The relationship between thaumatin, a sweet protein and thaumatopain, a cysteine protease, from the arlis of Thaumatococcus daniellii.

Thaumatin, an intensely sweet protein, is the major proteinaciouscompnentinthe arils ofThaumatococcusdaniellii [l].Thaumatinisamemberofafamilyofatleastfiveisoforms, separable on cation exchange chromatography and all with a molecular weight of approx. 22 kDa and possessing eight disulphide bridges. Thaumatin I and II are derived from distinct genes, the other subforms may result from post-translational modifications or may also represent separate gene products. It had previously been suggested that thaumatin possessed cysteine protease activity [2]. but we have shown that this activity is due to thaumatopain. a papain-like protease [3]. Thaumatopain is the second most abundant protein in aril extracts, and together, thaumatin and thaumatopain constitute over 99% of the protein in this preparation. We have examined more closely the relationship between these two proteins. Application of aqueous aril extract to a Mono S cation exchange column effectively separates the thaumatins. and although the majority of proteolytic activity (due to thaumatopain) is eluted as a very cationic peak, significant activity co-elutes with the thaumatins (Figure 1). This activity is thiol-dependent. There are several explanations for this distribution: Thaumatin i s capable of hydrolysing ZFRNMec: The proteolytic activity in thaumatin preparations can be inhibited by levels of E64 that are much lower than the amount of thaumatin in this assay. [4]. Thaumatopain is associated with thawnatin in a complex that is stable to M o d chromatography: When material from fraction 16 is reapplied to Mono S the activity remains associated with thaumatin. When aril extract is incubated with 500kM DTT for 15 mins little shift in proteolytic activity is seen. Comparison ofreducing andnon-reducing SDS-PAGEgaveno evidence for a covalent complex between the two proteins. Initialstudies indicate thattheproteolytic activity andthaumatin are separable on hydrophobic interaction chromatography. There are thaumatopain sub-form: There is no correlation between the proteolytic activity seen in each fraction and the amount of the 30kDa protein (putatively thaumatopain). Thaumahisneither anactivatornoraninhibitorofthaumatopain and it is likely that these thaumatopain variants have very different specific activities. It appears that there are several thaumatopain variants in the arils. At present it is not known whether these variants are the result of post-translational modifications or represent other gene products. A role for thaumatopain might be to digest thaumatin to release amino acids for the germinating embryo. However, initialexperiments on intact arils, aqueous aril extract and isolated thaumatin and thaumatopain. have shown that thaumatin is refractory to thaumatopain. Further studies using different chromatographic and immunochcmical techniques are required to resolve the relationship between thaumatin and thaumatopain. 20