ABSTRACT
Immunochemical procedures were employed to analyse changes in contractile proteins during limb regeneration in the adult newt, Triturus. Methods for the isolation of myosin and actomyosin were modified for use in the newt. After these proteins were characterized biochemically they were shown to be antigenic in the rabbit.
The activity and specificity of anti-actomyosin and anti-myosin sera were determined by means of agar diffusion techniques. Purified actomyosin was found to be contaminated by myosin and a second component, a nucleoprotein. The diffusion coefficients increased in the order actomyosin, myosin, nucleoprotein. The injection of myosin into rabbits elicited the formation of antibodies which, when tested in agar diffusion, usually demonstrated a minimum of one band but occasionally, when contaminated by minor quantities of actomyosin, two bands were seen. Some modifications of existing procedures are described for the use of small reagent samples in Ouchterlony plates.
The development of muscle during limb regeneration was tested by the agar diffusion method. Actomyosin and myosin can first be detected in the regenerate at the stage of hand formation (‘palette’ stage). Thereafter there is an upsurge of synthesis of these proteins during the ‘early’ and ‘medium’ digital stages. The appearance of these contractile proteins probably coincided with the time of myofibril formation.
A rise in nucleoprotein preceded the appearance of muscle protein and later decreased to a concentration characteristic of adult tissues. All tissues which were tested immunologically contained the nucleoprotein component.
Infusions of rabbit serum and anti-muscle serum into the regenerate yielded a cytotoxic response which varied according to the serum used, the reaction to the immune serum being more severe. There was a temporary cessation of regeneration for a few days, the duration of which was related to the severity of the reaction. Observations of the histopathology of infused regenerates revealed sarcolysis attributable to the specific effects of the anti-muscle serum.
Autoradiograms of limbs infused with I131 gamma globulin demonstrated the course and disposition of labelled protein within the regenerate and stump. There was localization of material about muscle-cells, suggesting a possible specificity of the reaction. The epidermis appeared to be an active organ for the elimination of foreign material.