Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been identified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 · 10 3 )1 · 10 4 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.
Washed bottom-fraction particles (WBPs), that is, intact membrane-bound vesicles, prepared from the bottom fraction of centrifuged fresh latex, were shown to be active in in vitro rubber biosynthesis (RB) assays. The RB activity catalyzed by WBP enzymes was confirmed by enzyme parameter criteria. A washed bottom-fraction particle membrane (WBM), prepared from WBPs, was even more active in in vitro RB activity. Mg 2ϩ was a cofactor required for RB enzymes, and the cation chelators EDTA and EGTA inhibited the RB process. The temperature had a strong effect on RB stimulation during the heat pretreatment of the WBM before RB assays. A detergent also proved to a strong RB stimulator. The anionic detergent SDS, above the critical micelle concentration, was a strong stimulator of WBM activity. This was not observed with the nonionic detergents Triton X-100 and Tween 20. Various temperature ranges used for WBM preincubation showed a sharp rise in RB activity above 70°C. The temperature required for a sharp rise in RB was lowered substantially in the presence of SDS. It was down to 40°C with the combined preincubation but remained constant with an even higher RB activity. Greater rubber formation at a lower temperature was observed in the presence of SDS. A synergistic effect for RB stimulation appeared during the heat pretreatment of WBM in the presence of SDS.
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