Isolation,characterization and analysis of the agglutinative activity of a lectin from <Emphasis Type="Italic">Crotalaria spectabilis</Emphasis>

Lectins are proteins with ability to recognize specific carbohydrates. These are present in virtually all organisms and have increasing applications in biotechnology. Here, our aim was to purify lectins from seeds of Crotalaria spectabilis Roth and determine their agglutinative ability. In this study, 45 g of seeds were milled, their proteins were precipitated by acetone or ammonium sulfate and purified by exclusion and ion-exchange chromatography. An isolated lectin was submitted to tests for hemagglutination and inhibition of hemagglutinating activity by carbohydrates as well as tests for its response to chelating and reducing agents. Our results show that the apparent molecular weight (as determined by SDS-PAGE) of the lectin is 30 kDa, and the tests for inhibition of erythrocytes’ agglutinative activity by sugars were positive for d-galactose and N-acetyl-d-galactosamine. Data obtained with the chelating agent EDTA demonstrated the presence of divalent cations in the protein structure. However, the reducing agent 2-mercaptoethanol was unable to inhibit the protein’s bioactivity. The lectin agglutinated the blood groups A, B, AB and O, as well as bacterial lineages from the species Leptospira interrogans and Leptospira biflexa, indicating a prospective application in the diagnosis and treatment of leptospirosis.     

Cystathionine Metabolism in Methionine Auxotrophic and Wild-Type Strains of Saccharomyces cerevisiae

The role of cystathionine in methionine biosynthesis in wild-type and auxotrophic strains of Saccharomyces cerevisiae was studied. Homocysteine and cysteinerequiring mutants were selected for detailed study. Exogenously supplied cystathionine, although actively transported by all strains tested, could not satisfy the organic sulfur requirements of the mutants. Cell-free extracts of the wild-type, homocysteine, and cysteine auxotrophs were shown to cleave cystathionine. Pyruvic acid and homocysteine were identified as teh products of this cleavage. A mutant containing an enzyme which could cleave cystathionine to homocysteine in cell-free experiments was unable to use cystathionine as a methionine precursor in the intact organisms. The significance of this finding is discussed.     

Free radicals from plastic syringes.

We have found that disposable sterile plastic syringes can leach free radicals into the fluids they hold. The free radical we observe appears to be a nitroxide (aminoxyl radical) because in aqueous solution it produces a 1:1:1 three line spectrum with a hyperfine splitting constant of 16.9 G.     

Transition metals as catalysts of "autoxidation" reactions

Superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (.OH) produced from the "autoxidation" of biomolecules, such as ascorbate, catecholamines, or thiols, have been implicated in numerous toxicities. However, the direct reaction of dioxygen with the vast majority of biomolecules, including those listed above, is spin forbidden, a condition which imposes a severe kinetic limitation on this reaction pathway. Therefore, an alternate mechanism must be invoked to explain the "autoxidations" reactions frequently reported. Transition metals are efficient catalysts of redox reactions and their reactions with dioxygen are not spin restricted. Therefore it is likely that the "autoxidation" observed for many biomolecules is, in fact, metal catalyzed. In this paper we discuss: 1) the quantum mechanic, thermodynamic, and kinetic aspects of the reactions of dioxygen with biomolecules; 2) the involvement of transition metals in biomolecule oxidation; and 3) the biological implications of metal catalyzed oxidations. We hypothesize that true autoxidation of biomolecules does not occur in biological systems, instead the "autoxidation" of biomolecules is the result of transition metals bound by the biomolecules.     

Mechanism of hypoglycaemic action of methylenecyclopropylglycine.

Cytochrome b-245 from neutrophil plasma membranes contains two types of subunit with apparent molecular masses from gel electrophoresis in the presence of SDS of 23 kDa and 76-92 kDa. Radiation-inactivation analysis revealed a single-exponential decay process for the visible absorption of the haem chromophore in the membrane, corresponding to a molecular mass of 21 +/- 5 kDa for the haem-containing polypeptide chain. Sedimentation equilibrium of the cytochrome solubilized by the detergent Triton N101 showed that the protein was polydisperse, with a molecular mass of approx. 350 kDa for the smallest detectable species. In another detergent, n-octyl beta-O-glucopyranoside (octyl glucoside), the molecular mass of the haem-containing particle was found to be 20-30 kDa. Thus the quaternary structure of the protein breaks down in this detergent. The haem group is inferred to be attached to the smaller subunit.