02647nas a2200241   4500008004100000022001400041245013000055210006900185260009500254300001200349490000800361520173000369653002202099653002302121653001702144653002802161653001902189653002002208653001402228100001902242700002502261856011902286       2010                        eng d  a0006-291X00aMultistate unfolding of alpha-mannosidase from Canavalia ensiformis (Jack Bean): evidence for the thermostable molten globule0 aMultistate unfolding of alphamannosidase from Canavalia ensiform  a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC  a391-3970 v4033 a<p>The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II alpha-mannosidase from Canavalia ensiformis (Jack Bean) (Jb alpha-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jb alpha-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jb alpha-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 degrees C and stability of the structure up to 80 degrees C unlike the native Jack Bean alpha-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II alpha-mannosidase. (C) 2010 Elsevier Inc. All rights reserved.</p>10aalpha-Mannosidase10aCircular dichroism10afluorescence10aGuanidine hydrochloride10aMolten globule10aThermostability10aUnfolding1 aKumar, Avinash1 aGaikwad, Sushama, M.  uhttp://library.ncl.res.in/content/multistate-unfolding-alpha-mannosidase-canavalia-ensiformis-jack-bean-evidence-001666nas a2200229   4500008004100000022001400041245014200055210006900197260007300266300001400339490000700353520078800360653002101148653002201169653002501191653001501216653001501231653001801246100001901264700002501283856012801308       2011                        eng d  a0141-813000aJack bean alpha-mannosidase (Jb alpha-man): tolerance to alkali, chelating and reducing agents and energetics of catalysis and inhibition0 aJack bean alphamannosidase Jb alphaman tolerance to alkali chela  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcDEC  a1066-10710 v493 a<p>Investigations of the catalytic and structural transitions of jack bean alpha-mannosidase (Jb alpha-man) are described in the present paper. The enzyme was maximally stable at pH 5.0; however, when incubated in the pH range of 11.0-12.0, showed 1.3 times higher activity and also stability for longer time. The free amino group at or near the active site was probably involved in the stability and activation mechanism. The active site is constituted by the association of two unidentical subunits connected by disulfide linkages. The metalloenzyme has Zn(2+) ions tightly bound and chelation reduces the thermal stability of the protein. Energetics of catalysis and thermodynamics of inhibition of the enzyme were also carried out. (C) 2011 Elsevier B.V. All rights reserved.</p>10aAlkali tolerance10aalpha-Mannosidase10abeta-Mercaptoethanol10aEnergetics10aInhibition10aMetalloenzyme1 aKumar, Avinash1 aGaikwad, Sushama, M.  uhttp://library.ncl.res.in/content/jack-bean-alpha-mannosidase-jb-alpha-man-tolerance-alkali-chelating-and-reducing-agents-0