Purification and characterization of neutral invertase from chickpea nodules.

Neutral invertase from nodules of chickpea (Cicer arietinum L.) was isolated and purified by ammonium sulphate fractionation, gel filtration and DEAE-cellulose column chromatography. The purified enzyme was stable between 0 to 40 degrees C beyond which it was irreversibly denatured. Optimum temperature and pH of the enzyme were 37 degrees C and 7.0, respectively. K(m) for sucrose was 14.2 mM and Vmax was 4.8 mumole hr-1. The enzyme was inhibited by several metal ions. From the temperature effect on K(m) and Vmax values, the energy of activation (Ea), enthalpy change (delta H) and entropy change (delta S) of the enzyme were calculated to be 147 kJmol-1, -4.10 kJmol-1 and -2.33 JK-1mol-1, respectively. By employing photo-oxidation and chemical modification and by studying the effect of pH on K(m) and Vmax, the involvement of sulphydryl-, imidazole- and alpha-amino groups in the active site of the enzyme has been indicated.

Purification and kinetic characterization of chicken liver inorganic pyrophosphatase.

Soluble inorganic pyrophosphatase (EC 3.6.1.1) was isolated from chicken liver, RIR breed, to apparent homogeneity. The enzyme showed a molecular mass of 100 kDa as estimated by gel filtration and a subunit mass of 49 kDa on SDS-PAGE. The enzyme was very specific for pyrophosphate (PPi) and magnesium, and there was no measurable activity on replacing Mg2+ with Zn2+. At optimal conditions of assay, 50% of the enzyme activity was inhibited at 42 microM Ca2+, 70 microM fluoride and 0.91 mM Cd2+. There was a 50% inactivation of enzyme activity at 0.1 M guanidine hydrochloride (GuHCl). Kinetic analysis of GuHCl inactivation revealed 2 essential binding sites for this ligand. The enzyme showed allosteric behaviour with the substrate PPi and Mg2+. The apparent Hill coefficient of 1.47 and 1.48 for PPi and Mg2+, respectively indicate positive cooperatively. Hill plots also gave [S]0.5 of 0.177 mM and 2.5 microM for Mg2+ and PPi, respectively.

Colchicine binding to tubulin from brain homogenates in the presence of the sugars, glycols and metal ions: effect of nickel ions on the tubulin solubility

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions A1(III), Co(II), Ni(II), while compounds structurally related to glycero (glucose and sucrose) did not inhibition it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and NI (II)] favored tubulin sedimentation when these latterwere centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion madeinsoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and streteched structures, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein