Separation of SH-modified myosin subfragment-1 (A1) isozyme into two distinct equimolar fractions by an affinity chromatography.
Our previous kinetic studies indicated that SH-modified myosin subfragment-1 A1 isozyme (S1(A1] contains at least two different types of active sites (Emoto, Y., Kawamura, T., & Tawada, K. (1985) J. Biochem. 98, 735-745). In those studies we have modified highly reactive SH-groups in S1(A1) with thimerosal. In this work, we separated the modified S1(A1) into two equimolar fractions by affinity chromatography with agarose-ADP. For the separation, Mg2+ in the elution buffer was indispensable. Although the two fractions appeared to have the same number of modified SH-groups per mol of S1, they had different enzymic and fluorescent properties. SH-modification with an excess of thimerosal for a much longer duration did not change any of the results: not the chromatographic profile, the properties of the two fractions, nor the number of modified SH-groups. Hence the two different populations were not generated by incomplete modification. After reduction with dithiothreitol, however, the differences between the two fractions disappeared. When we separately re-modified the reduced fractions and re-chromatographed them, in each case we again obtained two fractions, which had the same properties as the two fractions obtained from the original modification with thimerosal. These results demonstrate that the active site heterogeneity in SH-modified S1(A1) had no intrinsic origin in the unmodified S1: it was introduced by the SH-modification, but by an unknown mechanism(s) other than incomplete modification.
Emoto
Y
Y
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Kawamura
T
T
Tawada
K
K
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D005035:Ethylmercury Compounds / Q000494:pharmacology*; D007700:Kinetics; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013849:Thimerosal / Q000494:pharmacology*
D005035:Ethylmercury Compounds; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013849:Thimerosal; D000251:Adenosine Triphosphatases; D009218:Myosins; D004229:Dithiothreitol