Some binding properties of Omp T digested muscle tropomyosin.
Cleavage of vertebrate muscle tropomyosin by bacterial Omp T produces an amino-terminally truncated product (residues 7-284). The proteolysed protein, which is resolved from the parent by electrophoresis in the presence of sodium dodecylsulphate, can be generated from a variety of striated and smooth muscle tropomyosins, including ones from mammal, bird and fish. Edman-based sequencing and mass analysis confirm that the main site of chain hydrolysis is the peptide bond between Lys 6 and Lys 7. Loss of the hexapeptide, together with the blocking group, from tropomyosin weakens its affinity for troponin. Compared to wild type, the shortened forms of rabbit skeletal tropomyosin and Atlantic salmon fast skeletal tropomyosin, as well as the unacetylated (full-length) version of the latter, all display reduced affinity for both troponin and the amino-terminal fragment of troponin-T (residues 1-158), as judged by affinity chromatography. This is consistent with the view that the amino terminal region is required for full interaction with troponin-T. Truncated tropomyosin fails to bind to F-actin at micromolar concentration, as expected. Interestingly, binding is restored by troponin in the presence of either added Ca(2+) or EGTA. Digestion of muscle tropomyosin by Omp T, which can be carried out on quantitative amounts of protein, is concluded to yield a product that has useful biochemical applications.
Goonasekara
Charitha L
CL
Department of Biochemistry, Memorial University, 2 Livyers Loop, St. John's, NL, Canada A1B3X9.
Gallivan
Lisa J
LJ
Jackman
Donna M
DM
Heeley
David H
DH
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D008239:Lysine / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D018272:Porins / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D012487:Salmonidae; D012754:Sharks; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*; D014336:Troponin / Q000737:chemistry