Cation effects on the conformations of muscle and non-muscle alpha-actinins.
We examined the effects of changing KCl concentration on the secondary structures of alpha-actinins using circular dichroism (CD), 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mM KH2PO4, pH 7.5, increasing KCl from 0 to 120 mM led to decreases in alpha-helix conformation for brain, platelet and heart alpha-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mM KCl, 0.65 mM calcium produced small changes in the CD spectra of both brain and platelet alpha-actinin, but had no effect on heart alpha-actinin. bisANS fluorescence of all three alpha-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mM KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any alpha-actinin. Digestion of brain, platelet and heart alpha-actinins with alpha-chymotrypsin showed an increase of proteolytic susceptibility in 120 mM KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mM KCl. The results suggest that alpha-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different alpha-actinins.
Wenegieme
E F
EF
Department of Chemistry, Texas Christian University, Fort Worth 76129, USA.
Naren
A P
AP
Bobich
J A
JA
D000185:Actinin / Q000187:drug effects*; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000187:drug effects*; D002122:Calcium Chloride / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology*; D002942:Circular Dichroism; D006868:Hydrolysis; D015636:Magnesium Chloride / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000187:drug effects*; D011189:Potassium Chloride / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D001798:Blood Proteins; D002412:Cations; D009419:Nerve Tissue Proteins; D015636:Magnesium Chloride; D000185:Actinin; D011189:Potassium Chloride; D002122:Calcium Chloride