Involvement of Hevea Latex Organelle Membrane Proteins in the Rubber Biosynthesis Activity and Regulatory Function

Abstract

Centrifugation of fresh Hevea rubber latex yields three distinct fractions. The sediment bottom fraction (BF) content of membrane-bound organelles is ca. 20 vol.-% of latex. Prolonged storage or delayed use of fresh latex will result in disintegration and loss of the bottom fraction. This is due to the osmotically sensitive BF rupture and its membrane debris being tightly bound to the top rubber particles (RP) phase. The BF membrane was found to be highly active for rubber biosynthesis (RB), in contrast to previous reports that describe RB only occurring on the RP surface. It was clearly shown that washed BF membrane (WBM) was much more active than fresh RP for RB activity. WBM was highly activated by SDS for RB in a biphasic manner, but SDS strongly inhibited the RP. Probably WBM micelle formation resulted in a highly increased active surface area for RB. C55-PP (UPP) was a very active allylic for WBM in RB function, but inactive for RP. Serial acetone extraction of WBM proteins showed a distinct profile of the fractions with different RB activity. WBM isolated proteins suspended in 2% sodium dodecyl sulfate (SDS) with an RB activity equal to that of intact WBM was with the 20% acetone protein fraction. The 60 and 80% fractions were inactive. Combining the 20 with 80% fractions showed a complete inhibition of RB activity. Complete RB loss was also found when WBM was mixed with the 80% fraction, indicating that WBM has both an enzyme system and a factor for regulation of the RB activity in a well controlled metabolic function for the latex RB process.

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

“…For the isolation of native and functional rubber particles we followed the general procedure described for H. brasiliensis [27,43] with the following modifications. Latex was harvested from petioles or roots of 20 week old T. koksaghyz plants, if not stated otherwise, by dissecting the tissue with a razor blade and transferring the expelling latex into an equal volume of ice-cold rubber extraction buffer (100 mM Tris.Cl (pH 7.8), 350 mM sorbitol, 10 mM NaCl, 5 mM MgCl 2 , 5 mM DTT) and centrifuging (12,000 × g , 20 min, 4°C).…”

NONTEMPLATE-DEPENDENT POLYMERIZATION PROCESSES: Polyhydroxyalkanoate Synthases as a Paradigm

“…The top fraction phase that contains the rubber particles was the focus of most investigators. Very recent biochemical and EM studies, however, have established that latex in this fractionated form is unstable (43). These studies also suggest that the bottom faction is required for initiation of polymer synthesis.…”

“…In fact, most of the studies thus far reported begin with rubber particles. This starting point was based on early biochemical studies that demonstrated that fresh latex could be separated by centrifugation into three phases (43). The bottom fraction (20% of the latex) contains membrane-bound organelles, called lutoids and Frey-Wyssling particles.…”

“…These methods are still used today for granule purification. As noted above, especially in the case of the rubber synthase, the method of isolation is crucial, and this is an area where additional studies are essential (43). Proteins associated with the granules have only been partially identified.…”

Proteomics analysis of latex from Hevea brasiliensis (clone RRIM 600)

“…Two different major proteins have been identified in rubber particles at molecular masses of 14.6 and 24 kDa. These proteins were classified as proteolipids (Hasma, 1987;Wititsuwannakul et al, 2004) and were identified as latex allergens (i.e. Hev b1 and Hev b3) (Czuppon et al, 1993;Alenius et al, 1993;Yeang et al, 1996).…”