Functional analysis of rat acidic calponin.
Recombinant acidic calponin, a member of the calponin family, interacted with F-actin, but not with microtubules, desmin filaments, tropomyosin, calmodulin, S100 and phosphatidylserine (PS) vesicles with significant affinity. The bindings of acidic calponin to F-actin occurred in a concentration-dependent manner and were saturated at a molar ratio of about 1 acidic calponin to 1-2 actin molecules. The apparent Kd value of acidic calponin to F-actin was calculated to be 1.6 x 10(5) M(-1). Chemical cross-linking experiments indicated that a 1:1 molar covalent complex of acidic calponin and actin monomer was produced as in the case of basic calponinactin binding. No significant morphologic change of F-actin was observed by the addition of acidic calponin. Acidic calponin had little effect on actomyosin Mg2+-ATPase activity unlike basic calponin. Basic calponin partially competed with acidic calponin for binding to F-actin. Domain mapping with V8 protease revealed that acidic calponin binding site resided within the C-terminal 16 kDa fragment of actin, where the binding of basic calponin also occurs. However, both calponins showed reversal effects on fluorescence intensity of pyrene-labeled F-actin. Fragments of acidic calponin with 30 and 22 kDa, lacking the C-terminal acidic tail, were bound to F-actin. Interestingly, both the fragments became bound to PS vesicles, but not to other components. Circular dichroism studies showed that limited digestion of acidic calponin resulted in about 30% decrease of alpha-helix and beta contents. The present results suggest that acidic calponin is functionally distinct from basic calponin and expresses a novel characteristic after removal of the acidic tail region.
Fujii
Toshihiro
T
Department of Kansei Engineering, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan. fujiit1@giptc.shinshu-u.ac.jp
Yabe
Sachiko
S
Nakamura
Kouta
K
Koizumi
Youichi
Y
D000199:Actins / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006868:Hydrolysis; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000096985:Calponins
D000199:Actins; D017301:Ca(2+) Mg(2+)-ATPase; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D008840:Microfilament Proteins; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D000096985:Calponins