A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation.
In the first of this three paper series, an in vitro latex coagulation was shown to arise from aggregation of rubber particles (RP) and lutoid membranes. RP aggregation was shown to be induced by a specific Hevea latex lectin-like protein (HLL) present on the lutoid membrane. In this second paper, a binding protein (BP) ligand counterpart for HLL was identified. This RP-HLLBP, having a specific interaction, with HLL was isolated from RP and characterized. The protein was extracted from the small RP in the presence of a surfactant (0.2% Triton-X-100) and further purified to homogeneity. Purification steps included acetone precipitation, heat-treatment, and column chromatography. The presence of RP-HLLBP was monitored by its ability to compete with erythrocytes in the hemagglutination inhibition (HI) assay. The purified RP-HLLBP had an HI titre of 1.37 microgml(-1), a pI value of 5.4, optimum activity at pH 5-8 and was thermostable up to 60 degrees C. On SDS-PAGE a single glycoprotein with M(r) of 24 kDa was detected while on native PAGE the major Mr was about 120 kDa. The purified RP-HLLBP was shown to inhibit latex coagulation. Chitinase, but no other glycosidase tested, abolished its HI action and inhibited HLL-induced RP aggregation in a competitive dose dependent manner. This indicated the presence of, and role for, N-acetylglucosamine residues in the binding recognition. The Hevea latex lectin-like protein can thus be referred to as a Hevea latex lectin. Based on protein identification by peptide mass fingerprinting, the RP-HLLBP was confirmed to be the small rubber particle protein (SRPP). This work has unambiguously determined the role of an intrinsic RP glycoprotein (RP-HLLBP or SRPP) as a key component in formation of the rubber latex coagulum.
Wititsuwannakul
Rapepun
R
Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand. wrapepun@yahoo.com
Rukseree
Kamonchanok
K
Kanokwiroon
Kamonwan
K
Wititsuwannakul
Dhirayos
D
D028482:Hevea / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry* / Q000302:isolation & purification; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D037121:Plant Lectins / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013696:Temperature
D007840:Latex; D008024:Ligands; D008565:Membrane Proteins; D037121:Plant Lectins