Calorimetric determination of inactivation parameters of micro-organisms

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Evaluation of the heat inactivation of Escherichia coli and Lactobacillus plantarum by differential scanning calorimetry

Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and reversibility after heat treatment of transitions associated with various cellular components of Escherichia coli and Lactobacillus plantarum. The reversibility and the change in the thermal stability of individual transitions are evaluated by a second temperature scan after preheating in the DSC to various temperatures between 40 and 130 degrees C. The viability of bacteria after a heat treatment between 55 and 70 degrees C in the DSC is determined by both plate count and calorimetric data. The fractional viability values based on calorimetric and plate count data show a linear relationship. Viability loss and the irreversible change in DSC thermograms of pretreated whole cells are highly correlated between 55 and 70 degrees C. Comparison of DSC scans for isolated ribosomes shows that the thermal stability of E. coli ribosomes is greater than that of L. plantarum ribosomes, consistent with the greater thermal tolerance of E. coli observed from viability loss and DSC scans of whole cells.     

A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.     

Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 M to 2.5 M. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 M and 1.45 M-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 M-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3, H4) complex has only one peak near 1.45 M-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 M-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 M-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 X 10(6) M-1 at 1.0 M-NaCl (20 degrees C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)] much greater than chicken erythrocyte DNA greater than poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).     

Characterization of two triacylglycerol lipase activities in pig post-heparin plasma.

Two triacylglycerol lipase activities were characterized after partial purification from pig post-heparin plasma. These two lipase activities were eluted sequentially with a NaCl gradient from columns containing Sepharose with covalently linked heparin. The first lipase activity, which was eluted at 0.75M-NaCl, was not inhibited at 28 degrees C in the presence of 1M-NaCl and was not further activated by plasma apolipoproteins. The absence of this lipase activity from post-heparin plasma from hepatectomized pigs indicates that the liver plays a role in the synthesis of this enzyme. A second lipase activity, which was eluted at 1.2M-NaCl, was inhibited when assayed in the presence of 1.0M-NaCl and was activated 14-fold by an apolipoprotein isolated from human very-low-density lipoprotein. The characteristics are identical with those of lipoprotein lipase purified from pig adipose tissue.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Thermal stability and conformational changes of transglutaminase from a newly isolated Streptomyces hygroscopicus

Thermal stability and conformational changes of transglutaminase (TGase) from a newly isolated Streptomyces hygroscopicus were investigated in this study. The inactivation kinetics of the microbial transglutaminase (MTGase) was fitted using one-step inactivation model. It was much more stable under 40 degrees C. The half-lives for the MTGase at 50 degrees C and 60 degrees C were only 20 min and 8 min, respectively. Spectroscopic studies of the enzyme suggested conformational transition from ordered secondary structural elements (alpha/beta-protein) to unordered structure during thermal denaturation. Some polyols could improve the thermal stability of the enzyme. Among the polyols examined, the prolonged half-lives of 40 min at 50 degrees C and 20 min at 60 degrees C were gained by adding 10% glycerol. The results of differential scanning calorimetric (DSC) analysis showed a distinct transition peak with a significant greater Tm and DeltaH for the MTGase mixed with polyols in comparison with the control, which indicated that the polyols could maintain the natural structure of the enzyme to some extent. The SDS-PAGE electrophoresis of cross-linked casein confirmed that the stabilizers could protect the MTGase from thermal denaturation.     

Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed. The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.     

The influence of hydrostatic pressure and urethane on the thermal inactivation of bacteriophage

In Difco nutrient broth, containing 0.5 per cent NaCl, pH 6.6, Escherichia coli phages T1, T2, and T5 were inactivated at 66 degrees C., and T7 at 60 degrees C., at nearly the same rate. In each case the rate of destruction was not uniform but more or less decreased with time of heating. With T2 there was an initial increase in number of infective centers after heating for several minutes at 66 degrees C. Hydrostatic pressures up to 10,000 pounds per square inch retarded the thermal destruction of T1, T2, and T5, but accelerated that of T7, while small concentrations of urethane accelerated the rate of each. The rate of inactivation was increased by the addition of 0.005 M phosphate, and was decreased by 0.005 M MgCl(2) in all but T7, whose rate was unaffected by this amount of Mg. The influence of Ca was similar to that of Mg. The addition of 0.005 M MgCl(2) to the broth medium resulted in a first order rate of destruction of T5 at either normal or increased pressure, and with as well as without urethane. Analysis of data obtained under these conditions indicated that the thermal inactivation proceeds with a volume increase of activation of 113 cc. per mol, and with a heat and entropy of 170,000 calories and 425 E. U., respectively, in the rate-limiting reaction. In the presence of 0.1 M urethane the heat and volume change of activation are apparently slightly greater. The relation between concentration of urethane and the amount of acceleration in rate of destruction at normal pressure and 66 degrees C. indicated that the total rate involves at least two first order rate processes: the thermal inactivation itself and a urethane-catalyzed reaction, the latter involving the combination of an average of 2.3 molecules of urethane in the activated state of the bacteriophage molecule whose destruction results in loss of phage activity.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

Response of Campylobacter jejuni to sodium chloride.

Studies were done to provide more comprehensive information on the response of Campylobacter jejuni and nalidixic acid-resistant, thermophilic Campylobacter (NARTC) to sodium chloride at 4, 25, and 42 degrees C. Three strains of C. jejuni were studies, and all could grow at 42 degrees C in the presence of 1.5% NaCl, but not 2.0% NaCl. At the same temperature, NARTC could grow in 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than was C. jejuni. Both C. jejuni and NARTC grew poorly in the absence of added NaCl and grew best in the presence of 0.5% NaCl at 42 degrees C. At 25 degrees C, NaCl concentrations of 1.0 to 2.5% were protective to NARTC, but the same concentrations of salt generally enhanced the rate of death of C. jejuni. At 4 degrees C, both C. jejuni and NARTC were sensitive to 1.0% or more NaCl; however, the rate of death at this temperature was substantially less than that which occurred at 25 degrees C. A 3 log10 decrease of cells occurred in 4.5% NaCl after 1.2 to 2.1 days at 25 degrees C, and a similar reduction in cells took approximately 2 weeks at the same salt concentration and 4 degrees C. Although C. jejuni grows best in the presence of 0.5% NaCl, the presence of NaCl at concentrations as low as 1.0% may retard growth or increase rate of death; hence, it is advisable that growth media used for recovering or enumerating this organism contain 0.5% NaCl, but not 1.0% or more NaCl.     

The effect of salt concentration and pH on the heat resistance of Escherichia coli in tryptic soy broth

The effect of the acid and the osmotic stress on the heat resistance of Escherichia coli (EC1 and EC2) was studied at 63 degrees C in tryptic soy broth adjusted to various pHs (2.5, 4.5 and 6) and various NaCl concentrations (2, 4 and 8%). In the second study, the effect of pretreatment on thermotolerance of E. coli cells was determined. The heat resistance of both strains was low at pH 2.5, but strain EC1 was more resistant than strain EC2. On the contrary, the heat resistance increased with increasing the pH values. Addition of NaCl (2%) to TSB medium, was involved in the protection of cells against heat inactivation, this protective effect was, however, not observed by increasing the NaCl concentration up to 8%. The combined effect of the pH and NaCl on the thermal resistance of both strains was significantly lower at pH 2.5 and NaCl 8%, the number of viable cells decreased from approximately 10(8) CFU/ml to an undetectable number within 20 min for strain EC1 and 15 min for strain EC2, respectively. This study indicates that heat resistance of strain EC1 was enhanced after acid or thermal adaptation. Heat resistance of strain EC2 was, however, enhanced only after thermal adaptation. For both strains no relationship was found between salt adaptation and the ability to resist thermal stress.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Ovarian adenosine 3'':5''-cyclic monophosphate-dependent protein kinase(s). Regulation by choriogonadotropin and lutropin in rat ovarian cells.

Choriogonadotropin and lutropin have been found to activate cyclic AMP-dependent protein kinase in ovarian cells isolated by collagenase dispersion from immature rats. The stimulatory effect of gonadotropins was dependent on both hormone concentration and incubation time. Choriogonadotropin at 1 mug/ml fully stimulated the protein kinase activity within 5 min of incubation, and this effect was specific for choriogonadotropin and lutropin-like activity. In addition, protein kinase activity has been characterized with respect to salt sensitivity, cyclic AMP binding, and its responsiveness to gonadotropins and other peptide hormones. Ovarian protein kinase was susceptible to high salt concentrations. The addition of 0.3-1.0 M-NaCl in incubation medium increased the activity ratio with a concomitant decrease in cycle AMP-dependence. The salt effect on protein kinase was observed both from hormone-treated and untreated cells. The hormone-stimulated and unstimulated protein kinase activity was completely stable in the absence of NaCl. No change in the activity ratio was observed when cellular extracts were assayed for protein kinase activity either immediately or after 2 h in the absence of added salt. Gel filtration in the absence of NaCl of cellular extracts prepared from choriogonadotropin-treated and untreated cells showned only a single peak of protein kinase activity that was sensitive to exogenously added cyclic AMP. By contrast, when 0.5 M-NaCl was included in the column buffer, the chromatography of untreated extract showed two peaks of protein kinase activity. The first peak was sensitive to added cyclic AMP, whereas the second peak was insensitive to it. Under identical experimental conditions, protein kinase from gonadotropin-treated cells showed, on gel filtration, only one peak of activity that was totally insensitive to added cyclic AMP. DEAE-cellulose column chromatography of a 20000 g supernatant fraction resulted in a peak of kinase activity that eluted in approx. 0.15 M-NaCl, similar to the similar to the elution of type II protein kinases as described by Corbin et al. (1975) (J. Biol. Chem. 250, 218-225). Choriogonadotropin stimulation produced a decrease in the capacity of protein kinase to bind exogenous cyclic [3H]AMP, with a concomitant increase in the kinase activity ratio. These results are consistent with the notion that cyclic AMP, GENERATED IN SITU Under hormonal stimulation, binds tot he regulatory subunit of protein kinase with subsequent dissociation of the active catalytic subunit from the holoenzyme.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Evaluation of the Heat Inactivation of Escherichia coli and Lactobacillus plantarum by Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and reversibility after heat treatment of transitions associated with various cellular components of Escherichia coli and Lactobacillus plantarum. The reversibility and the change in the thermal stability of individual transitions are evaluated by a second temperature scan after preheating in the DSC to various temperatures between 40 and 130°C. The viability of bacteria after a heat treatment between 55 and 70°C in the DSC is determined by both plate count and calorimetric data. The fractional viability values based on calorimetric and plate count data show a linear relationship. Viability loss and the irreversible change in DSC thermograms of pretreated whole cells are highly correlated between 55 and 70°C. Comparison of DSC scans for isolated ribosomes shows that the thermal stability of E. coli ribosomes is greater than that of L. plantarum ribosomes, consistent with the greater thermal tolerance of E. coli observed from viability loss and DSC scans of whole cells.