Speeds of Actin Translocation in Vitro by Myosins Extracted from Single Rat Muscle Fibres of Different Types

Abstract

As skeletal muscle fibres mostly express a single myosin isoform, they are a potential source of pure myosin isoforms. A technique is described that allows extraction and identification of pure myosin isoforms from single fibres, and testing of such myosins in an in vitro motility assay (IVMA). The results show that the extraction procedure does not alter myosin function and support the view that single fibres are reliable sources of purified myosin isoforms for IVMA.

Functional heterogeneity of mammalian single muscle fibres: do myosin isoforms tell the whole story?

“…Efficiency changes with velocity reaching a maximum at a velocity similar to the optimal velocity for power. Velocity is in fibre lengths (L) per second in all panels locity of actin filaments were qualitatively comparable with differences observed in the V o of fibres that contained the same myosin isoforms: a linear relationship was found between V o and the sliding velocity of actin filaments propelled by myosin in in vitro motility assays (V ivma ) of the same myosin [15]. These findings confirm that myosin isoforms are the main determinant of the velocity of actin sliding both when myosin works within the sarcomere structure and when it works isolated in a reconstituted contractile system in which only actin and myosin are present.…”

“…Very recently a new and simple technique was independently developed by two research groups and this has enabled the routine comparison of skeletal myosin isoforms in IVMA. The approach consists of extracting enough myosin to use in IVMA from single muscle fibres, which generally contain only one myosin isoform [15,42]. As in single fibre studies, the myosin isoform can be identified by electrophoresis.…”

“…As in single fibre studies, the myosin isoform can be identified by electrophoresis. Using myosin extracted from single fibres, the actin sliding velocity of myosin isoforms from rat [15] and human [14] muscles can be compared. In rat myosin isoform 1 propelled actin filaments at lower speed than myosin 2B.…”

Resistance training of long duration modulates force and unloaded shortening velocity of single muscle fibres of young women

“…The assay protocol was derived with minor modifications from a procedure previously described in detail ( Laemmli, 1970;Canepari et al, 1999). Briefly, small muscle bundle was weighed, cut and put in the extraction buffer 2X (mM: KCl, 800; KH 2 PO 4 , 200; K 2 HPO 4 , 100; Na 4 P 2 O 7 , 20; MgCl 2 , 8; NaN 3 , 1; EDTA, 50; NaATP, 4; dithiothreitiol (DTT), 4; phenylmethylsulphonic fluoride, 1 mg l À1 ; pH 6.5).…”

What Limits the Velocity of Fast-skeletal Muscle Contraction in Mammals?

“…32 Muscle fibres containing the same myosin isoform were pooled and myosin was extracted as described. 33 Myosin was collected after centrifugation and HMM was obtained by a proteolytic digestion of myosin as described 34 with modification applied by Canepari et al 35 To obtain HMM, myosin was dissolved in a solution containing 10 mM Mops (pH 7.2), 500 mM KCl, 2 mM MgCl 2 and 5 mM DTT so as to reach a protein concentration of 12 mg/ml. a-Chymostrypsin was added to a concentration of 12 ng/ml.…”