Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.     

Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium

NADP-isocitrate dehydrogenase [isocitrate:NADP(sup+) oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg(sup2+) is the true substrate. The V(infmax) depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK(infes) (pK of the enzyme-substrate complex) of 6.9 and a (Delta)H(infion) (activation enthalpy) of -2 (plusmn) 0.4 kcal mol(sup-1) (ca. 8 (plusmn) 2 kJ mol(sup-1)). The enzyme showed maximum activity at 60(deg)C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP(sup+) were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40(deg)C following a first-order kinetics. The presence of 12 mM Mg(sup2+) plus 10 mM dl-isocitrate led to 100% protection of enzyme activity against inactivation at 60(deg)C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg(sup2+) was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.     

Natural disturbance and treefalls in a pine-oak forest on the Peninsula of Baja California,Mexico

The magnitude of natural disturbances by treefalls and their spatial occurrence were studied in a pine-oak forest of Sierra de La Laguna, located at the southern part of the Peninsula of Baja California in Mexico.Twenty transects covering 4 ha, perpendicular to north- and south-facing slopes were sampled. The percentages of rocky outcrops, slope, orientation, and gap size created by treefalls were recorded. The mode of tree death, treefall direction, trunk length, and basal diameter were also measured. Data were analyzed using principal component analysis, one-way ANOVA, multiple comparison tests by contrasts, and chi-square independence tests.Results suggest that exogenous disturbances have an important effect in this community. A mean number of 80 gaps per ha was recorded comprising 18.1% of the observed area. Gap sizes were fitted to a lognormal distribution as 2.3±1.4 and patches were found to be created by single treefalls. Analysis of dead material shows that there are significant differences between numbers of fallen trees on N vs S facing slopes, and that forms of dead trees are significantly associated with species. Snapped trees represent 39.5% of treefalls, dead standing trees 26.4%, uprooted trees 20.5% and cut-down trees 0.9%. The frequency of occurrence of various forms of the dead trees suggests that the cause of mortality is primarily due to the high incidence of tropical hurricanes, and secondly to the combined effect of wind and natural fire which occurs during the rainy summer season.     

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.     

Mild protease treatment as a small-scale biochemical method for mitochondria purification and proteomic mapping of cytoplasm-exposed mitochondrial proteins

Because of its importance in basic biology and medicine, great efforts are being devoted to unraveling of the genuine mitochondrial proteome, which is the dynamic protein complement that the organelle uses to maintain its structure and functionality. Several proteomic investigations have now clearly shown that all the purification approaches we have at our disposal suffer from the problem of co-purification; therefore, it is very difficult to distinguish novel mitochondrial proteins from those that are just contaminants of the preparation. The question is further complicated by the fact that the mitochondrial proteome depends on the tissue source. Density gradient centrifugation is the most widespread purification method for obtaining highly pure mitochondrial fractions. The main disadvantage of these methods is the low yield of purified mitochondria that precludes their use in low-scale purifications. Here, we have treated small aliquots of crude mitochondria from mouse liver and from cultured hepatocytes (HEPA1-6) with trypsin under mild proteolysis conditions and have evaluated the suitability of this reaction as a small-scale purification approach. The protease removed several cytoplasmic and endoplasmic reticulum proteins, together with a fraction of mitochondrial proteins that we hypothesize to be associated with the cytosolic face of the outer mitochondrial membrane. The peculiar topology of these mitochondrial proteins could be indicative of their functional roles. Finally, our study represents an application of advanced mass spectrometry technology to the evaluation of biochemical approaches for the treatment of mitochondria.     

Protein carbonylation and adipocyte mitochondrial function

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.     

Solid character of membrane ceramides: a surface rheology study of their mixtures with sphingomyelin

The compression and shear viscoelasticities of egg-ceramide and its mixtures with sphingomyelin were investigated using oscillatory surface rheology performed on Langmuir monolayers. We found high values for the compression and shear moduli for ceramide, compatible with a solid-state membrane, and extremely high surface viscosities when compared to typical fluid lipids. A fluidlike rheological behavior was found for sphingomyelin. Lateral mobilities, measured from particle tracking experiments, were correlated with the monolayer viscosities through the usual hydrodynamic relationships. In conclusion, ceramide increases the solid character of sphingomyelin-based membranes and decreases their fluidity, thus drastically decreasing the lateral mobilities of embedded objects. This mechanical behavior may involve important physiological consequences in biological membranes containing ceramides.     

Induction of intracellular and extracellular beta-galactosidase activity in Phycomyces blakesleeanus

The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose.     

Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.