Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) 相似文献   

Similarities and differences in rubber biochemistry among plant species.

This report reviews aspects of the biochemical regulation of rubber yield and rubber quality in three contrasting rubber-producing species, Hevea brasiliensis, Parthenium argentatum and Ficus elastica. Although many similarities are revealed, considerable differences also exist in enzymatic mechanisms regulating biosynthetic rate and the molecular weight of the rubber biopolymers produced. In all three species, rubber molecule initiation, biosynthetic rate and molecular weight, in vitro, are dependent upon substrate concentration and the ratio of isopentenyl pyrophosphate (IPP, the elongation substrate, or monomer) and farnesyl pyrophosphate (FPP, an initiator), but these parameters are affected by intrinsic properties of the rubber transferases as well. All three rubber transferases are capable of producing a wide range of rubber molecular weight, depending upon substrate concentration, clearly demonstrating that the transferases are not the prime determinants of product size in vivo. However, despite these commonalities, considerable differences exist between the species with respect to cosubstrate effects, binding constants, effective concentration ranges, and the role of negative cooperativity in vitro. The P. argentatum rubber transferase appears to exert more control over the molecular weight it produces than the other two species and may, therefore, provide the best prospect for the source of genes for transformation of annual crop species.The kinetic data, from the three contrasting rubber-producing species, also were used to develop a model of the rubber transferase active site in which, in addition to separate IPP and allylic-PP binding sites, there exists a hydrophobic region that interacts with the linear portion of allylic-PP initiator proximal to the pyrophosphate. Substrate affinity increases until the active site is traversed and the rubber interior of the rubber particle is reached. The kinetic data suggest that the hydrophobic region in H. brasiliensis and F. elastica is about 1.8 nm long but only 1.3 nm in P. argentatum. The estimates are supported by measurements of the rubber particle monolayer membrane using electron paramagnetic resonance spectroscopy.     

Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica

The rubber-producing tree, Ficus elastica (the Indian rubber tree), requires the same substrates for rubber production as other rubber-producing plants, such as Hevea brasiliensis (the Brazilian or Para rubber tree), the major source of commercial natural rubber in the world, and Parthenium argentatum (guayule), a widely studied alternative for natural rubber production currently under commercial development. Rubber biosynthesis can be studied, in vitro, using purified, enzymatically active rubber particles, an initiator such as FPP, IPP as the source of monomer, and a metal cofactor such as Mg2+. However, unlike H. brasiliensis and P. argentatum, we show that enzymatically active rubber particles purified from F. elastica are able to synthesize rubber, in vitro, in the absence of added initiator. In this paper, we characterize, for the first time, the kinetic differences between initiator-dependent rubber biosynthesis, and initiator-independent rubber biosynthesis, and the effect of cofactor concentration on both of these processes.     

Regulation of rubber biosynthetic rate and molecular weight in Hevea brasiliensis by metal cofactor

Metal ion cofactors are necessary for prenyltransferase enzymes. Magnesium and manganese can be used as metal ion cofactor by rubber transferase (a cis-prenyltransferase) associated with purified rubber particles. The rubber initiation rate, biosynthetic rate, and molecular weight produced in vitro from Hevea brasiliensis rubber transferase is regulated by metal ion concentration. In addition, varies significantly with [Mg(2+)]. decreases from 8000 +/- 600 microM at [Mg(2+)] = 4 mM to 68 +/- 10 microM at [Mg(2+)] = 8 mM and increases back to 970 +/- 70 microM at [Mg(2+)] = 30 mM. The highest affinity of rubber transferase for IPP.Mg occurred when [Mg(2+)] = A(max) (metal concentration that gives highest IPP incorporation rate). A metal ion is required for rubber biosynthesis, but an excess of metal ions interacts with the rubber transferase inhibiting its activity. The results suggest that H. brasiliensis could use [Mg(2+)] as a regulatory mechanism for rubber biosynthesis and molecular weight in vivo.     

Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzyme stereospecificity

A prenyltransferase purified from the commercial rubber tree, Hevea brasiliensis, that elongates existing cis-polyisoprene rubber molecules also catalyzes the formation of all trans-farnesyl pyrophosphate (t,t-FPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In assays of the latter activity trans-geranyl pyrophosphate is the only other product identified. In contrast to this limited addition of IPP to DMAPP, we measured 7000 additions of isoprene per rubber molecule in a previous titration of active allylic ends of rubber molecules by purified prenyltransferase (Light, D. R., and Dennis, M. S. (1989) J. Biol. Chem. 264, 18589-18597). In order to confirm that purified prenyltransferase extensively elongates rubber molecules, doubly labeled [1-14C]isopentenyl [U-32P]pyrophosphate ([14C,32P]IPP) was synthesized. Using this reagent we show that both prenyltransferase purified from H. brasiliensis and prenyltransferase purified from avian liver (FPP synthase) add greater than 15 isoprene units to existing rubber molecules, consistent with the previous titration data. For confirmation that the prenyltransferase purified from H. brasiliensis adds isoprene units to rubber to make cis-polyisoprene, chirally tritiated [14C]IPP ([14C,2S-3H]IPP) was synthesized. Retention of the tritium label in FPP synthesized from [14C,2S-3H]IPP and DMAPP, geranyl pyrophosphate, or neryl pyrophosphate by prenyltransferase from H. brasiliensis or avian liver confirms trans addition to these substrates. In contrast, when [14C,2S-3H]IPP is incubated with serum-free rubber particles and prenyltransferase purified from H. brasiliensis, avian liver, or yeast, no tritium is incorporated into the rubber particles indicating cis addition. Thus, rubber particles have the ability to alter the stereoselective removal of the 2R-prochiral proton in favor of the removal of the 2S-prochiral proton. This apparent inversion of carbon 2 of IPP during the proton abstraction step by rubber particles represents a novel example of a switch in enzyme stereospecificity. In addition to being enzymatically similar to other prenyltransferases, rubber transferase also appears to be related immunologically to FPP synthases, since polyclonal antibodies to the H. brasiliensis prenyltransferase cross-react with the purified yeast prenyltransferase. In order to investigate potential primers of greater molecular weight than that of FPP, cis-undecaprenyl pyrophosphate (C55PP) was synthesized. C55PP stimulates the incorporation of [14C]IPP into rubber particles suggesting that it may prime new rubber molecules. However, in contrast to DMAPP, C55PP is not incorporated into any detectable products when incubated with prenyltransferase and [14C]IPP in the absence of rubber particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase.

Rubber transferase, a cis-prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4-7) and alpha-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species-specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes.     

Microstructure of Purified Rubber Particles

Purified rubber particles from Hevea brasiliensis (Brazilian rubber tree), Parthenium argentatum (guayule), Ficus elastica (Indian rubber tree), and Euphorbia lactiflua were examined and compared using conventional scanning electron microscopy (SEM), field-emission SEM, cryo-SEM, and transmission electron microscopy (TEM). Rubber particles of all four species were spherical; they varied in size and had a uniform homogeneous material, the rubber core, surrounded by a contiguous monolayer (half-unit) membrane. Frozen-hydrated and/or untreated particles from H. brasiliensis and P. argentatum deformed and fused readily, whereas those from F. elastica and E. lactiflua retained their spherical shapes. These results indicate that the surface components of the H. brasiliensis and P. argentatum particles are more fluid than those of F. elastica or E. lactiflua. When fixed in aldehyde, F. elastica particles retained their spherical exterior shapes but had hollow centers, whereas H. brasiliensis and P. argentatum particles completely collapsed. In aldehyde-osmium tetroxide-fixed material, the rubber core of F. elastica was poorly preserved in some particles in which only a small amount of the rubber core remained adhering to the monolayer membrane, leaving a hollow center. Euphorbia lactiflua particles were well preserved in terms of retaining the rubber core; however, the membrane was not as easily discernible as it was in the other three species. Both H. brasiliensis and P. argentatum were well preserved following fixation; their cores remained filled with rubber, and their monolayer membranes were defined. The addition of potassium permanganate to the fixation-staining regime resulted in higher-contrast micrographs and more well defined monolayer membranes.     

Identification of natural rubber and characterization of rubber biosynthetic activity in fig tree

Natural rubber was extracted from the fig tree (Ficus carica) cultivated in Korea as part of a survey of rubber producing plants. Fourier transform infrared and (13)C nuclear magnetic resonance analysis of samples prepared by successive extraction with acetone and benzene confirmed that the benzene-soluble residues are natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of fig tree was about 4%, whereas the rubber content in the bark, leaf, and fruit was 0.3%, 0.1%, and 0.1%, respectively. Gel-permeation chromatography revealed that the molecular size of the natural rubber from fig tree is about 190 kD. Similar to rubber tree (Hevea brasiliensis) and guayule (Parthenium argentatum Gray), rubber biosynthesis in fig tree is tightly associated with rubber particles. The rubber transferase in rubber particles exhibited a higher affinity for farnesyl pyrophosphate than for isopentenyl pyrophosphate, with apparent K(m) values of 2.8 and 228 microM, respectively. Examination of latex serum from fig tree by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major proteins of 25 and 48 kD in size, and several proteins with molecular mass below 20 and above 100 kD. Partial N-terminal amino acid sequencing and immunochemical analyses revealed that the 25- and 48-kD proteins were novel and not related to any other suggested rubber transferases. The effect of EDTA and Mg(2+) ion on in vitro rubber biosynthesis in fig tree and rubber tree suggested that divalent metal ion present in the latex serum is an important factor in determining the different rubber biosynthetic activities in fig tree and rubber tree.     

A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.     

Identification and comparison of natural rubber from two Lactuca species

Renewed interest in the identification of alternative sources of natural rubber to Hevea brasiliensis has focused on the Compositae family. In our search for Compositae models for rubber synthesis, we extracted latex from stems of two lettuce species: Lactuca serriola, prickly lettuce, and Lactuca sativa cv. Salinas, crisphead lettuce. Both species contained cis-1,4-polyisoprene rubber in the dichloromethane-soluble portions of their latex, and sesquiterpene lactones in their acetone-soluble portions. The rubber from both species and their progeny had molecular weights in excess of 1,000,000g/mol, and polydispersity values of 1.1. Rubber transferase activity was detected across a range of farnesyl diphosphate initiator concentrations, with decreased activity as initiator concentrations exceeded putative saturation. These results add lettuce to the short list of plant species that produce high molecular weight rubber in their latex. Due to the genomic and agronomic resources available in lettuce species, they provide the opportunity for further dissection of natural rubber biosynthesis in plants.     

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Heveabrasiliensis and Parthenium argentatum.Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz-DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz-DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.     

产胶植物橡胶转移酶的研究进展

天然橡胶合成中,橡胶转移酶催化异戊二烯焦磷酸的多聚化过程,这一过程对天然橡胶的品质及产量至关重要。橡胶转移酶及其性质、橡胶生物合成分子机理及橡胶的分子量大小决定机制是亟待解决的重要科学问题。本文介绍了以巴西橡胶树为主的产胶植物橡胶转移酶的性质和生物学功能,对橡胶转移酶的分离与鉴定及其活性调节等研究进展进行了综述。     

The micromorphology and protein characterization of rubber particles in Ficus carica,Ficus benghalensis and Hevea brasiliensis

Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.     

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase,and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.     

New aspects of rubber biosynthesis

New aspects of rubber biosynthesis. Following a review of the site of rubber biosynthesis in Hevea brasiliensis and Parthenium argentalum, evidence is given for the initiation of polyisoprene molecules from (ranMerpenoid precursors including geranylgeranyl pyrophosphate. All franj-¹⁴C-geranylgeraniol has been isolated from incubations of H. brasiliensis latex serum with ¹⁴C-isopentenyl pyrophosphate. Gel-filtration chromatography of the serum yields very small rubber particles of high biosynthetic activity, and two proteinaceous fractions. One of these increases the biosynthesis of rubber and may contain the enzyme, isopentenyldiphosphate δ-isomerase, whilst the other appears to inhibit rubber formation. The nature and molecular weight of the rubber formed in vitro is discussed and a mechanism for the de novo formation of rubber particles is suggested.     

Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

不同提取液对巴西橡胶树橡胶粒子蛋白质提取和分离的影响

橡胶粒子是巴西橡胶树（Hevea brasiliensis）乳管细胞内进行橡胶生物合成的亚细胞结构;对橡胶粒子的蛋白质组学研究,可为揭示天然橡胶生物合成机理提供线索。采用5种提取液分别提取橡胶粒子蛋白,并对橡胶粒子蛋白进行SDS—PAGE和16-BAC／SDS—PAGE电泳分离及MALDITOF／TOF串联质谱分析,证明不同提取液抽提的橡胶粒子蛋白具有不同组成,发现分子量较高的橡胶延伸因子家族蛋白更难从橡胶粒子上被洗脱和提取。通过检索橡胶树转录组数据库,鉴定了3个新的橡胶粒子蛋白,即醌氧化还原酶、含蓖麻毒素B链凝集素结构域蛋白及枯萎／脱水相关蛋白。本研究建立的橡胶粒子蛋白质提取和分离方法,为进一步鉴定低丰度和具有重要功能的橡胶粒子蛋白提供了参照体系。     

Roles of Mg2+ in TPP-dependent riboswitch

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.