Interaction of Myosin Subfragment 1 with Forms of Monomeric Actin
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- 作者：Edda Ballweber ; Peter Kiessling ; Dietmar Manstein ; and Hans Georg Mannherz
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：March 18, 2003
- 年：2003
- 卷：42
- 期：10
- 页码：3060 - 3069
- 全文大小：233K
- 年卷期：v.42,no.10(March 18, 2003)
- ISSN：1520-4995
文摘
The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequesteringproteins 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 toagarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retainedby this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonucleaseI (DNase I) or thymosin 
4 demonstrated S1 binding to these actin complexes. A KD of 5 × 10-8 M forS1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicatebinding 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 indicatedits interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complexwas directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitiveto ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable tosignificantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant ofDictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within aninsertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase Icomplex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higheraffinity than wild-type S1 and was less sensitive to mono- and divalent cations.
4 demonstrated S1 binding to these actin complexes. A KD of 5 × 10-8 M forS1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicatebinding 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 indicatedits interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complexwas directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitiveto ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable tosignificantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant ofDictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within aninsertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase Icomplex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higheraffinity than wild-type S1 and was less sensitive to mono- and divalent cations.