Interaction of myosin subfragment 1 with forms of monomeric actin.
The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequestering proteins was tested by a number of different techniques such as affinity absorption, chemical cross-linking, fluorescence titration, and competition procedures. For affinity absorption, actin was attached to agarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retained by this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonuclease I (DNase I) or thymosin beta4 demonstrated S1 binding to these actin complexes. A K(D) of 5 x 10(-8) M for S1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicate binding of S1 to actin in complex with gelsolin segment 1 (G1) or vitamin D-binding protein (DBP). However, fluorescence competition experiments and analysis of tryptic cleavage patterns of S1 indicated its interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complex was directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitive to ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable to significantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant of Dictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within an insertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase I complex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higher affinity than wild-type S1 and was less sensitive to mono- and divalent cations.
Ballweber
Edda
E
Department of Anatomy and Embryology, Ruhr-University, Bochum, Germany.
Kiessling
Peter
P
Manstein
Dietmar
D
Mannherz
Hans Georg
HG
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D004023:Dictyostelium / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D009994:Osmolar Concentration; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014357:Trypsin / Q000737:chemistry; D014809:Vitamin D-Binding Protein / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000199:Actins; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D015879:Myosin Subfragments; D015800:Protozoan Proteins; D011721:Pyrenes; D014809:Vitamin D-Binding Protein; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D003850:Deoxyribonuclease I; D014357:Trypsin; D008274:Magnesium; D005022:Ethyldimethylaminopropyl Carbodiimide