Characterization of Cold- and High-Pressure-Active Polygalacturonases from a Deep-Sea Yeast,Cryptococcus liquefaciens Strain N6

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0–10 °C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 °C with a very small activation volume of ?1.1 ml/mol. At 10 °C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme–substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme–substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Activation parameters of the blue shift (Shibata shift) subsequent to protochlorophyllide phototransformation

The Shibata shift was analyzed in flash irradiated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates in the pressure range of 0.1 to 500 MPa, at temperatures of 20, 30 and 40 degrees C. The kinetics of the blue shift (called Shibata shift in case of intact leaves) was followed by repeated recording of fluorescence emission spectra after phototransformation. At 20 degrees C, above 100 MPa, the blue shift slowed down remarkably. Two components of the blue shift could be distinguished, one was pressure-dependent and the other was almost pressure-independent. The pressure-independent component can be associated with minor conformational changes of the NADPH:protochlorophyllide oxidoreductase (POR) enzyme, followed by molecular movements of the newly formed chlorophyllide molecules. The calculated activation volume of the pressure-dependent component was 43+/-11 cm(3) mol(-1) at 20 degrees C. This value reflects major molecular reorganizations in the lipid system of the membrane and in the chlorophyllide-protein complexes, and corresponds to changes of the tertiary structure of proteins which can proceed directly or indirectly via structural changes of the membrane lipids. The process was inhibited by 300 and 400 MPa at 30 and 40 degrees C, respectively. The activation volume reduced to 35+/-1.5 cm(3) mol(-1) at 40 degrees C. The decrease of the activation volume with increasing temperature indicates that the blue shift requires loosened lipid structures. The activation energy of the blue shift (measured between 10 and 40 degrees C at atmospheric pressure) was 100+/-20 kJ/mol, indicating that the structural change involves rearrangement of strong molecular interactions.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

Regulation of translation in rabbit reticulocytes and mouse L-cells; comparison of the effects of temperature.

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10 degrees C and 40 degrees C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10-24 degrees C; 22 kcal/mole, 24-40 degrees C), as did protein elongation and release (35 kcal/mole, 10-25 degrees C; 12 kcal/mole, 25-40 degrees C). However, the level of polysomes remained essentially unchanged between 0 degrees C and 42 degrees C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L-cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5-6 amino acids/second at 36 degrees C.     

Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 degrees C and at various pressure values. The activation energy values were 181+/-8 kJ mol(-1) and 188+/-6 kJ mol(-1) for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 degrees C giving values around 100-125 ml mol(-1) which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Purification and characterization of a psychrophilic catalase from Antarctic Bacillus

Catalase from Bacillus sp. N2a (BNC) isolated from Antarctic seawater was purified to homogeneity. BNC has a molecular mass of about 230 kDa and is composed of four identical subunits of 56 kDa. The catalase showed optimal activity at 25 degrees C and at a pH range of 6-11. The enzyme could be inhibited by azide, hydroxylamine, and mercaptoethanol. These characteristics suggested that BNC is a small-subunit monofunctional catalase. The activation energy of BNC was 13 kJ/mol and the apparent kcat/Km values were 3.6 x 10(6) and 4 x 10(6) L.mol(-1).s(-1) at 4 and 25 degrees C, respectively. High catalytic efficiency of BNC at low temperatures enables this bacterium to scavenge H2O2 efficiently. BNC exhibited activation energy, catalytic efficiency, and thermostability comparable with some mesophilic homologues. Such similarity of enzymatic characteristics to mesophilic homologues, although uncommon among the cold-adapted enzymes in general, has also been observed in other psychrophilic small-subunit monofunctional catalases.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Temperature dependence of Kedem-Katchalsky membrane transport coefficients for mature mouse oocytes in the presence of ethylene glycol.

Ethylene glycol (EG) is the emerging cryoprotectant of choice for preservation of mammalian embryos but has not been widely used for oocyte preservation. Techniques for oocyte cryopreservation need to be improved before they can be incorporated into routine clinical practice. Hence the permeability characteristics of oocytes in the presence of EG have been determined in order to facilitate the design of cryopreservation protocols using this cryoprotectant. Individual mouse oocytes were held using negative pressure applied to the zona pellucida by means of a micropipet. Each oocyte was perfused with 1 ml 1.5 mol L(-1) EG at 30, 19, or 10 degrees C, a total of 10 oocytes being perfused at each temperature. The osmotic response of each oocyte before, during and after perfusion was recorded on videotape. Measurements of mean cell diameter across three axes were used to calculate oocyte volume, assuming them to be spherical, and, using mathematical modeling, values for hydraulic conductivity (L(p)) were found to be 0.91 +/- 0.05, 0.51 +/- 0.02, and 0.18 +/- 0.01 microm min(-1) atm(-1); cryoprotectant permeability (P(EG)) was 0.24 +/- 0.01, 0.09 +/- 0.005, and 0.03 +/- 0.004 microm s(-1); and reflection coefficient (sigma) was 0.98 +/- 0.005, 0.96 +/- 0.01, and 0.97 +/- 0.01 at 30, 19, and 10 degrees C, respectively. The activation energy (E(a)) of L(p) was 14. 0 kCal mol(-1) and of P(EG) was 16.4 kCal mol(-1).     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

Functional reconstitution of the integral membrane enzyme, isoprenylcysteine carboxyl methyltransferase, in synthetic bolalipid membrane vesicles

Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (also known as bolalipids) have been prepared to determine (1) the influence of molecular structure on the physical properties of bolalipid membranes and (2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. Three bolalipids were synthesized: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 degrees C; C32phytBAS Tm = 14 degrees C; and C20BAS Tm = 17 degrees C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 25 degrees C, were 1.5 x 10(-8) and 1.8 x 10(-9) cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS/E. coli polar lipid, C20BAS/POPC, C32phytBAS/E. coli polar lipid, and C32phytBAS/POPC. Ste14p activity was lost in vesicles composed of 75-100 mol % C20BAS and 0-100 mol % C32BAS but retained in vesicles with 0-50 mol % C20BAS and 0-100 mol % C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol % C20BAS and 100 mol % C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 A. Because Ste14p activity was not affected by either the gel-to-liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75-100 mol % C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.     

Nitrogen removal performance using anaerobic ammonium oxidation at low temperatures

An anaerobic ammonium oxidation (anammox) process for ammonia-rich wastewater treatment has not been reported at temperatures below 15 degrees C. This study used a gel carrier with entrapped anammox bacteria to obtain a stable nitrogen removal performance at low temperatures. In a continuous feeding test, a high nitrogen conversion rate (6.2 kg N m(-3) day(-1)) was confirmed at 32 degrees C. Nitrogen removal activity decreased gradually with decreasing operation temperature; however, it still occurred at 6 degrees C. Nitrogen conversion rates at 22 and 6.3 degrees C were 2.8 and 0.36 kg N m(-3) day(-1), respectively. Moreover, the stability of anammox activity below 20 degrees C was confirmed for more than 130 days. In batch experiments, anammox gel carriers were characterized with respect to temperature. The optimum temperature for anammox bacteria was found to be 37 degrees C. Furthermore, it was clear that the temperature dependence changed at about 28 degrees C. The apparent activation energy in the temperature range from 22 to 28 degrees C was calculated as 93 kJ mol(-1), and that in the range from 28 to 37 degrees C was 33 kJ mol(-1). This value agrees with the result of a continuous feeding test (94 kJ mol(-1), between 6 and 22 degrees C). The nitrogen removal performance demonstrated at the low temperatures used in this study will open the door for the application of anammox processes to many types of industrial wastewater treatment.     

Spontaneous lipid peroxidation in rabbit and mouse epididymal spermatozoa: dependence of rate on temperature and oxygen concentration

The rate of spontaneous lipid peroxidation, as measured by formation of malonaldehyde (MA), was determined as a function of O2 concentration and temperature in mouse and rabbit spermatozoa released from the cauda epididymidis. The peroxidation rate was linear in O2 concentration in the suspending medium up to 210 microM (the concentration at PO2 of ambient air at 34 degrees C) for sperm from both species over the temperature range 34-40 degrees C. This is the range over which the reaction is measurable for both species: below 34 degrees C, the rates become too slow to be measured accurately for rabbit sperm by our methods, while above 40 degrees C the rates for mouse sperm become too rapid. This narrow range is characteristic of a high activation energy (EA) for the peroxidation process. Values of EA were calculated from plots of kox versus (T)-1, where kox is a second order rate constant with the units (10(8) cells/ml)-1 min-1. It is defined by the equation: vma = kox (Sp) (O2), where vma is the rate of malonaldehyde production, (Sp) is concentration of sperm cells and (O2) is the O2 concentration in the suspending medium. For mouse sperm, EA was calculated to 78.7 kcal/mol (329 KJ/mol); for rabbit sperm, the value was 77.6 kcal/ml (324 KJ/mol). These high EAs and consequent steep dependence of the spontaneous lipid peroxidation rates on temperature favor long sperm life in the epididymis at around 32 degrees C and low PO2 in these scrotal animals, while allowing for a relatively short life at 37 degrees C at higher PO2 in the oviduct.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

Reaction of N-acetylglucosamine oligosaccharides with lysozyme. Temperature, pH, and solvent deuterium isotope effects; equilbrium, steady state, and pre-steady state measurements*.

Temperature jump and stopped flow methods were used to study at pH 7.0 the temperature dependence of elementary steps of the reactions of lysozyme with the beta(1 yields 4)-linked trimer, tetramer, and hexamer of N-acetylglucosamine. The steady state rate of cleavage of the hexasaccharide was determined as a function of temperature (5 degrees-40 degrees) and pH(2 to 8) in H-2O solution and as a function of pD(2.5 to 9.5) at 40 degrees in D-2O solution. The apparent enthalpies of the two ionizations of apparent pK 3.8 and 6.7 observed in measurements of k are 0 to 2 kcal/mol. The energy of activation determined for the pH optimum is 21.5 kcal/mol. The solvent deuterium isotope effect measured for k at the pH (pD) optimum is 1.5 And reflects isotope effects on pre-equilibrium steps and on the rate-determining step. Transfer from H-2O to D-2O solution produces 0.2 to 0.4 kcal/mol more negative free energies of saccharide binding and no changes in the enthalpies of binding. Pre-steady state, steady state, and equilibrium measurements indicate a pathway for the reaction of lysozyme with hexasaccharide. The results define for this mechanism the complete free energy profile and an essentially complete enthalpy profile. Three of the five observable ES complexes are present at nearly equal concentrations. The free energies of the transition states are within a range of 3 kcal. The enthalpies of productive enzyme-substrate complexes are about 5 kcal/mol greater than the enthalpies of nonproductive complexes. Changes in tryptophan fluorescence were observed for each elementary step, and changes in pK of Glu-35 for the isomerizations of nonproductive and productive complexes. The signal changes during formation of nonproductive complexes are the same for the oligosaccharides (ClcNAc)3 to (GlcNAc)6. The changes for productive complexes are similar but not identical with saccharides (GlcNAc)4 to (GlcNAc)6. Correlations of the present data with previous crystallographic and solution measurements indicate the structures of productive and nonproductive ES complexes and suggest that full interaction of the substrate with the enzyme active site is established in the rate-determining step.