Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Further properties of the polyamine oxidase from oat seedlings

After purification, the polyamine oxidase from the leaves of oat seedlings grown in the dark appeared to be homogeneous on electrophoresis. The MW determined by density gradient centrifugation was 119 000. The enzyme would not oxidise diaminodipropylamine and neither diaminodipropylamine nor diaminopropane were inhibitors at concentrations up to 1 mM. With spermidine as substrate, the energy of activation was 19.7 kJ/mol and activity was reduced to 50% on heating for 10 min at 50°. With spermine as substrate, activity was increased up to 3-fold in the presence of M sodium chloride. This stimulation was not observed with spermidine as substrate The enzyme was also stimulated by sodium phosphate and sodium citrate at high concentrations. The pH for optimal stability was 6.5, the same as the pH for maximum activity with both spermidine and spermine as substrates. For spermidine and spermine the K_ms were 8 × 10 ^?6 M and 2 × 10 ^?6 M respectively. Loss of activity on storage of leaves at ? 15° was ca 5 % per week and in extracts the loss was ca 10 % per week.     

Apparent differences in tryptophan hydroxylation by cockroach (periplaneta americana) nervous tissue and rat brain

Tryptophan hydroxylation in cockroach (Periplaneta americana) nervous tissue was measured and compared to the hydroxylation of tryptophan in rat brain. Tryptophan hydroxylation in both tissues requires a pterine cofactor, and is inhibited by p-chlorophenylalanine. The molecular weight of the protein responsible for hydroxylation of tryptophan in cockroach nervous tissue obtained from gel filtration was estimated to be 54,000.The pH optima and enzyme kinetics differed greatly between the two hydroxylases. Hydroxylation of tryptophan by the enzyme obtained from cockroach tissues incubated with dimethyltetrahydropterine had a pH optimum of about 5.8–5.9 and a K_m in crude enzyme preparations of 2.6 × 10⁻⁶ M and is activity was substrate inhibited above 10⁻⁴ M tryptophan. Hydroxylation of tryptophan by the enzyme obtained from rat brain incubated with dimethyltetrahydropterine had a pH optimum of about 6.5–7.0, a K_m of about 6.7 × 10⁻⁴ M and exhibited no substrate inhibition at tryptophan concentrations up to 2 × 10⁻³ M.When incubated with biopterin, the presumed natural cofactor, the hydroxylase from cockroach tissues had a K_m of about 6.8 × 10⁻⁵ M and no substrate inhibition occurred at tryptophan concentrations up to 2 × 10⁻³ M. Under the same conditions rat hydroxylase had a K_m of 1.1 × 10⁻⁵M and substrate inhibition occurred above 10⁻⁴ M tryptophan.Unlike the mammalian situation, administration of tryptophan peripherally did not change the 5-hydroxytryptamine concentration in cockroach nervous tissue, but did increase tryptophan levels. The low V_max values of the cockroach hydroxylase and the inability of administered tryptophan to elevate 5-hydroxytryptamine levels suggest that in the cockroach hydroxylation of tryptophan itself may be the limiting factor in the biosynthesis of 5-hydroxytryptamine.     

Spectral characteristics of human fetal adrenal microsomes.

Investigations of human fetal adrenal gland microsomes indicated that a carbon monoxide binding pigment had an absorption maximum of 446 to 448 nm. This pigment, upon heat treatment at 37°C was degraded to the form of cytochrome p-420. NADPH reduced cytochrome p-450 slowly and completely. Typical concentrations of 0.75 and 0.16 nmoles/mg protein cytochrome P-450 and b₅, respectively, were observed. Reduced ethylisocyanide spectra were similar to those of rat hepatic microsomes with absorption maxima at 430 as well as 454 nm. Typical type I spectral changes were observed with progesterone, 17-α-OH-progesterone, pregnenolone and androstenedione when these steroids were added to the sample cuvettes. Androstenedione exhibited an apparent spectral dissociation constant (K_S) of 5×10⁻⁶M pregnenolone and progesterone exhibited higher affinities with apparent dissociation constants of 1.1×10⁻⁷M and 1.8×10⁻⁷M, respectively. The maximal absorbance change induced by androstenedione was lower (Emax = 0.027 per mg protien) than the changes in absorbance maxima induced by pregnenolone or progesterone (Emax = 0.060 and 0.047 per mg protein, respectively) when saturating concentrations of these steroids were added to the sample cuvettes. Ethylmorphine and aminopyrine (10⁻³M final concentrations) did not exhibit observable spectral changes; however, type II spectra could be elicited with aniline and nicotinamide and apparent dissociation constants of 3.5×10⁻²M and 2.5×10⁻²M, respectively, were obtained.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Partial purification of nitrilase from Chinese cabbage

Nitrilase was purified ca 28-fold from Chinese cabbage seedlings. K_m values of 5.2 × 10^?4 and 2.6 × 10^?3 M were obtained for indoleacetonitrile (IAN) and 3-cyanopyridine (3-CP) as substrates. For hydrolysis of 3-CP, the maximal velocity was 44 times higher than for the natural substrate IAN. The pH optimum is at 7.5. IAA concentrations from 10^?6 to 10^?3 M did not inhibit the partially purified enzyme. Nitrilase activity was investigated during development of seedlings grown under continuous light. Roots with hypocotyls exhibited only slightly lower activity than cotyledons based on fresh weight, although their specific activity was ca 5 times higher. Etiolated seedlings showed a very similar distribution of nitrilase activity. The significance of the results for IAA biosynthesis is discussed.     

Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

Purification and properties of d-xylulose reductase from Pachysolen tannophilus

d-Xylulose reductase (EC 1.1.1.9) from Pachysolen tannophilus IFO 1007 was purified by Sephadex G-100 gel chromatography with three columns and DEAE cellulose chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. It was most active at pH 9.1–10.0 and 55°C, and stable at pH 7–9 and below 25 °C. Its activity was stimulated by NH₄Cl,NaCl,MgCl₂,KCl, glutathione, cysteine and glycine, and inhibited remarkably by SH inhibitor such as lead acetate, HgCl₂ and AgNO₃. It oxidized xylitol, sorbitol, ribitol and glycerine but not mannitol, inositol, arabitol and erythritol. Its K_m values of enzyme against xylitol, sorbitol and ribitol were 1.1 × 10⁻² M, 3.0 × 10⁻² M and 5.0 × 10⁻² M, respectively. Its molecular weight was determined to be 120,000 by Sephadex G-200 column chromatography, and that of its subunit was 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

Exploitation of micellar medium for photochemical-spectrofluorimetric flow-injection determination of fenvalerate

A photochemical flow-injection methodology for the fluorimetric determination of fenvalerate, a nonfluorescent pyrethroid pesticide, is proposed. The sample was irradiated by ultraviolet light inside a reaction coil in a nonstop mode and the photodegradation products were monitored by spectrofluorimetry at λ_exc=277 nm, λ_em=329 nm. The exploitation of a micellar medium resulted in a pronounced fluorescence enhancement with a concomitant tenfold sensitivity improvement. Several surfactants were evaluated, and best results were obtained with sodium dodecylsulphate (SDS). The calibration plot was linear within 2.0×10⁻⁶ and 1.0×10⁻⁵ mol l⁻¹ fenvalerate. The proposed system is very stable, handles 30 samples h⁻¹ and requires only 60 μmol SDS per determination. The detection limit is 5×10⁻⁷ mol l⁻¹. The method was evaluated in the determination of fenvalerate in tap water.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

Novel nonclassical inhibitors of glycinamide ribonucleotide formyltransferase: 10-Formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid

Several new 10-formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid have been synthesized by a novel and convenient enamine alkylation procedure. Two of these compounds (10a and 10b) were shown to be very powerful inhibitors of L. casei (10a, IC₅₀ = 8 × 10⁻⁶ M ; 10b, IC₅₀ = 7 × 10⁻⁶ M ) and recombinant mouse (10a, IC₅₀ = 3.4 × 10⁻⁵ M ; 10b, IC₅₀ = 2.8 × 10⁻⁵ M ) glycinamide ribonucleotide formyltransferase (GARFT). These IC₅₀ values are comparable to the classical GARFT inhibitor (6R)-DDATHF (IC₅₀, L. casei 2.3 × 10⁻⁶M ; recombinant mouse 2.3 × 10⁻⁵ M ) under identical assay conditions. For both compounds, the inhibition of L. casei GARFT increased with time of incubation, but not markedly with the recombinant mouse enzyme. Due to their potential ability to interfere with purine biosynthesis and to penetrate microbial cells the new nonclassical GARFT inhibitors reported here may be useful for the treatment of infections caused by microorganisms that are sensitive and resistant to conventional antimicrobial agents.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

A comparison of the 8-hydroxydeoxyguanosine,chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes

We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxydeoxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH₃HgCl) and mercuric chloride (HgCl₂) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH₃HgCl or HgCl₂ increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 × 10⁻⁵ M (HgCl₂) or 2 × 10⁻⁶ M (CH₃HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. CH₃HgCl was about 4-fold more potent than HgCl₂. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH₃HgCl at a concentration that was about 50% of the amount of HgCl₂ required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl₂- and/or CH₃HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.     

Immobilization of β-glucosidase from Scytalidium lignicola on Chitosan

Chitosan was found to be a better support than alginate beads for immobilization of β-glucosidase from Scytalidium lignicola. The optimum concentration of glutaraldehyde for enzyme immobilization was 0.2%. Immobolized β-glucosidase was more able in the pH range of 3–6. Immobilized β-glucosidase retained about 70% of its activity at 50%C after 72 h of incubation while free enzyme lost most of its activity. The log of activity retained vs time was a straight line with free enzyme but was curved for immnobilized enzyme. Lineweaver-Burk plots of free and immoblized β-glucosidase gave K_m values of 2 × 10⁻⁴ M and 5.5 × 10⁻⁴ M for p-nitrophenyl β-d-glucopyranoside, respectively. Addition of immobilized β-glucosidase to a saccharification system gave a 30% increase in reducing sugar availability compared to free enzyme addition and was at least 4 times reusable without appreciable loss in enzyme activity.     

Chlorpromazine increases sodium permeability across the isolated toad skin

The effects of Chlorpromazine (Cpz) on the potential difference (PD), short-circuit current (SCC), and total conductance (G_T) on Pleurodema thaul skin and on the skin response to dopamine were analysed. Cpz applied to the serosal surface in concentrations ranging from 1.25 × 10⁻⁵ to 1.25 × 10⁻⁴ M significantly increased the PD, the SCC and the GT. The effect of Cpz was abolished by BaCl₂ but not by alpha or beta adrenergic receptor antagonists. Cpz decreased the skin response to noradrenaline and to angiotensin 11. Dopamine (5 × 10⁻⁷ M to 5 × 10⁻⁶M) also induced a significant increase in the PD, SCC and G_T. This response was antagonized by propranolol but not by dibenamine. Additive effects of dopamine and Cpz were also found. The amiloride test showed that Cpz decreased E_Na., the driving force of sodium and increased g_na, which represents active sodium conductance. These results are consistent with the hypothesis that Cpz increases transport across the isolated toad skin by increasing mucosal and serosal permeability. The results also suggest that Cpz decreases membrane cabnodulin availability.