The isolation and characterization of troponin T from the slow and fast myotomal muscles of Atlantic salmon

Waddleton, Deena Maureen (1997) The isolation and characterization of troponin T from the slow and fast myotomal muscles of Atlantic salmon. Masters thesis, Memorial University of Newfoundland.

[English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (4MB)

Abstract

1. Subunits of troponin (Tn) were isolated and characterized from the fast and slow swimming muscles of Atlantic salmon (Salmo salar). Troponin C (TnC) from both slow and fast muscle was determined, by electrophoretic analysis to be present as single and distinct isoforms. Three isoforms of fast troponin T (TnT 1F, 2F and 3F) and two from slow muscle (TnT 1S and 2S) were detected. These proteins were determined to be TnT by the following criteria: copurification with whole Tn, SDS PAGE, amino acid composition, partial protein sequence data, immunoreaction with an anti-TnT antibody and affinity chromatography with immobilized tropomyosin (TM). The TnTs were specific to the corresponding muscle type. -- 2. All TnTs, with the exception of TnT 1F were N-terminally blocked. Partial protein sequence data was obtained for all of the isoforms. The various isoforms of TnT possess similar amino acid compositions with the exception of their proline contents. The higher molecular weight isoforms (TnT 1F and 1S) contain higher amounts of proline than the lower molecular weight forms (TnT 2F, 3F and 2S). All TnTs contain one tryptophan, with the exception of TnT 1S which contains two. None of the isoforms contain cysteine. TnT 2F and 3F contained phospho-serine while TnT 1F, 1S and 2S did not. -- 3. A full length TnT clone was isolated from a brown trout (Salmo trutta) slow muscle cDNA library and sequenced. The clone is believed to correspond to TnT 1S based on identity (94-112) with protein sequence data derived from a CNBr fragment of TnT 1S and similar amino acid composition data. The nucleotide sequence encodes 278 amino acids with a predicted molecular mass of 32362 and a negative charge at neutral pH. The N-terminal region is highly acidic (26 acidic residues occur in the first 55 amino acids). In addition, all of the 13 proline residues are located within the first 73 amino acids. The central part of the molecule contains a large number of charged residues, while the C-terminus is positively charged. -- 4. During exploratory sequencing of the slow cDNA library, a full length clone corresponding to actin was isolated and sequenced. Comparative sequence analyses revealed that this clone encodes a striated muscle class II α-actin. The nucleotide sequence encodes 377 residues with an acidic pI and a predicted molecular mass of 41852.

Actions (login required)

View Item