Multiple forms of thaumatin : structure and biosynthesis
The aim of the investigation was to determine the structural and biosynthetic relationships between the various forms of the sweet protein, thaumatin, which are found in the tropical plant, Thaumatococcus danielli. Three thaumatins, Tu, TI and TII were isolated from the aril tissue of Thaumatococcus fruits in, apparently, homogenous forms and the structural relationships between them were Investigated using various protein analytical techniques. Antibodies to theumatin II were raised and used to measure the immunological relatedness of the three proteins by quantitative precipitin and micro-complement fixation methods. The evidence suggests that the thaumatins have almost identical amino acid sequences and conformations. The observed differences in isoelectric points are consistant with the view that the thaumatins represent forms of the same protein which differ only in their relative amide contents. Studies on the content of Thaumatococcus fruits at different stages of maturation showed that the amount of thaumatin increased during development and that in the ripe fruit up to 60% of the protein in the aril tissue was thaumatin. Interestingly fruits from the Ashanti region of Ghana have roughly similar TI and TII contents together with much lower amounts of TO whilst Kadjebe region material does not contain any TII but has correspondingly larger amounts of TI and TO. The hypothesis that the thaumatin forms arise by interconversion of a common precursor was tested. No interconversion was observed under conditions of high salt concentration or high pH or in the presence of aril protein extracts. Polysomes and RNA preparations isolated from aril tissue directed protein synthesis in wheat germ and nuclease-treated reticulocyte lysate systems. SDS P.A.G.E. analysis of the radiolabelled product precipitated by anti-TII showed that only a small proportion (0.05%) of the total protein synthesised was thaumatin. The proportion of thaumatin synthesised was similar inplus the case of poly(A) RNA preparations from ripe and unripe Ashanti and ripe Kadjebe region fruits. Poly(A) minus RNA preparations directed synthesis of a similar proportion of thaumatin to total protein as poly(A) plus RNA. Various methods were used to determine whether the immunoprecipitable product was a single or a mixture of thaumatins but this problem remained unresolved.