In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

Mutations in the Res subunit of the EcoPI restriction enzyme that affect ATP-dependent reactions

The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes. (C) 1997 Academic Press Limited.

Barriers to plantation activities in different agro-ecological zones of Southern India

This paper analyses environmental and socio-economic barriers for plantation activities on local and regional level and investigates the potential for carbon finance to stimulate the increased rates of forest plantation on wasteland, i.e., degraded lands, in southern India. Building on multidisciplinary field work and results from the model GCOMAP, the aim is to (1) identify and characterize the barriers to plantation activities in four agro-ecological zones in the state of Karnataka and (2) investigate what would be required to overcome these barriers and enhance the plantation rate and productivity. The results show that a rehabilitation of the wasteland based on plantation activities is not only possible but also anticipated by the local population and would lead to positive environmental and socio-economic effects at a local level. However, in many cases, the establishment of plantation activities is hindered by a lack of financial resources, low land productivity and water scarcity. Based on the model used and the results from the field work, it can be concluded that certified emission reductions such as carbon credits or other compensatory systems may help to overcome the financial barrier; however, the price needs to be significantly increased if these measures are to have any large-scale impact.

Growth inhibitory, apoptotic and anti-inflammatory activities displayed by a novel modified triterpenoid, cyano enone of methyl boswellates

Triterpenoids are pentacyclic secondary metabolites present in many terrestrial plants. Natural triterpenoids have been reported to exhibit anti-inflammatory and anti-carcinogenic activities. Here, we show that modifications of ring A of boswellic acid (2 cyano, 3 enone) resulted in a highly active growth inhibitory, anti-inflammatory, pro-differentiative and anti-tumour triterpenoid compound called cyano enone of methyl boswellates (CEMB). This compound showed cytotoxic activity on a number of cancer cell lines with IC50 ranging from 0.2 to 0.6 mu M. CEMB inhibits DNA synthesis and induces apoptosis in A549 cell line at 0.25 mu M and 1 mu M concentrations, respectively. CEMB induces adipogenic differentiation in 3T3-L1 cells at a concentration of 0.1 mu M. Finally, administration of CEMB intra-tumourally significantly inhibited the growth of C6 glioma tumour xenograft in immuno-compromised mice. Collectively, these results suggest that CEMB is a very potent anti-tumour compound.

Thermodynamic analysis of enzyme catalysed reactions: new insights into the Michaelis-Menten equation

A simple thermodynamic analysis of the well-known Michaelis-Menten equation (MME) of enzyme catalysis is proposed that employs the chemical potential mu to follow the Gibbs free energy changes attending the formation of the enzyme-substrate complex and its turnover to the product. The main conclusion from the above analysis is that low values of the Michaelis constant KM and high values of the turnover number k(cat) are advantageous: this supports a simple algebraic analysis of the MME, although at variance with current thinking. Available data apparently support the above findings. It is argued that transition state stabilisation - rather than substrate distortion or proximity - is the key to enzyme catalysis.

Affinity-Chromatographic Procedure For The Purification Of The Enzyme From Mung-Bean (Phaseolus, Aureus) Seeds And Conformational-Changes On Its Interaction With Ortho-Phosphate

Flavokinase was purified, for the first time from a plant source [mung bean (Phaseolus aureus)] by affinity chromatography in the presence of orthophosphate and by using C-8 ATP-agarose (ATP linked through the C-8 position to beaded agarose), Cibacron Blue and riboflavin--Sepharoses. An altered substrates-saturation pattern was observed in the presence of K2HPO4. The conformational changes of the enzyme in the presence of K2HPO4 were monitored by fluorescence spectroscopy. These results highlight the regulatory nature of this enzyme.

Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and non-hydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.

Structural characterization of angiotensin I-converting enzyme in complex with a selenium analogue of captopril

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin-angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356-1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 angstrom resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein-selenolate interaction. These new structures of tACE-SeCap and AnCE-SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.

Angiotensin converting enzyme inhibitors in the treatment of hypertension

Angiotensin converting enzyme (ACE) catalyses the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). The ACE activity directly related to hypertension as Ang II is the blood pressure regulating hormone. Therefore, ACE inhibitors are a major class of antihypertensive drugs. Captopril, chemical name, was the first orally active ACE inhibitory antihypertensive drug, discovered in 1977. Since then, a number of such drugs have been synthesized. Enzyme-inhibitor bound crystal structural studies reveal a great deal of understanding about the interactions of the inhibitors at the active site of ACE. This can be helpful in the rational design of ACE inhibitors. With the advancement of the combination therapy, it is known that ACE inhibitors having antioxidant activity can be beneficial for the treatment of hypertension. This study describes the development of ACE inhibitors in the treatment of hypertension. Importance of ACE inhibitors having antioxidant activity is also described.

Activities of oxygen and lead in Pb-Ag O solutions

The reversible e.m.f. of galvanic cells: stainlesssteel,Ir,Pb+PbO|CaO+ZrO2|Ag+Pb+PbO,Ir,stainlesssteel,I and Pt,Ni+NiO|CaO+ZrO2|O(Pb+Ag),Cermet,Pt,II incorporating solid oxide electrolytes were measured as a function of alloy composition. In lead-rich alloys, the temperature dependence of the e.m.f. of cell I was also investigated. Since the solubility of oxygen in the alloy is small, the relative partial molar properties of lead in the binary Ag + Pb system can be calculated from the e.m.f. of this cell. The Gibbs free energies obtained in this study are combined with selected calorimetric data to provide a complete thermodynamic discription of liquid Ag + Pb Alloys. The activity coefficient of oxygen in the whole range of Ag + Pb alloys at 1273 K have been obtained from the e.m.f. of cell II; and these are found to deviate positively from Alcock and Richardson's quasichemical equation when the average co-ordination number of all the atoms is assigned a value of 2.

Prediction of activities of oxygen in dilute quaternary solutions using binary data

Equations are developed for predicting the activity coefficients of oxygen dissolved in ternary liquid alloys. These are extensions of earlier treatments, and are based on a model in which each oxygen atom is assumed to make four bonds with neighboring metal atoms. It is also postulated that the strong oxygen-metal bonds distort the electronic configuration around the metal atoms bonded to oxygen, and that the quantitative reduction of the strength of bonds made by these atoms with all of the adjacent metal atoms is equivalent to a factor of approximately two. The predictions of the quasichemical equation which is derived agree satisfactorily with the partial molar free energies of oxygen in Ag-Cu-Sn solutions at 1200°C reported in literature. An extension of this treatment to multicomponent solutions is also indicated.

An improved method for calculating activities from distribution equilibria

The method of Gibbs-Duhem integration suggested by Speiser et al. has been modified to derive activities from distribution equilibria. It is shown that, in general, the activities of components in melts with a common anion can be calculated, without using their standard Gibbs energies of formation, from eqUilibrium ratios and the knowledge of activities in the metal phase. Moreover, if systems are so chosen that the concentration of one element in the metal phase lies in the Henry's law region (less than 1 %), information on activities in the metal phase is not required. Conversely, activities of elements in an alloy can be readily calculated from equilibrium distribution ratios alone, if the salt phase in equilibrium contains very small amounts of one element. Application of the method is illustrated using distribution ratios from the literature on AgCI-CuCI, AgBr-CuBr, and CuDo.5 -PbD systems. The results indicate that covalent bonding and van der Waals repulsive interactions in certain types of fused salt melts can significantly affect the thermodynamic properties of mixing.

Activities of indium in solid solutions of Cu+In, Au+In and Cu+Au+In alloys

Thin foils of Cu, Au and Cu + Au alloys embedded in indium sesquioxide were equilibrated with controlled streams of CO-CO2 mixtures. The equilibrium concentrations of indium in the foils were determined by neutron activation analysis. The corresponding chemical potentials of indium were calculated from the standard free energies of formation of carbon monoxide, carbon dioxide, and indium oxide. It was found that the size difference between the solute and the solvent does not make significant contributions to the solute—solute interaction energy in the α-phase. The chemical potential of indium at one at.% concentration is 8.6 Kcals more negative in gold than in copper at 900°K. The variation of this chemical potential with alloy composition in Cu + Au system was in good agreement with Alcock and Richardson's quasichemical equation. The agreement is strengthened by the accurate knowledge of the co-ordination number in these substitutional solid solutions from X-ray diffraction studies.