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Functional Characterisation of Dictyostelium Myosin II with Conserved Tryptophanyl Residue 501 Mutated to Tyrosine

  • R. Batra and D. J. Manstein
Published/Copyright: June 1, 2005
Biological Chemistry
From the journal Volume 380 Issue 7-8

Abstract

We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.

:
Published Online: 2005-06-01
Published in Print: 1999-07-01

Copyright © 1999 by Walter de Gruyter GmbH & Co. KG

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https://doi.org/10.1515/BC.1999.126

Articles in the same Issue

  1. Paul Nurse Felix Hoppe-Seyler Lecturer 1999
  2. Cyclin Dependent Kinases and Regulation of the Fission Yeast Cell Cycle
  3. Paper of the Year 1998
  4. Autonomous Regulation in Mammalian Mitochondrial DNA Transcription
  5. Prospects for the Precise Engineering of Plant Genomes by Homologous Recombination
  6. The Glycosphingolipidoses from Disease to Basic Principles of Metabolism
  7. The Dual Role of Lipopolysaccharide as Effector and Target Molecule
  8. A Unified Mechanism of Enzymatic Synthesis of Two Calcium Messengers: Cyclic ADP-Ribose and NAADP
  9. The Tranquilizing Injection of Yersinia Proteins: A Pathogens Strategy to Resist Host Defense
  10. IL-6 Type Cytokine Receptor Complexes: Hexamer, Tetramer or Both?
  11. Genetically Engineered and Synthetic Allergen Derivatives: Candidates for Vaccination against Type I Allergy
  12. Molecular Farming of Recombinant Antibodies in Plants
  13. Chimeric Restriction Enzymes: What Is Next?
  14. Viroids with Hammerhead Ribozymes: Some Unique Structural and Functional Aspects with Respect to Other Members of the Group
  15. Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi
  16. Role of Mitochondria in Parkinson Disease
  17. Mitochondria Harbouring Mutant mtDNA a Cuckoo in the Nest?
  18. Mutant p53: Gain-of-Function Oncoproteins and Wild-Type p53 Inactivators
  19. The Role of Chemokines in Cutaneous Allergic Inflammation
  20. Mutations of Calcium Channel beta Subunit Genes in Mice
  21. Agonist-Stimulated Pathways of Calcium Signaling in Pancreatic Acinar Cells
  22. Some of the Early Events Underlying Th2. Cell Maturation and Susceptibility to Leishmania major Infection in BALB/c Mice
  23. Universal and Unique Features of Kinesin Motors: Insights from a Comparison of Fungal and Animal Conventional Kinesins
  24. Elementary Steps in Protein Folding
  25. Molecular Reaction Mechanisms of Proteins Monitored by Time-Resolved FTIR-Spectroscopy
  26. Sugars as Signal Molecules in Plant Seed Development
  27. Diphosphoinositol Polyphosphates: The Final Frontier for Inositide Research?
  28. A Role of Poly (ADP-Ribose) Polymerase in NF- B Transcriptional Activation
  29. Processing of Artificial Peptide-DNA-Conjugates by the Mitochondrial Intermediate Peptidase (MIP)
  30. The Two SH2-Domain-Containing Inositol 5-Phosphatases SHIP1 and SHIP2 Are Coexpressed in Human T Lymphocytes
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  35. A Nonspecific, Single-Stranded Nuclease Activity with Characteristics of a Topoisomerase Found in a Major Grass Pollen Allergen: Possible Biological Significance
  36. Functional Characterisation of Dictyostelium Myosin II with Conserved Tryptophanyl Residue 501 Mutated to Tyrosine
  37. Mitochondrial Nitric Oxide Synthase Regulates Mitochondrial Matrix pH
  38. Directed Evolution of an Esterase from Pseudomonas fluorescens. Random Mutagenesis by Error-Prone PCR or a Mutator Strain and Identification of Mutants Showing Enhanced Enantioselectivity by a Resorufin-Based Fluorescence Assay
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