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At least 127 records · Page 7Linked to original sources
PubMed1996

Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

Aldehyde-Lyases↗
PubMed1999

Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis.

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. We have isolated and characterized a cDNA clone that encodes a protein tightly bound on a small rubber particle. We named this protein small rubber particle protein (SRPP). Prior to this study, this protein was known as a latex allergen, and only its partial amino acid sequence was reported. Sequence analysis revealed that this protein is highly homologous to the rubber elongation factor and the Phaseolus vulgaris stress-related protein. Southern and Northern analyses indicate that the protein is encoded by a single gene and highly expressed in latex. An allergenicity test using the recombinant protein confirmed that the cloned cDNA encodes the known 24-kDa latex allergen. Neither ethylene stimulation nor wounding changed the transcript level of the SRPP gene in H. brasiliensis. An in vitro rubber assay showed that the protein plays a positive role in rubber biosynthesis. Therefore, it is likely that SRPP is a part of the rubber biosynthesis machinery, if not the rubber polymerase, along with the rubber elongation factor.

Allergens↗
PubMed2000

Genes expressed in the latex of Hevea brasiliensis.

Rubber (cis-1,4-polyisoprene), an important raw material for many industrial uses, is synthesized in the latex of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. We postulated that the genes uniquely or preferentially expressed in the latex may be important for rubber biosynthesis. We constructed cDNA libraries from the latex of H. brasiliensis to investigate the genes expressed in the latex by single-run partial sequencing of the cDNA clones. Sequence analyses identified 245 expressed sequence tags (ESTs), of which 57% showed homology to previously described sequences in public databases. About 16% of the database-matched ESTs encode rubber biosynthesis-related proteins such as rubber elongation factor (REF) and small rubber particle protein (SRPP). The second most frequent transcripts next to rubber biosynthesis-related genes were defense genes and protein metabolism-related genes (12.6% each). About 27% of the database-matched ESTs had sequence homology with genes of unknown function. Among the redundantly expressed genes, REF was the most frequently expressed (6.1%), followed by SRPP (3.7%) and HbLAR (2.9%). Northern blot analyses showed that ten (71%) of the 14 ESTs studied were expressed at a higher level in latex than in leaves.

Journal Article↗
PubMed1992

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis.

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg(2+) diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg(2+). These results suggest the presence of a Mg(2+)/H(+) antiporter. The maximum Mg(2+)/H(+) exchange rate was observed at pH 8.5. The K(m) values for Mg(2+) (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K(+) and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg(2+)/H(+) exchange was electroneutral. Mg(2+)/H(+) exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K(m) for Mg(2+) are similar to those reported for the Mg(2+)/2Na(+) antiporter in animals cell. These data are consistent with the existence of a Mg(2+)/2H(+) antiporter in a plant tonoplast.

Journal Article↗
PubMed1994

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells.

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.

Journal Article↗
PubMed1995

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction.

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.

Journal Article↗
PubMed1991

The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes.

The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant.

Journal Article↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis.

Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Journal Article↗
PubMed1999

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.

Aldehyde-Lyases↗
PubMed1995

Identification of profilin as an IgE-binding component in latex from Hevea brasiliensis: clinical implications.

Considering the high occurrence of profilin as an allergen in many plant species, the assumption was made that profilin might be an allergen in Hevea brasiliensis, a member of the latex producing Euphorbiaceae family. Using IgE-binding inhibition by purified profilins we demonstrated that profilin is an IgE-binding component in the cytosolic fraction of natural latex and, to a lower extent, in the rubber fraction. Thirty-five out of 36 sera containing IgE to ragweed-profilin reacted with profilin from latex, indicating structural homologies between profilins from latex and ragweed. A large percentage (59%) of these sera were found to be positive in CAP latex assay. The preincubation of these sera with purified ragweed profilin greatly inhibited the CAP latex. Because profilin is also present in banana extract, it is likely to be involved in cross-sensitivity to banana and latex. In a group of 19 individuals allergic to latex only two had anti-profilin IgE antibodies. Profilin was barely detectable on glove extract immunoblots, whereas some sera from patients allergic to latex reacted with a 15 kDa allergen which was not profilin. Consequently, IgE antibodies to latex-profilin is a questionable factor for sensitization of occupationally-exposed patients; however, sensitization to profilin should be taken into account when interpreting the results of latex IgE antibody assays.

Allergens↗
PubMed1995

Prohevein from the rubber tree (Hevea brasiliensis) is a major latex allergen.

BACKGROUND: There is general agreement that proteins eluting from different natural rubber latex products can cause immediate type hypersensitivity reactions in latex-allergic patients. However, there is as yet no consensus as to what are the most important allergens in natural rubber latex. OBJECTIVE: We wanted to purify and characterize at the primary structure level three natural latex proteins, suggested to represent significant allergens. METHODS: Proteins were purified from ultracentrifuged bottom fraction of natural rubber latex using high performance liquid chromatography gel filtration and reversed phase chromatography. Purified proteins were subjected to tryptic cleavage, peptide separation and amino acid sequencing. Immunoblotting was used to demonstrate IgE antibodies to the purified proteins in sera from latex-allergic patients. RESULTS: A 20 kDa protein was identified by amino acid sequencing as prohevein, a major protein in the rubber tree Hevea brasiliensis, and a 30 kDa natural rubber latex protein as hevamine, another essential rubber tree protein. A third, previously undescribed natural rubber latex protein, showed high homology to several plant endo-1,3-beta-glucosidases. In immunoblotting, the purified prohevein bound IgE antibodies from 24/29 (83%) sera of latex-allergic patients including positive results in 4/6 latex-allergic children with spina bifida or other congenital anomalies. The purified prohevein elicited positive skin-prick test reactions in all six latex-allergic patients showing IgE to prohevein. The purified 36 kDa protein bound IgE from 6/29 (21%) latex-allergic sera, and the purified hevamine from only 1/29 patient sera. CONCLUSION: The observed high frequency of IgE antibodies to prohevein suggests that this protein is a major natural rubber latex allergen.

Adolescent↗
PubMed1997

HLA class II antigens DR4 and DQ8 are associated with allergy to hevein, a major allergen of Hevea latex.

In this study we investigated the relationship between HLA class II alleles and the IgE-specific immune response to the 4.7 kDa polypeptide hevein of Hevea brasiliensis, a major latex allergen, 51 individuals with immediate-type latex allergy and 90 controls were examined for the polymorphisms in exon 2 of HLA-DRB1, 3, 4, 5 and DQB1 by sequence-specific oligonucleotide probe typing. 35 (69%) out of 51 latex-sensitized subjects showed positive hevein-specific IgE values. Analysis of the HLA data among these 35 subjects revealed increased phenotype frequencies for DR4 (22/35, 63%) and DQ8 (18/35, 51%) when compared with those in the 16 hevein-negative but latex-positive subjects (DR4: 2/16, 13%, p = 0.0009, Pc = 0.047; DQ8: 0/16, p = 0.0003, pc = 0.018) and with healthy controls (DR4: 22/90, 24%, p = 0.00012, pc = 0.013; DQ8: 16/89, 18%, p = 0.0003, pc = 0.036). Finally the DR4-DQ8 haplotype frequency was significantly elevated in hevein-positives when compared with hevein-negatives (51% vs. 0, p = 0.0003, pc = 0.034) or controls (51% vs. 18%, p = 0.0002, pc = 0.045) The present data suggest DR4 and DQ8 to be operating jointly as susceptibility factor for the allergy to hevein.

Adolescent↗
PubMed1985

Solubilization and purification of the ATPase from the tonoplast of Hevea.

The tonoplast-bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100-fold with two ammonium sulfate precipitation steps and a G-200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 110 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54-kDa and the 66-kDa polypeptides exist in multiple copies. No close resemblance to the membrane-bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody cross-reaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase. Many properties of the tonoplast-bound ATPase such as pH-dependence, substrate specificity, ion-dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP-hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, which are strong inhibitors of membrane-bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.

Adenosine Triphosphatases↗
PubMed1991

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex.

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N-terminal sequence of the lysozyme/chitinase of Parthenocissus quinquefolia. The half-cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevamine and cucumber chitinase are similar to those between class I and class II chitinases from tobacco and other plant species.

Amino Acid Sequence↗
PubMedDate not supplied

Atomic resolution crystal structure of hydroxynitrile lyase from Hevea brasiliensis.

The X-ray crystal structure of native hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) has been determined at 1.1 A resolution. It refined to a final R of 11.5% for all data and an Rfree of 14.4%. The favorable data-to-parameter ratio at atomic resolution made the refinement of individual anisotropic displacement parameters possible. The data also allowed a clear distinction of the alternate orientations of all histidine and the majority of asparagine and glutamine side chains. A number of hydrogen atoms, including one on the imidazole of the mechanistically important His-235, became visible as peaks in a difference electron density map. The structure revealed a discretely disordered sidechain of Ser-80, which is part of the putative catalytic triad. Analysis of the anisotropy indicated an increased mobility of residues near the entrance to the active site and within the active site.

Aldehyde-Lyases↗
PubMed1989

Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis.

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.

Allergens↗
PubMed1989

Amino acid sequence of rubber elongation factor protein associated with rubber particles in Hevea latex.

The amino acid sequence of rubber elongation factor, a recently discovered protein tightly bound to rubber particles isolated from the commercial rubber tree Hevea brasiliensis, is presented. The role of this protein in rubber elongation and its interaction with prenyltransferase and rubber particles have been discussed in the preceding paper in this series (Dennis, M. S., and Light, D. R. (1989) J. Biol. Chem. 264, 18608-18617). Trypsin, Staphylococcus protease, chymotrypsin, acetic acid, and hydroxylamine cleavage were used to generate peptide fragments that were isolated by reverse phase high pressure liquid chromatography and analyzed by amino acid composition and automated Edman degradation. Each digest contained one blocked peptide identified as the amino terminus. The blocked amino-terminal peptide from the tryptic digest was analyzed by amino acid composition, fast atom bombardment mass spectrometry (molecular ion 1659.9), subdigested with Staphylococcus protease for partial sequence analysis, and finally deblocked with bovine liver acyl-peptide hydrolase removing an acetylalanine to allow analysis by Edman degradation. Rubber elongation factor is 137 amino acids long, has a molecular mass of 14,600 daltons, and lacks four amino acids: cysteine, methionine, histidine, and tryptophan. The NH2 terminus is highly charged and contains only acidic residues (5 of the first 12 amino acids). The first four amino acids are highly represented in other known NH2-terminally acetylated proteins. Comparison of the sequence of rubber elongation factor with other known sequences does not reveal significant sequence similarities that would suggest an evolutionary relationship.

Acetates↗
PubMed1987

[Demonstration of a pyrophosphate-fructose 6-phosphate 1-phosphotransferase in the latex from Hevea brasiliensis].

A pyrophosphate: fructose-6-phosphate 1-phosphotransferase activity (EC 2.7.1.90) has been characterized in cytosol from Hevea brasiliensis latex. It is Mg+ dependent enzyme, and the cation has an optimal effect between 2.5 to 3 mM for a concentration of 1 mM of pyrophosphate and 10 mM of fructose-6-phosphate. It is activated by catalytic content of fructose-2,6-diphosphate. Its potential activity is higher than 40% of that of ATP dependent phosphofructokinase (EC 2.7.1.11). Its optimum pH is between 7.5-7.6; then, the enzyme affinity is 0.3 mM for pyrophosphate and 3.5 mM for fructose-6-phosphate. It is suggested that the transferase plays a role in the pyrophosphate metabolism and the increasing of the energetic efficiency of glycolysis and so takes a significant part in the biochemical mechanisms involved in the latex yield.

Cytosol↗