Biomedical subjects

J R Lepock

Publications and source records attributed to J R Lepock.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effect of the organic Ca2+ channel blocker D-600 on sarcoplasmic reticulum Ca2+ uptake in skeletal muscle.

Experiments were undertaken to study the possibility that the calcium channel blocker D-600 (gallopamil), which penetrates into muscle cells (20), facilitates excitation-contraction coupling in skeletal muscle (7) by a direct effect on the sarcoplasmic reticulum (SR). The effects of D-600 were studied on single phasic muscle fibers, either intact or split open. D-600 potentiated twitches in intact fibers at concentrations lower than those reported in whole muscles. In split fibers, the force produced by caffeine-induced Ca2+ release from the SR was reversibly inhibited by 5 microM D-600, when added to the Ca2+ loading solution. This inhibitory effect was inversely related to temperature, and it was dose dependent. When D-600 was added after Ca2+ loading and before caffeine exposure, or during the caffeine exposure itself, it did not inhibit Ca2+ release, but rather increased the development of force. We conclude that, apart from the blocking effect that D-600 may have on the voltage sensor, the drug penetrates into the myoplasm and affects excitation-contraction coupling by inhibiting the SR Ca2+ pump. This may be the consequence of a conformational change in the transmembrane Ca2+ binding domain of the ATPase.

Animals↗

PubMed1997

Preferential activation of HSF-binding activity and hsp70 gene expression in Xenopus heart after mild hyperthermia.

We have examined the effect of mild hyperthermia on the pattern of heat shock transcription factor (HSF) binding activity, heat shock protein 70 (hsp70) and hsp30 gene expression and protein denaturation in selected tissues of adult Xenopus namely, heart, hind limb muscle, eye, liver and spleen. In these studies it was found that heart tissue was the most thermally sensitive of all of the tissues examined since maintenance of adult frogs at 26 degrees C resulted in a preferential activation of HSF binding. Thus, heart has a lowered set point temperature for HSF activation compared to the other tissues examined. At 30 degrees C HSF activation was observed in all of the tissues examined. Heart HSF activation at 26 degrees C was correlated with an increase in hsp70 mRNA and Hsp70 protein accumulation. At 28 degrees C the largest amount of hsp70 and hsp30 mRNA accumulation was detected in heart and skeletal muscle compared to other tissues while hsp70 mRNA accumulation was relatively low in spleen and hsp30 mRNA accumulation was not detectable in eyes, liver and spleen. Incubation of adult frogs at 30 degrees C resulted in enhanced hsp70 and hsp30 mRNA accumulation in all of the tissues. Finally, we have used differential scanning calorimetry (DSC) to compare the temperatures at which protein denaturation occurs in heart and liver tissue. The onset of protein denaturation (T0) occurred approximately 8.5 degrees C lower in heart compared to liver. Also the midpoint of the DSC profile (T1/2) was approximately 10.4 degrees C lower in heart than in liver. Thus, heart proteins are generally more thermolabile than proteins in liver tissue. Taken together these data suggest that heart is more sensitive than the other tissues examined with respect to moderate increases in environmental temperature.

Animals↗

PubMed1996

The role of a conserved water molecule in the redox-dependent thermal stability of iso-1-cytochrome c.

Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule (Wat166) at an adjacent site (Berghuis, A. M., Guillemette, J. G., McLendon, G., Sherman, F., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 236, 786-799; Berghuis, A. M., Guillemette, J. G., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 235, 1326-1341; Rafferty, S. P., Guillemette, J. G., Berghuis, A. M., Smith, M., Brayer, G. D., and Mauk, A. G. (1996) Biochemistry, 35, 10784-10792). We used differential scanning calorimetry (DSC) to determine the change in heat capacity (DeltaCp) and the temperature dependent enthalpy (DeltaHvH) for the thermal denaturation of both the oxidized and reduced forms of the iso-1 cytochrome c variants. The relative stabilities were expressed as the difference in the free energy of denaturation (DeltaGD) between the wild type and mutant proteins in both redox states. The disruption of the hydrogen bonding network results in increased stability for all of the mutant proteins in both redox states with the exception of the reduced Y67F variant which has approximately the same stability as the reduced wild type protein. For the oxidized proteins, DeltaGD values of 1.3, 4.1, 1.5, and 5.8 kcal/mol were determined for N52A, N52I, Y67F, and Y67F/N52I, respectively. The oxidized proteins were 8.2-11.5 kcal/mol less stable than the reduced proteins due to a redox-dependent increase in the entropy of unfolding.

Cytochrome c Group↗

PubMed1996

Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase.

Eukaryotic Cu,Zn superoxide dismutases (CuZnSODs) are antioxidant enzymes remarkable for their unusually stable beta-barrel fold and dimer assembly, diffusion-limited catalysis, and electrostatic guidance of their free radical substrate. Point mutations of CuZnSOD cause the fatal human neurodegenerative disease amyotrophic lateral sclerosis. We determined and analyzed the first crystallographic structure (to our knowledge) for CuZnSOD from a prokaryote, Photobacterium leiognathi, a luminescent symbiont of Leiognathid fish. This structure, exemplifying prokaryotic CuZnSODs, shares the active-site ligand geometry and the topology of the Greek key beta-barrel common to the eukaryotic CuZnSODs. However, the beta-barrel elements recruited to form the dimer interface, the strategy used to forge the channel for electrostatic recognition of superoxide radical, and the connectivity of the intrasubunit disulfide bond in P. leiognathi CuZnSOD are discrete and strikingly dissimilar from those highly conserved in eukaryotic CuZnSODs. This new CuZnSOD structure broadens our understanding of structural features necessary and sufficient for CuZnSOD activity, highlights a hitherto unrecognized adaptability of the Greek key beta-barrel building block in evolution, and reveals that prokaryotic and eukaryotic enzymes diverged from one primordial CuZnSOD and then converged to distinct dimeric enzymes with electrostatic substrate guidance.

Amino Acid Sequence↗

PubMed1996

Cholesterol increases the thermal stability of the Ca2+/Mg(2+)-ATPase of cardiac microsomes.

The effect of membrane cholesterol on the thermal inactivation of Ca2+/Mg(2+)-ATPase activity of bovine cardiac microsome was measured and compared to the thermal denaturation profiles of the microsomes as measured by differential scanning calorimetry (DSC). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Both thermal inactivation of Ca2+/Mg(2+)-ATPase activity and thermal denaturation were shifted to higher temperatures in microsomes enriched with cholesterol (37 +/- 5 micrograms cholesterol/mg protein, cholesterol/phospholipid molar ratio 0.31) compared to control microsomes (15 +/- 3 micrograms cholesterol/mg protein, molar ratio 0.12). Thermal inactivation was measured by two methods: first, measuring activity at room temperature as a function of heating to elevated temperatures at 1 K/min, where inactivation temperatures (T1, temperature of half activity) were 58.9 +/- 0.3 degrees C for control membranes and 59.9 +/- 0.1 degrees C for cholesterol-enriched membranes, respectively. Second, measuring ATPase activity as a function of time at constant temperature, where T1 values of 57.6 +/- 0.5 degrees C and 59.2 +/- 0.5 degrees C were determined for control and cholesterol-enriched membranes, respectively. DSC profiles of microsomal membranes consisting of a number of overlapping peaks were obtained. A well resolved component (transition C) was observed with a transition temperature (T 1/2) of 58.2 degrees C. This T 1/2, which is a measure of conformational stability, correlates with the T1 for Ca2+/Mg(2+)-ATPase activity and is 1.9 +/- 0.6 K higher in cholesterol-enriched membranes. Thus, the increased resistance to inactivation appears to be due to increased conformational stability of the protein induced by cholesterol, demonstrating that a change in lipid composition can influence the stability of an integral membrane protein in a natural membrane. The increased stability is of sufficient magnitude to account for the previously observed correlation between cholesterol content and resistance to heat shock in several cell lines.

Animals↗

PubMed1996

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface.

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.

Binding Sites↗

PubMed1996

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins.

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0 degrees C, are present in both the nucleus and cytoplasm.

3T3 Cells↗

PubMed1996

A two-step reversible-irreversible model can account for a negative activation energy in an Arrhenius plot.

Arrhenius plots of the rate of inactivation (killing) of mammalian cells and Drosophila embryos have "apparent negative" activation energies at low temperatures. This can be explained by assuming that the rate-limiting event resulting in killing is a two-step process or mechanism, where the first step is reversible and the second irreversible. Two examples, consistent with this model, are suggested as possible mechanisms of hypothermic killing: (i) a membrane lipid liquid crystalline-to-gel transition followed by a metabolic block or event which kills the cell and (ii) cold denaturation of a protein followed by protein aggregation.

Animals↗

PubMed1996

Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer.

Differential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic transitions with midpoints (Tm) at 59 degrees C and 63 degrees C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60 degrees C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm = 66 degrees C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 beginning at about 30 degrees C (Tm = 41 degrees C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20 degrees C, sensitive at 40 degrees C and resistant when returned to 20 degrees C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c, a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing temperature over the same range as the 41 degrees C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that matches the supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.

Amino Acid Sequence↗

PubMed1996

Astrophysical and biological constraints on radiopanspermia.

We have carried out a series of calculations involving bacteria and viruses embedded in dust grains, which are ejected from our solar system by radiation pressure and travel through space to other star systems. Under many conditions this type of panspermia is impractical, primarily because the ultraviolet (UV) radiation of the present Sun inactivates the micro-organisms. However, if the organisms are shielded by an absorbing material like carbon and if ejection takes place in the red-giant phase of a one solar mass star like our Sun, there is a significant probability that the micro-organisms can reach another star system alive (i.e. with only sub-lethal damage from UV and ionizing radiation). In addition to panspermia with viable micro-organisms, it is possible to seed the Galaxy with inactivated ones whose DNA and RNA fragments may provide the initial information necessary to start biological evolution in favourable environments.

Astronomical Phenomena↗

PubMed1995

Use of thermal analysis to distinguish magnesium and calcium stimulated ATPase activity in isolated transverse tubules from skeletal muscle.

The presence of calcium stimulated adenosine triphosphatase (Ca2+,Mg(2+)-ATPase) activity in isolated transverse tubule (t-tubule) membranes is distinguished from magnesium adenosine triphosphatase (Mg(2+)-ATPase) activity on the basis of differing thermal stabilities. The Mg(2+)-ATPase is the major protein component of the t-tubule membrane, and it can be difficult to discriminate between the low levels of Ca2+ stimulated ATPase activity found in isolates of t-tubules compared to the much higher Mg(2+)-ATPase activity. Thermal analysis reveals different inactivation temperatures (Ti) for the proteins responsible for ATP dependent calcium transport (Ti = 49 degrees C) and Mg(2+)-ATPase activity (Ti = 57 degrees C) in isolated t-tubule membranes. The differential scanning calorimetry profile of t-tubule membranes consists of three major components with transition temperatures (Tm) of 51 degrees C, 57 degrees C and 63 degrees C. Denaturation of the component with Tm = 57 degrees C correlates with inactivation of Mg(2+)-ATPase activity, and denaturation of the Tm = 51 degrees C component correlates with the inactivation of Ca2+,Mg(2+)-ATPase activity and calcium transport. The functions of the t-tubule membrane component or components that denature with Tm = 63 degrees C have yet to be identified. The lack of stimulation of calcium transport in isolated t-tubules by oxalate, the impermeability of isolated t-tubules to oxalate, and experiments performed on t-tubules with defined amounts of sarcoplasmic reticulum (SR) added suggest that contamination of the isolated t-tubules by SR is unlikely to account for the level of Ca2+,Mg(2+)-ATPase activity detected. The presence of a Ca2+,Mg(2+)-ATPase in the t-tubule membrane would provide a mechanism that may be involved in the partial removal of calcium that is accumulated in the junctional space during muscle relaxation or calcium that is released from the terminal cisternae of sarcoplasmic reticulum during excitation-contraction coupling.

Animals↗

PubMed1995

Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor.

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.

Animals↗

PubMed1995

Evidence for two modes of hypothermia damage in five cell lines.

The Arrhenius plots of inactivation (killing) rates of five mammalian cell lines, V-79 Chinese hamster lung, mouse L-929, mouse neuroblastoma, human 18LU, and human erythrocytes, exposed to hypothermia contain a break somewhere between 5 and 10 degrees C caused by a change from positive to negative slope, which corresponds to the minimum inactivation rate. This implies that there are two distinct mechanisms of hypothermic damage above and below the minimum inactivation rate temperature in a system that is uncomplicated by previous or simultaneous hypoxia. Hence, having two distinct mechanisms for hypothermia damage is not unique to the Chinese hamster cell line. This suggests that the optimum aerobic hypothermic storage temperature for human cells and tissues is not 0 degree C, but somewhere in the range of 5 to 10 degrees C.

Animals↗

PubMed1995

Factors influencing survival of mammalian cells exposed to hypothermia. VI. Effects of prehypothermic hypoxia followed by aerobic or hypoxic storage at various hypothermic temperatures.

The Arrhenius plot of inactivation (killing) rates of V-79 Chinese hamster cells exposed to hypothermia in air-equilibrated (aerobic) medium contains a break at about 8 degrees C, which corresponds to the minimum inactivation rate, implying that there are distinct hypothermic damage mechanisms above (range I, 8 to 25 degrees C) and below (range II, 0 to 8 degrees C) 8 degrees C. Prehypothermic hypoxia (PHH) for 75 min at room temperature sensitizes cells to subsequent aerobic hypothermia at both 5 and 10 degrees C (range II and I). However, PHH followed by severe hypoxia (0.03 microM oxygen in the medium) protected cells during 10 degrees C (range I) storage by increasing the shoulder, but not the slope, of the cell survival curve compared to the PHH plus 10 degrees C aerobic hypothermia case. On the other hand, PHH plus severe hypoxia during 5 degrees C storage (range II) protected cells by decreasing the slope, but not the shoulder, of the cell survival curve compared to the PHH plus 5 degrees C aerobic hypothermia case. Furthermore, PHH plus severe hypoxia during 5 degrees C storage was not significantly worse than aerobic storage without PHH at 5 degrees C. With or without severe hypoxia, 10 degrees C storage is preferable to 5 degrees C storage in this cell line. Extrapolated to organ storage, the results may imply that if warm ischemia (PHH) has occurred, subsequent hypoxic hypothermic perfusion storage may be preferable to aerobic hypothermic perfusion storage.

Aerobiosis↗

PubMed1994

Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The site of interaction of dibucaine with the Ca(2+)-ATPase of rabbit sarcoplasmic reticulum, an ion-transporting membrane protein, was investigated by determining the effect of dibucaine on the denaturation of the transmembrane domain and the aqueous domain containing, respectively, the high-affinity Ca2+ binding sites and the site of ATP hydrolysis. In the absence of Ca2+, a single irreversible denaturation transition with Tm approximately equal to 49 degrees C is observed for the Ca(2+)-ATPase by differential scanning calorimetry (DSC). In the presence of Ca2+, but not Mg2+, Sr2+, or Ba2+, a new high-temperature transition is observed that has been shown to be due to stabilization of the transmembrane region [Lepock, J. R., Rodahl, A. M., Zhang, C., Heynen, M. L., Waters, B., & Cheng, K. H. (1990) Biochemistry 29, 681-689]. The maximum stabilization corresponds to a shift in Tm of 13.8 degrees C, and Hill analysis indicates that the Ca2+ binding site yielding stabilization has a Kd = 2.5 x 10(-4) M with a cooperativity (n) of 1. Thus, stabilization is due to Ca2+ binding not to the high-affinity sites but to one of the previously observed sites of low or intermediate affinity, which must be located in the transmembrane or stalk subdomains. Dibucaine has little effect on the Tm of the aqueous domain, but it decreases the Tm of the transmembrane domain with Kd approximately equal to 4.1 x 10(-4) M and a cooperativity of approximately 1.6, implying that destabilization is due to the binding of dibucaine to sites of intermediate or moderately high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock.

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.

Animals↗

PubMed1992

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

PubMed1992

Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.

Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37 degrees C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50 degrees C). A reversible transition at approximately 15 degrees C and an irreversible transition at approximately 35 degrees C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20 degrees C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37 degrees C in 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗