegel J., Szent-Györgyi A. G. Amino acid sequence of myosin essential light chain from the scallop Aquipecten irradians. Biochemistry. 1986 Nov 18;25(23):7651–7656. doi: 10.1021/bi00371a056. [DOI] [PubMed] [Google Scholar]
  7. Cross R. A., Cross K. E., Sobieszek A. ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi. EMBO J. 1986 Oct;5(10):2637–2641. doi: 10.1002/j.1460-2075.1986.tb04545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eccleston J. F., Trentham D. R. The interaction of chromophoric nucleotides with subfragment 1 of myosin. Biochem J. 1977 Apr 1;163(1):15–29. doi: 10.1042/bj1630015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goody R. S., Hofmann W. Stereochemical aspects of the interaction of myosin and actomyosin with nucleotides. J Muscle Res Cell Motil. 1980 Mar;1(1):101–115. doi: 10.1007/BF00711928. [DOI] [PubMed] [Google Scholar]
  10. Hardwicke P. M., Wallimann T., Szent-Györgyi A. G. Light-chain movement and regulation in scallop myosin. Nature. 1983 Feb 10;301(5900):478–482. doi: 10.1038/301478a0. [DOI] [PubMed] [Google Scholar]
  11. Itaya K., Ui M. A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta. 1966 Sep;14(3):361–366. doi: 10.1016/0009-8981(66)90114-8. [DOI] [PubMed] [Google Scholar]
  12. Jackson A. P., Bagshaw C. R. Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin. Biochem J. 1988 Apr 15;251(2):515–526. doi: 10.1042/bj2510515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kendrick-Jones J., Szentkiralyi E. M., Szent-Györgyi A. G. Regulatory light chains in myosins. J Mol Biol. 1976 Jul 15;104(4):747–775. doi: 10.1016/0022-2836(76)90180-7. [DOI] [PubMed] [Google Scholar]
  14. Munson K. B., Smerdon M. J., Yount R. G. Cross-linking of myosin subfragment 1 and heavy meromyosin by use of vanadate and a bis(adenosine 5'-triphosphate) analogue. Biochemistry. 1986 Nov 18;25(23):7640–7650. doi: 10.1021/bi00371a055. [DOI] [PubMed] [Google Scholar]
  15. Okamoto Y., Sekine T., Grammer J., Yount R. G. The essential light chains constitute part of the active site of smooth muscle myosin. Nature. 1986 Nov 6;324(6092):78–80. doi: 10.1038/324078a0. [DOI] [PubMed] [Google Scholar]
  16. Prince H. P., Trayer H. R., Henry G. D., Trayer I. P., Dalgarno D. C., Levine B. A., Cary P. D., Turner C. Proton nuclear-magnetic-resonance spectroscopy of myosin subfragment 1 isoenzymes. Eur J Biochem. 1981 Dec;121(1):213–219. doi: 10.1111/j.1432-1033.1981.tb06451.x. [DOI] [PubMed] [Google Scholar]
  17. Rodbell M., Birnbaumer L., Pohl S. L., Krans H. M. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanylnucleotides in glucagon action. J Biol Chem. 1971 Mar 25;246(6):1877–1882. [PubMed] [Google Scholar]
  18. Rossomando E. F., Jahngen J., Eccleston J. F. A fluorometric-high-performance liquid chromatographic assay procedure for several enzymatic activities using formycin analogs of adenosine 5'-mono, 5'-tri, and cyclic 3',5'-monophosphate as substrates. Anal Biochem. 1981 Sep 1;116(1):80–88. doi: 10.1016/0003-2697(81)90325-0. [DOI] [PubMed] [Google Scholar]
  19. Schaub M. C., Watterson J. G. Symmetry and asymmetry in the contractile protein myosin. Biochimie. 1981 Apr;63(4):291–299. doi: 10.1016/s0300-9084(81)80117-4. [DOI] [PubMed] [Google Scholar]
  20. Shoham M., Steitz T. A. Crystallographic studies and model building of ATP at the active site of hexokinase. J Mol Biol. 1980 Jun 15;140(1):1–14. doi: 10.1016/0022-2836(80)90353-8. [DOI] [PubMed] [Google Scholar]
  21. Trentham D. R., Eccleston J. F., Bagshaw C. R. Kinetic analysis of ATPase mechanisms. Q Rev Biophys. 1976 May;9(2):217–281. doi: 10.1017/s0033583500002419. [DOI] [PubMed] [Google Scholar]
  22. Trybus K. M., Taylor E. W. Transient kinetics of adenosine 5'-diphosphate and adenosine 5'-(beta, gamma-imidotriphosphate) binding to subfragment 1 and actosubfragment 1. Biochemistry. 1982 Mar 16;21(6):1284–1294. doi: 10.1021/bi00535a028. [DOI] [PubMed] [Google Scholar]
  23. Vibert P., Craig R. Structural changes that occur in scallop myosin filaments upon activation. J Cell Biol. 1985 Sep;101(3):830–837. doi: 10.1083/jcb.101.3.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vibert P., Szentkiralyi E., Hardwicke P., Szent-Györgyi A. G., Cohen C. Structural models for the regulatory switch of Myosin. Biophys J. 1986 Jan;49(1):131–133. doi: 10.1016/S0006-3495(86)83622-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ward D. C., Cerami A., Reich E., Acs G., Altwerger L. Biochemical studies of the nucleoside analogue, formycin. J Biol Chem. 1969 Jun 25;244(12):3243–3250. [PubMed] [Google Scholar]
  26. Ward D. C., Reich E. Conformational properties of polyformycin: a polyribonucleotide with individual residues in the syn conformation. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1494–1501. doi: 10.1073/pnas.61.4.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ward D. C., Reich E., Stryer L. Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives. J Biol Chem. 1969 Mar 10;244(5):1228–1237. [PubMed] [Google Scholar]
  28. Wells C., Bagshaw C. R. Calcium regulation of molluscan myosin ATPase in the absence of actin. Nature. 1985 Feb 21;313(6004):696–697. doi: 10.1038/313696a0. [DOI] [PubMed] [Google Scholar]
  29. Wells C., Bagshaw C. R. Segmental flexibility and head-head interaction in scallop myosin. A study using saturation transfer electron paramagnetic resonance spectroscopy. J Mol Biol. 1983 Feb 15;164(1):137–157. doi: 10.1016/0022-2836(83)90090-6. [DOI] [PubMed] [Google Scholar]
  30. Wells C., Bagshaw C. R. The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins. J Muscle Res Cell Motil. 1984 Feb;5(1):97–112. doi: 10.1007/BF00713154. [DOI] [PubMed] [Google Scholar]
  31. Wells C., Warriner K. E., Bagshaw C. R. Fluorescence studies on the nucleotide- and Ca2+-binding domains of molluscan myosin. Biochem J. 1985 Oct 1;231(1):31–38. doi: 10.1042/bj2310031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. White D. C., Zimmerman R. W., Trentham D. R. The ATPase kinetics of insect fibrillar flight muscle myosin subfragment-1. J Muscle Res Cell Motil. 1986 Apr;7(2):179–192. doi: 10.1007/BF01753419. [DOI] [PubMed] [Google Scholar]
  33. Yount R. G., Ojala D., Babcock D. Interaction of P--N--P and P--C--P analogs of adenosine triphosphate with heavy meromyosin, myosin, and actomyosin. Biochemistry. 1971 Jun 22;10(13):2490–2496. doi: 10.1021/bi00789a010. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES