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Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32⁄grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of mono-meric actin with either ATP or ADP as a bound nucleotide. | ễFEBS Journal Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin Anastasia V. Pivovarova1 Sofia Yu. Khaitlina2 and Dmitrii I. Levitsky1 3 1 A. N. Bach Institute of Biochemistry Russian Academy of Sciences Moscow Russia 2 Institute of Cytology Russian Academy of Sciences St Petersburg Russia 3 A. N. Belozersky Institute of Physico-ChemicalBiology Moscow State University Moscow Russia Keywords actin differential scanning calorimetry DNase-I binding loop proteolytic cleavage thermal unfolding Correspondence D. I. Levitsky A. N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky Prospect 33 119071 Moscow Russia Fax 7 495 954 2732 Tel 7 495 952 1384 E-mail levitsky@inbi.ras.ru. Received 10 June 2010 revised 14 July 2010 accepted 16 July 2010 doi 10.1111 j.1742-4658.2010.07782.x Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNasel-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases subtilisin or ECP32 grimelysin ECP respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride however in all cases the thermal stability of the cleaved F-actins was much lower than that of intact F-actin and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure both in monomers and in the filament subunits and suggest that the thermal stability of actin depends at least partially on the conformation of the .
