Studies on the synthesis of cytochrome P-450 and cytochrome P-448 in rat liver

The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat germ cell-free system using anti- cytochrome P-450 and anti-cytochrome P-448 antibodies. The major mature forms synthesized in vivo correspond to a molecular weight of 47,000 for cytochrome P-450 and 53,000 for cytochrome P-448. Translation of poly(A)-containing RNA from phenobarbital-treated rats in the wheat germ cell-free system reveals that the cell-free product immunoprecipitated with anti-cytochrome P-450 antibody has a molecular weight close to 47,000. In the case of 3-methylcholanthrene, the cell- free product immunoprecipitated with anti-cytochrome P-448 antibody shows a molecular weight around 59,000. Significant conversion of the 59,000 species to the 53,000 species can be demonstrated when the translation is carried out in the presence of microsomal membranes isolated from rat liver. Phenobarbital and 3-methylcholanthrene enhance the translatable messenger.

oxidation of catechol in plants iv. purification and properties of the 3,4,3',4'-tetrahydroxydiphenyl-forming enzyme system from tecoma leaves

Acetone powders prepared from leaf extracts of Tecoma stans L. were found to catalyze the oxidation of catechol to 3,4,3',4'-tetrahydroxydiphenyl. Fractionation of the acetone powders obtained from Tecoma leaves with acetone, negative adsorption of the acetone fraction with tricalcium phosphate gel, and chromatography of the gel supernatant on DEAE-Sephadex yielded a 68-fold purified enzyme with 66% recovery. The enzyme had an optimum pH around 7.2. It showed a temperature optimum of 30° and the Km for catechol was determined as 2 x 10-4 m. The purified enzyme moved as a single band on polyacrylamide gel electrophoresis. Its activity was found to be partially stimulated by Mg2+. The reaction was not inhibited by o-phenanthroline and agr,agr'-dipyridyl. The purified enzyme was highly insensitive to a range of copper-chelating agents. It was not affected appreciably by thiol inhibitors. The reaction was found to be suppressed to a considerable extent by reducing agents like GSH, cysteine, cysteamine, and ascorbic acid. The purified enzyme was remarkably specific for catechol. Catalase affected neither the enzyme activity nor the time course of the reaction. Hydrogen peroxide was not formed as a product of the reaction.

Studies on nucleotidases in plants - Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect. The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The Km (0.2 m ) and V (3.2) of the native enzyme increased on renaturation to 1.8 m and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn2+.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe2+ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.

Studies on nucleotidases in plants Dimerization of the crystalline mung bean nucleotide pyrophosphatase by 5-adenosine monophosphate and the properties of the dimerized enzyme

The addition of AMP to the crystalline and homogeneous mung bean nucleotide pyrophosphatase [EC 3.6.1.9]altered its electrophoretic mobility. AMP was tightly bound to the enzyme and was not removed on passage through a column of Sephadex G-25 or on electrophoresis. The molecular weight of the native and AMP-modified enzymes were 65,000 and 136,000, respectively. The properties of the native enzyme such as the pH (9.4) and temperature (49 °C) optima, inhibition by EDTA, reversal of EDTA-inhibition by Zn2+ and Co2+, were not altered on dimerization by AMP. The AMP-modified enzyme had a linear time-course of reaction, unlike the native enzyme which exhibited a biphasic time-course of reaction. The AMP-modified enzyme was irreversibly denatured by urea. AMP concentrations larger than 100 μM inhibited linearly the activity of the AMP-modified enzyme. ADP and ATP inhibited the activity in a sigmoidal manner. Km and V of the native and AMP-modified enzymes were, 0.25 mImage and 0.58 mImage ; and 3.3 and 2.5, respectively.

Purification and properties of Image -mandelate-4-hydroxylase from Pseudomonas convexa

An inducible Image -mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of Image -mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe2+, and O2 for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with Image -mandelate, NADPH, and ferrous sulfate and Km values for these substrates were found to be 1 × 10−4, 1.9 × 10−4, and 4.7 × 10−5 Image , respectively. The enzyme is very specific for Image -mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.

Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of −181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.

Polyvanadate-stimulated NADH oxidation by plasma membranes-The need for a mixture of deca and meta forms of vanadate

Polyvanadate solutions obtained by extracting vanadium pentoxide with dilute alkali over a period of several hours contained increasing amounts of decavanadate as characterized by NMR and ir spectra. Those solutions having a metavanadate:decavanadate ratio in the range of 1-5 showed maximum stimulation of NADH oxidation by rat liver plasma membranes. Reduction of decavanadate, but not metavanadate, was obtained only in the presence of the plasma membrane enzyme system. High simulation of activity of NADH oxidation was obtained with a mixture of the two forms of vanadate and this further increased on lowering the pH. Addition of increasing concentrations of decavanadate to metavanadate and vice versa increased the stimulatory activity, reaching a maximum when the metavanadate:decavanadate ratio was in the range of 1-5. Increased stimulatory activity can also be obtained by reaching these ratios by conversion of decavanadate to metavanadate by alkaline phosphate degradation, and of metavanadate to decavanadate by acidification. These studies show for the first time that both deca and meta forms of vanadate present in polyvanadate solutions are needed for maximum activity of NADH oxidation.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Chemistry and biology of DNA methyltransferases

Recognition of a specific DNA sequence by a protein is probably the best example of macromolecular interactions leading to various events. It is a prerequisite to understanding the basis of protein-DNA interactions to obtain a better insight into fundamental processes such as transcription, replication, repair, and recombination. DNA methyltransferases with varying sequence specificities provide an excellent model system for understanding the molecular mechanism of specific DNA recognition. Sequence comparison of cloned genes, along with mutational analyses and recent crystallographic studies, have clearly defined the functions of various conserved motifs. These enzymes access their target base in an elegant manner by flipping it out of the DNA double helix. The drastic protein-induced DNA distortion, first reported for HhaI DNA methyltransferase, appears to be a common mechanism employed by various proteins that need to act on bases. A remarkable feature of the catalytic mechanism of DNA (cytosine-5) methyltransferases is the ability of these enzymes to induce deamination of the target cytosine in the absence of S-adenosyl-L-methionine or its analogs. The enzyme-catalyzed deamination reaction is postulated to be the major cause of mutational hotspots at CpG islands responsible for various human genetic disorders. Methylation of adenine residues in Escherichia coli is known to regulate various processes such as transcription, replication, repair, recombination, transposition, and phage packaging.

Thermodynamics of Monosaccharide and Disaccharide Binding to Erythrina corallodendron Lectin

Isothermal titration calorimetry measurements of the binding of 2′-fucosyllactose, lactose, N-acetyllactosamine, galactopyranose, 2-acetamido-2-deoxygalactopyranoside, methyl α-N-dansylgalactosaminide (Me-α-DNS-GalN), methyl α-D-galactopyranoside, methyl β-D-galactopyranoside, and fucose to Erythrina corallodendron lectin (ECorL), a dimer with one binding site per subunit, were performed at 283-286 and 297-299 K. The site binding enthalpies, ΔHb, with the exception of Me-α-DNS-GalN, are the same at both temperatures and range from −47.1 ± 1.0 kJ mol−1 for N-acetyllactosamine to −4.4 ± 0.3 kJ mol−1 for fucose, and the site binding constants range from 3.82 ± 0.9 × 105 M−1 for Me-α-DNS-GalN at 283.2 K to 0.46 ± 0.05 × 103 M−1 for fucose at 297.2 K. The binding reactions are mainly enthalpically driven except for fucose and exhibit enthalpy-entropy compensation. The binding enthalpies of the disaccharides are about twice the binding enthalpies of the monosaccharides in contrast to concanavalin A where the binding enthalpies do not double for the disaccharides. Differential scanning calorimetry measurements show that denaturation of the ECorL dimer results in dissociation into its monomer subunits. The binding constants from the increase in denaturation temperature of ECorL in the presence of saccharides are in agreement with values from isothermal titration calorimetry results. The thermal denaturation of ECorL occurs around 333 K, well below the 344-360 K denaturation temperature of other legume lectins of similar size and tertiary structure, undoubtedly due to the difference in its quaternary structure relative to other legume lectins. This is also apparent from the independent unfolding of its two domains.

Involvement of Synthesis and Phosphorylation of Nuclear Protein Factors That Bind to the Positivecis-Acting Element in the Transcriptional Activation of the CYP2B1/B2 Gene by Phenobarbitonein Vivo

The synthesis and phosphorylation of protein factor(s) that bind to the positivecis-acting element (−69 to −98 nt) of the CYP2B1/B2 gene have been examinedin vivoin the rat. Treatment of rats with cycloheximide, a protein synthetic inhibitor, suppresses basal as well as phenobarbitone-induced levels of CYP2B1/B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged by gel shift assays. Treatment of rats with 2-aminopurine, a general protein kinase inhibitor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 mRNA, cell-free transcription of a minigene construct containing the positive element, pP450e179DNA, and binding of nuclear proteins to the positive element. Treatment of rats with okadaic acid, a protein phosphatase inhibitor, mimics the effects of phenobarbitone, but only partially. Thus, both phenobarbitone and okadaic acid individually enhance binding of the nuclear protein(s) to the positive element, cell-free transcription of the minigene construct, and phosphorylation of the not, vert, similar26- and 94-kDa proteins binding to the positive element. But unlike phenobarbitone, okadaic acid is not an inducer of CYP2B1/B2 mRNA or its run-on transcription. Thus, phenobarbitone-responsive positive element interactions constitute only a minimal requirement, and okadaic acid is perhaps not able to bring about the total requirement for activation of CYP2B1/B2 gene transcription that should include interaction between the minimal promoter and further upstream elements. An intriguing feature is the antagonistic effect of okadaic acid on phenobarbitone-mediated effects on CYP2B1/B2 mRNA levels, cell-free and run-on transcription, and nuclear protein binding to the positive element. The reason for this antagonism is not clear. It is concluded that phenobarbitone treatment enhancesin vivothe synthesis and phosphorylation of protein factors binding to the positive element and these constitute a minimal requirement for the transcriptional activation of the CYP2B1/B2 gene.

Mapping the common antigenic determinants in avidin and streptavidin

An epitope scan analysis of the whole sequence of avidin and core streptavidin using polyclonal antibodies to these two antigens reveal the presence of multiple common epitopes in both the proteins. These antigenic determinants consist mostly of either identical or similar residues. The antibody recognition sites in both antigens are shown to be localized to homologous regions.

A Novel Invertase from a Thermophilic FungusThermomyces lanuginosus:Its Requirement of Thiol and Protein for Activation

Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungus,Thermomyces lanuginosus,was unusually unstable bothin vivoandin vitro.The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b)N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction. In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds. Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation. A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called “PRIA” for theprotein which helps inrestoringinvertaseactivity. The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form. A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations. The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source. The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry.