Effect of heat treatment on the ATPase activity of various sarcoplasmic reticulum preparations

Summary Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum (SR) preparations is activated after a short period of preincubation at temperatures between 40 and 45°C, but for temperatures higher than 48°C pronounced denaturation is observed. Heat denaturation is decreased if Mg²⁺ or K⁺ are present during heat treatment.Acknowledgments. This work was supported by research grants from Instituto de Alta Cultura (Grant No. CB/2) and the Calouste Gulbenkian Foundation. We are grateful to Drs.A. P. Carvalho andV. M. C. Madeira for many discussions and suggestions during the course of this work.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Temperature dependence of neurotransmitter release in the antarctic fish Pagothenia borchgrevinki

The quantal contents of endplate potentials from extraocular muscles of an antarctic fish Pagothenia borchgrevinki were measured over a range of temperatures. Quantal release was maximal at about 5 degrees C but showed little dependence on temperature between -2 degrees C and 10 degrees C. Above 10 degrees C quantal content declined until release ceased about 18 degrees C. In view of the fact that the ambient temperature at which these fish live is constant at -1.9 degrees C, the results suggest that Pagothenia borchgrevinki is only partially adapted to its environment despite 25 million years acclimatization.     

Response of the ampullae of Lorenzini to static combined electric and thermal stimuli in Scyliorhinus canicula.

The effect of long-lasting electric currents on the Lorenzinian ampullae at constant temperatures between and 25 degrees C was investigated in the dogfish (Scyliorhinus canicula). Steady state neural impulse patterns in single afferent units were analyzed by plotting interval length histograms and computing mean values and standard deviations for currents between -100 and +100 nA. The mean values depended on temperature and on current strength; the relative standard deviations remained almost constant (ca. 20--30%). Negative currents, inserted at the orifice of the ampullary canal led to higher, and positive currents to lower, steady impulse rates in the whole temperature range investigated here. This static component of electrosensitivity again disappeared at higher currents (of 50 nA and more; electric overstimulation). The maximum static response was two orders of magnitude less than the maximum dynamic component of electroreception. The electrosensitivity depended on temperature: the ampullae were most sensitive to electric currents between 13 and 19 degrees C. The maximal neural activity at 19 degrees C was not shifted to higher or lower temperatures by electric stimulation. A constant equivalent of electric and thermal stimulation throughout the tested temperature and current range could not be found.     

Temperature acclimation of Mg2+Ca2+-myofibrillar ATPase from a cold-selective teleost, Salvelinus fontinalis: a compromise solution

Brook trout (Salvelinus fontinalis, Mitchill) were acclimated over 15 weeks to either +4 degrees C or +24 degrees C. The effects of temperature on myofibrillar Mg2+Ca2+-ATPase activities were investigated. In contrast to goldfish, temperature acclimation does not alter the kinetic properties of the brook trout myofibrillar ATPase. Activation energy (delta G not equal to) is lower and substrate turnover number is higher than values previously reported for cold-adapted stenotherms. Properties of brook trout ATPase appear to be a compromise enabling function across a broad temperature range. The different strategies of adapting to seasonal temperature variations are briefly discussed.     

Effects of temperature on the bioaccumulation of cadmium by the crayfish Ponstacus leptodactylus. Microanalysis by X-ray spectrography and secondary ion emission

Low temperatures (10 +/- 1 degrees C) induce a rapid entry of cadmium into the body across the gills and gut; higher temperatures (20 +/- 1 degrees C) induce a medium and long term absorption and storage of the metal in the digestive gland and its long term accumulation in the exoskeleton. From a methodical point of view, electron microprobe appears more suitable than secondary ion emission microanalysis for the detection of cadmium.     

Heterogeneity of HeLa cell DNA as evidenced by CsCl density gradient centrifugation

Summary HeLa cell DNA was analyzed through CsCl density gradient centrifugation and thermal denaturation. Neutral centrifugation without ionic treatment allowed the resolution of a main peak with density 1.699 g/ml and of 3 satellites DNAs with densities 1.683, 1.710 and 1.720. First derivatives of melting curves showed the presence of 4 DNA families, whose G+C content calculated from T_m values corresponded almost exactly to the G+C content expected from the previously densities. The extraction method seems particularly suitable for quantitative separation of DNA classes.     

Cholecystokinin-octapeptide-induced hyperthermia in guinea-pigs

Intracerebroventricular administration of cholecystokinin-octapeptide (CCK-8) at room temperature (21 degrees C) induced dose-related hyperthermia in guinea-pigs and also produced hyperthermia at low (10 degrees C) and high (30 degrees C) ambient temperatures. CCK-8-induced hyperthermia was direct and not mediated by prostaglandins, norepinephrine, cyclic AMP or naloxone-sensitive receptors.     

Alteration in thermal stability of ribosomes from Drosophila melanogaster with age.

Thermal analysis of high salt (0.5 M) washed ribosomal monomers from young and old male Drosophila melanogaster revealed an 8 degrees C downshift in the mean temperature of denaturation (Tm). Moreover, there was observed a marked loss in the ability of ribosomes extracted from older flies to reassociate upon cooling. These observations suggest that age-dependent alterations in the structural integrity of the rRNA-r protein complex could, at least in part, be responsible for the diminished capacity for protein synthesis in this species with advancing age.     

Prostaglandin E1-induced fever in rabbits pretreated with p-chlorophenylalanine.

The prostaglandin E1-induced fever was neither potentiated nor attenuated at all levels of the ambient temperatures (2, 22 and 32 degrees C) studied after pretreatment of the conscious rabbits with p-chlorophenylalanine, when compared to the untreated control.     

Effects of thyrotrophic-releasing hormone (TRH) on thermoregulation in the rat

At ambient temperatures (Ta) of both 8 and 22 degrees C, intraventricular administration of TRH (10-80 microgram) produced a dose-dependent hypothermia in rats. The hypothermia was due to both decreased metabolic heat production and cutaneous vasodilatation. In contrast, at 30 degrees C Ta, TRH increased metabolic heat production (due to behavioral excitation) and led to hyperthermia.     

Dissociation-constants of metat-ion-complexes with alkaline phosphatase from pig kidney.

Using metal-ion buffers it was possible to remove Zn2+, Mg2+ and Mn2+ ions of pig kidney alkaline phosphatase reversibly. The dissociation constants obtained are KEMg: 4 X 10(-7) M, KEMn: 4 X 10(-8) M and KEZn: 8 X 10(-13) M (22 degrees C, pH: 9.6, mu: 0.07).     

Effect of temperature on the activity of AMP-deaminase from rat heart.

In the presence of 1 mM ATP, the plots of Michaelis dependence of the reaction catalysed by AMP-deaminase from rat heart were hyperbolic at all temperatures between 10 and 40 degrees C. Calculation of the energy of activation for ATP-activated enzyme and of the enzyme-substrate complex formation is presented.     

Differential scanning calorimetry of chromaffin granule membranes.

Differential scanning calorimetry thermograms of native chromaffin granule membranes exhibit several peaks in the 15-35 degrees C region. Extraction of cholesterol increases the size of the melting peaks. Addition of Ca2+ ions does not seem to influence the lipid transitions.     

Dantrolene and the effect of temperature on the spontaneous release of transmitter at the frog neuromuscular junction

The characteristic effect of temperature on m.e.p.p. frequency at the amphibian neuromuscular junction is unaltered by the presence of Dantrolene (an agent that is believed to reduce the efflux of Ca2+ from intracellular stores) or by changes in [Ca2+)o. It is concluded that temperature affects the release system directly, with a transition temperature at about 16 degrees C.     

Pimozide and p-chlorophenylalanine blockade in DL-amphetamine and pargyline-treated rats held at two environmental temperatures.

In rats, treated with DL-amphetamine + monoamine oxidase inhibitor, and held at an ambient temperature of 28.5 degrees C, hyperthermia was completely eliminated by treatment with pimozide + p-chlorophenyl-alanine. The same drugs markedly reduced the hypothermic effects in rats treated similarly at 4 degrees C. Results implied that serotoninergic and dopaminergic neurones were involved in the thermoregulatory effects of amphetamine.     

Effects of hypothalamic injections of 5,6-dihydroxytryptamine on thermoregulation in rats.

5,6-Dihydroxytryptamine, a serotonin depletor, infused directly into the anterior hypothalamus of rat's brain, produced an increase in both heat production and heat loss (as indicated by changes in peripheral circulation) at temperatures of 8, 15 and 22 degrees C. The rectal temperature of these treated rats remained constant.     

Über Struktur und Aktivität der den H2O2-Zerfall katalysierenden Cu2+-Komplexe. — VI. Differenzierung von nativer RNS und nativer DNS bzw. denaturierter DNS auf Grund der katalytischen Eigenschaften

Summary The catalysis of H₂O₂ decomposition by Cu²⁺-complexes of RNA and DNA has been investigated. It is shown that both complexes decompose H₂O₂, but only the Cu²⁺-RNA-system shows peroxidative activity too, e.g. only in this case the nucleotide bases are degraded. Thermal denaturation of DNA also leads to a Cu²⁺-complex with peroxidative activity, the latter being dependent on the degree of denaturation.

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V. Mitteilung:S. Petri, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 1778 (1966).

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12. Mitteilung überMetallionen und H ₂ O ₂; 11. Mitteilung:H. Ch. Curtius, P. Anders, R. Zell, H. Sigel undH. Erlenmeyer, Helv. chim. Acta49, 2256 (1966).     

Biomolecular stability and life at high temperatures

It is not clear what the upper temperature limit for life is, or what specific factors will set this limit, but it is generally assumed that the limit will be dictated by molecular instability. In this review, we examine the thermal stability of two key groups of biological molecules: the intracellular small molecules/metabolites and the major classes of macromolecules. Certain small molecules/metabolites are unstable in vitro at the growth temperatures of the hyperthermophiles in which they are found. This instability appears to be dealt with in vivo by a range of mechanisms including rapid turnover, metabolic channelling and local stabilisation. Evidence to date suggests that proteins have the potential to be stable at substantially higher temperatures than those known to support life, but evidence concerning degradative reactions above 100 degrees C is slight. DNA duplex stability is apparently achieved at high temperature by elevated salt concentrations, polyamines, cationic proteins, and supercoiling rather than manipulation of C-G ratios. RNA stability seems dependent upon covalent modification, although secondary structure is probably also critical. The diether-linked lipids, which make up the monolayer membrane of most organisms growing above 85 degrees C are chemically very stable and seem potentially capable of maintaining membrane integrity at much higher temperatures. However, the in vivo implications of the in vitro instability of biomolecules are difficult to assess, and in vivo data are rare.     

Response of the brown trout (Salmo trutta, L. 1766) and rainbow trout (Salmo gairdneri Richardson, 1836) to infection by a new rhabdovirus]

Intra peritoneal injection of 200 g Trout hold at + 12 degrees C with 2x10(8) p.f.u. of a new Rhabdovirus (strain 23.75) induces an interferon synthesis within 2 days. (IF titres reaching 2,000 units/ml). 1 month later, the surviving Fish exhibit a specific anti 23.75 neutralizing serum activity (titre 40 to 80) which has been demonstrated to be due to immunoglobulins. This seroneutralization is also recorded in 1/3 of the survivors of natural infection.