Mercer, Robert C. C. (2010) Slow skeletal muscle actin: confirmation and further insights. Masters thesis, Memorial University of Newfoundland.
- 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.
1) The 1° structures of three sarcomeric actin isoforms were inferred from cDNA sequences: Atlantic herring (Clupea harengus) fast skeletal muscle actin, Acc# GQ455648; Atlantic herring slow skeletal muscle actin, Acc# EF495203; and Atlantic Mackerel (Scomber scombrus) fast skeletal muscle actin, Acc# EF607093. The two herring skeletal muscle actin cDNAs possess different non-coding regions and are concluded to arise from different genes. -- 2) Sequence comparisons demonstrate that actin from a given type of skeletal muscle has been conserved across teleost evolution. Herring slow muscle actin and salmonid slow muscle actin (Salmo trutta; Acc# AF267426 [Mudalige et.al. 2007]) share a single substitution, Gin 354 Ala (herr:sal), while, in addition to this substitution the fast skeletal muscle actins from Atlantic herring and atlantic salmon (Salmo salar; Acc# AF304406 [Mudalige et.al. 2007]) contain Ser 160 Thr for a total of two substitutions. -- 3) There are thirteen inferred substitutions between fast and slow skeletal muscle actins isolated from Atlantic herring, six that are non-conservative in nature. This is a record for two vertebrate sarcomeric actins. The replacements (non-conservative underlined) are: residues: 2, 3, 103, 155, 160, 165, 278, 281, 310, 329, 358, 360 and 363. Most of the heterogeneity occurs after residue 100. Also of note is the exchange of Gin (fast actin) for Asp (slow actin) at position 360, which endows the slow type isoform with a greater negative charge in the neutral pH range. Comparison with the actin found in the skeletal muscles of the rabbit demonstrates 11 replacements (five non-conservative) with herring slow skeletal muscle actin and four (one non-conservative) when compared to herring fast skeletal muscle. -- 4) Two skeletal muscle actins of non-identical net-charge were detected by alkaline urea PAGE in the slow skeletal muscle of the following species - mackerel and smelt - but not in other teleosts (halibut and tiliapia), primitive fish (hagfish and lamprey), birds or amphibians. -- 5) Herring slow skeletal muscle G-actin (CaATP) is less resistant to thermal, and chemical, -induced denaturation (as monitored by electronic circular dichroism) than its fast muscle counterpart. Thermal melting temperatures (Tms) and [urea] unfolding midpoints, respectively, are: 50°C and 55°C; 3.5 M and 4.0 M. A rate of unfolding for slow skeletal muscle actin was determined, in the presence of 0.1 mM EDTA, to be 6.5x10⁴ sec⁻¹. The fast skeletal muscle isolated from Atlantic mackerel has a Tm of 55°C and a midpoint of unfolding in the presence of urea of 4.0 M. -- 6) Fast skeletal muscle G-actin from Atlantic mackerel contains a non-conservative substitution in common with slow skeletal muscle actin: Ser 155 Ala (herr fast: mack fast/herr slow). This finding rules out the identity of this residue as a contributor to the observed instability. -- 7) Herring slow skeletal muscle G-actin (CaATP) is less resistant to cleavage by subtilisin and thrombin than the fast muscle counterpart. Subtilisin cleavage between residues 48 and 49 was confirmed by Edman sequencing of Western blotted peptides. Of the two actin isoforms, only slow skeletal muscle actin could be cleaved by thrombin. The fast skeletal muscle actin isolated from mackerel, in spite of the present substitutions, behaves as the other fast skeletal muscle actins when subjected to subtilisin and thrombin exposure. -- 8) Fluorescence spectra of all G-actins (Ca - 2'-deoxy 3' O- (N'- Methylanthraniloyl) ATP) studied is characterized by emission maxima at 430 nm; demonstrating a virtual identity of the nucleotide binding pockets of the three isoforms. Emission intensity was reduced in the case of the slow muscle actin, consistent with it having a weaker affinity for the nucleotide.
|Item Type:||Thesis (Masters)|
|Additional Information:||Includes bibliographical references (leaves 121-151)|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Actin; Genetic regulation; Osteichthyes--Physiology; Striated muscle|
Actions (login required)