Bioenergetics of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Cells Grown at Alkaline Conditions

Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4–3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.     

A novel alkaline α-galactosidase gene is involved in rice leaf senescence

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG-Osh69 encoding alkaline α-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H₂O₂ and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal α-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards α-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence. The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Database under the accession number AF251068.     

Design, synthesis and anti-microbial activity of 1H-pyrazole carboxylates

In a SAR study, we have synthesized a few 1H-pyrazole carboxylate related microbicides using Vilsmeier reagent. The anti-microbial screening results of 1H-pyrazole-3-carboxylate are reported here for the first time. The effect of 1H-pyrazole carboxylates on the mycelial growth of plant pathogenic fungi is revealed. The first X-ray structure in the family of microbicidal 1H-pyrazole-4-carboxylates is presented.     

Recombinant alkaline serine protease II degrades scrapie isoform of prion protein

Summary An efficient Escherichia coli expression system for the production of mature-type alkaline serine protease II (mASP II) has been constructed. Complementary deoxyribonucleic acid-encoding mASP II was inserted into the inducible bacterial expression vector pGE-30. After introduction into E, coli, the plasmid was expressed by isopropyl-1-thio-β-d-galactopyranoside, and the recombinant product was purified using a Ni-nitrilotriacetic acid column The purified product had the expected NH₂-terminal sequence and showed a scrapie isoform of prion protein-degrading activity using hamster scrapie 263K prions as a substrate.     

Folding units govern the cytochrome c alkaline transition

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.     

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.     

Synthesis of a radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol: identification of the phosphorylation sites by N-terminal peptide sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol (DTT-S-C[3H(2)]CO(2)H, [3H]CM-DTT), was designed and synthesized at the micromole level by reaction of dithiothreitol with tritiated iodoacetic acid (I-C[3H(2)].CO(2)H). The reaction progress was followed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The usefulness of the synthesized reagent was evaluated in a series of experimental approaches. (i) The synthetic phosphopeptide, NSVS(P)EEGRGDSV, was derivatized by [3H]CM-DTT separated from excess reagent by RP-HPLC. The extent of derivatization was quantitated in terms of the mol of P-Ser/mol of peptide by 3H counting, and the location of the phosphoserine was defined by the N-terminal Edman degradation sequence analysis as being the fourth amino acid residue from the N terminus. (ii) A sample of trypsin-digested alpha-casein was derivatized with [3H]CM-DTT, peptides were separated by RP-HPLC, and aliquots of each fraction were counted for 3H label within the peptide map which rapidly pinpointed the original four phosphoserine-containing peptides. This demonstrated the utility of the synthesized radioactive thiol agent in rapid purification and identification of phosphopeptides from HPLC peptide mapping of proteolytic digests of phosphoproteins. (iii) The [3H]CM-DTT was also used to determine the extent of phosphorylation of phosphoproteins both qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography and quantitatively by 3H counting. The synthesized radioactive thiol reagent [3H]CM-DTT proved to be very efficient and sensitive and should be adaptable to a wide range of routinely utilized laboratory approaches in many fields of the biological sciences.     

Nature and mode of action of NAD and ATP dephosphorylating enzyme from Aspergillus terreus DSM 826

NAD and ATP were dephosphorylated by Aspergillus terreus extracts optimally at pH 8 and 40 °C. The data obtained indicate that one phosphohydrolase was involved in the cleavage of all the phosphate linkages of these two energy-carrying molecules, and also indicate that this enzyme can be classified as a non-specific alkaline phosphatase. This is based on the following criteria: during fractionation of the enzymes of the extracts, using Sephadex G-200 column chromatography, the recorded elution diagram showed only one phosphohydrolase activity peak and this peak was the same with NAD, ATP, inorganic pyrophosphate and phenyl phosphate as substrates; the activity profiles with these four substrates were similar; and these four substrates were hydrolyzed at almost constant relative rates. Moreover, the activities of the pooled fractions with these different substrates responded similarly on changing some experimental conditions, such as addition of fluoride to the reaction mixtures or exposing the enzyme preparation to temperatures above 40 °C. Chromatographic detection of the intermediates and the products formed during the progression of NAD and ATP dephosphorylation by the most purified fraction of this enzyme was found to be consistent with the following mode of its action: This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability

The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.     

Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg(2+) at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K(Mg(app.)) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg(2+)-free and Mg(2+)-containing enzymes by acrylamide showed almost identical dynamic quenching constant (K(sv) = 4.44 +/- 0.09 M(-1)), indicating that there is no gross conformational difference between the M3-free and the M3-Mg(2+) enzymes. However, Zn(2+) was found to have a high affinity with the M3 site (K(Zn(app.)) = 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k(obs)) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3'-Zn) having an extremely high affinity for the Zn(2+) (K*(Zn(app.)) = 0.33 microM). The conformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg(2+) activation and Zn(2+) inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg(2+), has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K(sv) = 3.86 +/- 0.04 M(-1)), suggesting a conformational change. Both Mg(2+) and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.