Degradation kinetics and physiological studies of organophosphates degrading microorganisms for soil bioremediation.

Organophosphate compounds are widely used in agricultural activities to optimize food production. Contamination of field soil by these compounds may result in detrimental effects on soil biota. The aim of the present study was to isolate microorganisms from field soils and evaluate the strains on ability to degrade organophosphates as single isolate and as a consortium. Isolated strains were identified using both biochemical and molecular techniques. Results revealed that, out of the 46 isolated strains, three isolates herein referred to as S6, S36 and S37 showed an average diazinon degradation rate of 76.4%, 76.7% and 76.8% respectively, of the initial dose (50 ppm) within 11 days of incubation in mineral medium. Notably, isolates S36 and S37 were more effective than S6 in degrading diazinon by 40% in soil aliquot after 11 days and therefore were evaluated on biochemical reactions and molecular identification. The isolates showed variable biochemical characteristics. However, both isolates possessed catalase enzyme, but lacked oxidase enzyme. Molecular characterization showed that, the closest species for S36 and S37 were Priestia megaterium and P. arybattia, respectively, based on 16S rRNA gene similarity (> 99%). Combination of the strains increased diazinon degradation ability by 45% compared to single strain treatment. Chlorpyrifos was the most highly degraded organophosphate, compared to phorate and cadusafos. Therefore it is expected that the pesticide-degrading bacteria could be a solution to soil health improvement and contribution to the production of safe agricultural products.

Generation of a free alpha-amino group by Raney nickel after 2-nitro-5-thiocyanobenzoic acid cleavage at cysteine residues: application to automated sequencing.

The selective reaction of SH containing proteins and peptides with NTCB (2-nitro-5-thiocyanobenzoic acid) has been reported (Degani, Y., & Patchornick, A. (1974) Biochemistry 13, 1; Jacobson, G.A., Schaffer, M.H., Stark, G.R., & Vanaman, T.C. (1973) J. Biol. Chem. 248, 6583). With this reagent, cysteinyl peptide bonds are selectively cyanylated and subsequently cleaved under alkaline conditions. In the present study we have successfully cleaved the beta-chains of guinea pig hemoglobin at the single cysteine and the peptides thus obtained were separated. However, the C-terminal peptide was blocked at its N terminal by a thiazolidine ring and hence could not be used for Edman degradation sequence analysis. Deblocking of this peptide was successfully done by Raney nickel in the buffer medium of pH 7.0, and also in water, at 50 degrees C for 6 to 10 h. The Raney nickel reagent is used in large excess by weight (at least ten times the weight of sulfur compound) over the compound to be desulfurized. Under these conditions, control experiments on cysteine, methionine, and some other amino acids showed that only the sulfur containing amino acids are degraded by Ni(H). Cysteine and methionine were rapidly converted to alanine and beta-aminobutyric acid, respectively. Gel electrophoresis of the iminothiazolidine peptide after exposure to Ni(H) showed no breakage of the chain.

Specific antibodies to hemoglobin A1 (anti-Glu) and hemoglobin S (anti-Val) in the guinea pig: immunologic and structural correlations.

Like goats and sheep, guinea pigs can produce, in response to human sickle cell hemoglobin (beta6 Glu leads to Val), an antibody population (anti-Val) that will bind sickle cell hemoglobin but not normal hemoglobin HbA. Unlike goats and sheep, guinea pigs can produce in response to human hemoglobin A1 an antibody fraction, anti-Glu, that will not react with human sickle cell hemoglobin. These anti-Glu antibodies have been isolated by affinity chromatography and their specificity confirmed by fluorescence-quenching titrations. The sequence of the first 10 amino acids of the beta-chain of guinea pig hemoglobin has been determined. This sequence differs from those of both hemoglobin HbA and sickle cell hemoglobin by two residues, those at positions 5 and 6. This explains the similarity of the immunogenicity of this site on the two human hemoglobins when administered to guinea pigs. Both goats and sheep are identical to hemoglobin A1 at the beta-6 position.

Evidence for a single essential thiol in the yeast hexokinase molecule.

In yeast hexokinase B, two thiols per monomer appeared to be essential when enzymic inactivation was produced by the concurrent alkylation of both of them, by several reagents including the affinity reagent N-bromoacetyl-2-D-galactosamine. However, it is shown that only one of these thiols is actually essential. Three of the four thiols present can be blocked by alkylation in the presence of a substrate in appropriate conditions, without loss of enzymic activity. Subsequently, in the absence of substrate, the affinity reagent reacts at the one remaining thiol, with complete inactivation. The same behavior can be obtained by reaction with iodoacetamide or by the formation of the -SCN group. The affinity reagent inactivates hexokinase B faster than does the isomeric glycosidic compound (glycosides being nonsubstrates), although the latter has twice the reactivity of the former toward glutathione. The reactions with alkylating agents, with or without substrate present, are used to classify the four thiols in the monomer. The temperature dependence of the alkylation of the essential thiol provides evidence for a transition in the molecule at about 31 degrees C. The inactive monomer containing the -SCN group can regenerate, by thiolysis, active enzyme with the thiol free. It can also perform an intramolecular cleavage of the chain. The latter reaction was used to locate the essential cysteine residue in the chain, at 80% of the length from the N terminus.

The antibody response in sheep vaccinated with experimental Nairobi sheep disease vaccines.

The antibody responses to experimental Nairobi sheep disease vaccines have been assayed. The responses to an inactivated methanol precipitated vaccine were comperable with those following infection with virulent virus. The responses to attenuated vaccines were inadequate to protect against challenge with virulent virus.

Essential and nonessential thiols of yeast hexokinase. Reactions with iodoacetate and iodoacetamide.

The reaction of yeast hexokinase with iodoacetate or iodoacetamide has been investigated in detail, using pure hexodinase B. Of the four thiols in each subunit of the molecule, two (the "apparently essential thiols") are alkylated rapidly at 35 degrees, and the enzymic activity is lost in parallel with their reaction. The other two thiols react subsequently to completion, but at a very much slower rate. In the conditions use, no other uptake of the reagent occurs elsewhere during these thiol alkylations. Electrophoretically homogeneous kialkylated and tetraalkylated protein species are formed, in the two stages of the reaction. The inactivating reaction at 35 degrees with the apparently essential thiols is second order. The rate constant increases with increasing pH, in the range pH 7.0-8.5, in a manner consistent with control of the reaction by a group with pKa of approximately 10. The absolute (pH independent) rate constant is of the same order as that for a normal thiol in model compounds. The availability of the apparently essential thiols appears to be associated with some conformational change in the molecule in the monomer form: it declines at high ionic strengths, is maximal at intermediate values where the dimer first dissociates, but is lowered in the dimer at very low ionic strengths. The reaction also shows a sharp temperature dependence: the dimer at 30 degrees (in constrast to 35 degrees) shows no availability of the apparently essential thiols. A similar transition to a state permitting fast inactivation is found with pH, above pH 8.5. The reaction of the two apparently essential thiols is strongly inhibited by glucose. ATP and ADP, and their Mg complexes, protect significantly, but less effectively than does glucose. The affinities of these substrates at the active site of the enzyme are measured in this protection system. These various reactions appear to be of value for identifying the cysteine-containing regions that are involved in the active center or in its maintenance in the structure.

Essential thiols of yeast hexokinase: alkylation by a substrate-like reagent.

It is demonstrated that N-bromoacetyl-D-galactosamine acts as a substrate-like reagent for yeast hexokinases A and B, producing affinity labeling. At the order of 10(-3) M reagent concentrations, rapid inactivation of the enzyme is produced: the kinetics are consistent with dependence upon a reversible inhibitor-enzyme initial complex, with a dissociation constant of 3.8 x 10(-3) M for hexokinase B at 35 degrees, pH 8.5. The glucose analog is 30-fold less effective, presumably due to self-protection. The inactivating reaction is an order of magnitude faster than that with bromoacetate. All the alkylation of hexokinase B was shown to occur at two thiol groups per subunit, associated stoichiometrically with inactivation. Unlike the reaction there of simple alkylators, two nonessential thiols per subunit are left unattacked when this inactivation reaction is complete. Protection against the affinity alkylation is exerted by the substrates glucose, mannose, fructose, glucose 6-phosphate, fructose 6-phosphate, ATP-Mg, and ADP-Mg, in proportion to their affinities for the active center. Free ATP also protects. Mg2+ alone has no influence, and Mn2+ gives a slight acceleration, when correction is made for a slow inactivation that occurs when the enzyme is incubated at 35 degrees with Mn2+ alone. Galactose, virtually a nonsubstrate, has no influence on the affinity alkylation, but N-acetylgalactosamine, a nonsubstrate and a weak inhibitor of the enzymic reaction, has an accelerating effect. An interpretation is made in terms of binding to a site that influences the active center. This affinity label should provide a means of isolating a peptide containing active-center-related groups.