Uptake and light activated esterification of P by isolated chloroplasts

1. 1. Esterification of ³²P₁ by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions.

2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors.

3. 3. Apparent K_m values for ADP and Mg²⁺ were found to be 40 and 250 μM, respectively. The K_m value for Mg²⁺ was increased by the presence of Ca²⁺. Two apparent values were observed for P₁ at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent K_m (P₁) values.

4. 4. Acceleration of the esterification resulting from the addition of ADP and P₁ to the medium indicated that these compounds were able to penetrate to the active site of esterification.

5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P₁ esterification without affecting the apparent K_m for ADP or P₁. The evidence suggests that Rib-5-P interferes with the uptake of P₁, and possibly ADP.

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.

Temperature and the regulation of activity of some mitochondrial enzyme systems in ecto- and endo-thermic animals

1. 1.|No obvious correlation was found between the temperature dependence of the apparent K_m values for either mitochondrial succinoxidase or NADH oxidase systems prepared from rat or rainbow trout liver tissue and the previously observed pattern of susceptibility of these same enzymes to thermal inactivation.

2. 2.|Apparent activation energies (E_A) have been calculated from the data and, although these are equivocal when considered in the above context, it is concluded that the possibility is not excluded that entropy may be a more important variable than enthalpy in the action of these enzymes in ectothermic animals.

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Reaction of myosin with salicylaldehyde II. Effect of salicylalation on the ATPase activity of myosin

The effect of salicylalation on the biological properties of myosin was studied.

1. 1. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The Mg²⁺-activated ATPase shows a maximal curve with 250–380% maximal activation when 25–70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca²⁺-activated ATPase shows a small increase with increasing salicylalation.

2. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PP_i does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PP_i are not different from those of the samples treated in their absence.

3. 3. Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole myosin.

Acetylene reduction with physiological electron donors by extracts and particulate fractions from nitrogen-fixing Azotobacter chroococcum

1. 1. Preparations were obtained from Azotobacter chroococcum which reduced acetylene to ethylene using physiological electron donors instead of sodium dithionite. These preparations fell into two categories: those which required catalytic amounts of benzyl viologen for acetylene reduction and those that did not.

2. 2. Acetylene reduction without benzyl viologen or sodium dithionite was observed only with particles that sedimented at 40 000 × g after disrupting bacteria in the French press or with preparations obtained by disrupting bacteria protected by a mixture of defatted bovine serum albumin-Ficoll-MgCl₂ with liquid N₂; supernatant fractions required benzyl viologen for acetylene reduction.

3. 3. Added ATP inhibited acetylene reduction by large particles; ATP and MgCl₂ were necessary for maximum acetylene reduction with bovine serum albumin-protected preparations.

4. 4. NADH and carbon substrates acted as electron donors but H₂ did not; NAD⁺ was necessary for maximum acetylene reduction with carbon substrates.

5. 5. Anaerobic conditions were necessary for maximum acetylene reduction in all cases.

Day-night variation of thermoregulatory responses to intraperitoneal electric heating in rats

1. 1.Hypothalamic temperature (T_hy), nonevaporative heat loss (R + C + K), evaporative heat loss (E), thermal conductance (k), metabolic heat production (M) and heat storage (S) of rats were simultaneously measured by direct and indirect calorimetry during internal heat loading (2 W per rat) with an intraperitoneal electric heater.

2. 2.The tests were made twice a day; once during the day (1000–1200 h) and once at night (2200–2400 h) at an ambient temperature of 24°C.

3. 3.The resting values of T_hy, colonic temperature, (R + C + K), E, M and heart rate, and the T_hy threshold for tail skin vasodilation (T_th) during internal heat load were significantly higher at night than during the day.

4. 4.The slopes showing the relationshiop between (R + C + K), k or M and T_hy were significantly steeper during the day than at night after T_hy exceeded T_th.

5. 5.The slopes showing the relationship between E or S and T_hy were not different during the day and at night.

6. 6.These results indicate that the responses of nonevaporative heat loss and heat production to internal heat load vary with the time of day in rats.

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

The effect of temperature changes on the spontaneous activity in the neural ganglia of the cockroach, Periplaneta americana

1. 1.|The effect of localized heating or protocerebrum (bp), the prothoracic (t₁), metathoracic (t₃) and the sixth abdominal (a₆) ganglion on the spontaneous neuronal firing rate in these ganglia was investigated in Periplaneta americana.

2. 2.|In almost every case heating the ganglion increased the firing rate. The most heat sensitive were bp, t₃ and t₁, a₆ was much less so.

3. 3.|In bp and t₁ the firing rate stayed on an increased level even 90 s after the thermal stimulus was over.

4. 4.|The differentiated thermosensitivity of the tested ganglia is discussed in terms of thermoregulatory behaviour of the cockroach.

Oxidative phosphorylation in yeast IX. Modification of the mitochondrial adenine nucleotide translocation system in the oxidative phosphorylation-deficient mutant op1

The properties of intact cells and isolated mitochondria of the op₁ mutant of Saccharomyces cerevisiae, which had been shown previously to be deficient in oxidative phosphorylation, have been studied further.

1. 1. When isolated mutant mitochondria were preincubated with substrate and phosphorylation was started by the addition of external ADP, low P/O ratios were obtained under standard conditions. The P/O ratios could be raised to normal values approaching 2 with citrate and succinate in the presence of unusually high concentrations of ADP. Under these conditions the Michaelis constant for ADP of respiration and phosphorylation was found to be 2.9 mM. When isolated mitochondria were added to a medium containing substrate and adenine nucleotide, the Michaelis constant for ADP was found to be lower, about 0.5 mM and maximal P/O ratios of only 0.8 were achieved.

2. 2. Adenine nucleotide translocation across the membrane of the mutant mitochondria was found to be different from that in wild-type mitochondria and dependent on the energy level in mitochondria. When the intramitochondrial nucleotide pool consisted mostly of ADP and AMP, the rate of adenine nucleotide translocation was approx. 30 times lower than in wild-type mitochondria and the Michaelis constants for ADP of the translocation process were similar in the two types of mitochondria, being lower than 10 μM. When the nucleotide pool was enriched in ATP, the translocation rate in mutant mitochondria was as high as in wild-type mitochondria but the Michaelis constant for external adenine nucleotide was more than 100 times higher in the former than in the latter.

3. 3. An examination of the effects of the uncoupler, oligomycin, valinomycin and nigericin on the translocation process in the mutant mitochondria provided additional evidence that varying energization of mutant mitochondria was responsible for the variations of the translocation rates and the Michaelis constants under different experimental conditions.

4. 4. The properties of the adenine nucleotide carrier of mutant mitochondria were studied and found to be different from those of wild-type mitochondria.

5. 5. It has been concluded that the modification of adenine nucleotide translocation across the mitochondrial membrane is responsible for oxidative phosphorylation deficiency in the op₁ mutant. The implications of these findings for the understanding of the adenine nucleotide translocation mechanism and the role of the translocation system in the control of cellular syntheses and growth are discussed.

How are neuronal thermosensitivity and lack of thermoreception related in the duck's hypothalamus? A tentative answer

1. 1.|In 15 conscious Pekin ducks, 40 “warm sensitive” hypothalamic neurons were identified according to their discharge rates at 40°C T_hy (F₄₀), local temperature coefficients (Δ/ΔT) and Q₁₀.

2. 2.|Q₁₀ and either F₄₀ or ΔF/ΔT were little or not related.

3. 3.|A positive correlation between F₄₀ and ΔF/ΔT was observed which was particularly close (r = 0.94 and 0.96) when the neurons were classified according to their Q₁₀ of <2 and >2.

4. 4.|The results suggest that neurons with positive temperature coefficients in the duck's hypothalamus mostly exhibit linear to exponential temperature-discharge relationships.

5. 5.|This is an contrast to observations on mammalian hypothalamic thermosensitive neurons and may relate to the absence of the thermosensory function in the duck's rostral brainstem.

Thermal parameters of four species of Gerbillurus

1. 1.Thermal parameter of the four Gerbillurus species measured in the laboratory were examined in relation to their micro-environments in a xeric habitat.

2. 2.Basal metabolic rates (BMR) were lower than predicted, while thermoneutral zonds (TNZ) were narrow and exceeded burrow temperatures.

3. 3.Body temperatures (T_b) were regulated over a range of ambient temperatures (T_a). Evaporative water loss was used as a short-term cooling mechanism to reduce hyperthermia above the TNZ.

4. 4.Conductance was low below the TNZ to reduce heat loss.

5. 5.Adaptation to low temperatures is important for gerbils when active at night.

6. 6.The adaptive significance of the thermal biology of Gerbillurus is discussed in relation to phylogeny, distribution, food availability and nocturnal activity.

Metabolic effects of thyroid hormones at a low temperature in the snake

1. 1.|The metabolic role of the thyroid gland was studied in intact snakes, Naja naja and Ptyas korros treated with tri-iodo-thyronine (T₃) and thyroxine (T₄) and in thyroidectomized (Tx) N. naja kept at 21°C by analyzing tissue composition and glycogen phosphorylase a activity.

2. 2.|Liver weight was unaffected by thyroid hormone injection in both species but decreases in liver glycogen followed T₃ or T₄ injection, and there was an increase in liver glycogen in N. naja. These changes in liver glycogen were accompanied by a decrease in glycogen phosphorylase a activity with T₃ injection. T₃ decreased muscle glycogen in Ptyas and Tx increased it in N. naja.

3. 3.|T₃ increased % liver lipid in Ptyas but not in Naja.

4. 4.|Between species, there were differences in liver weight, blood glucose level, cholesterol level and % muscle lipid.

5. 5.|The results showed that thyroid hormones affected carbohydrate and lipid metabolism at a low temperature of 21°C, although the significance is not known.

Fuscin, an inhibitor of mitochondrial SH-dependent transport-linked functions

1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.

2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[¹⁴C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.

3. 3. High affinity-fuscin binding sites (K_d = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.

4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;

5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (K_i in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (K_i = 5–10 μM); (c) the transport systems of phosphate (K_i ≈ 20 μM) and of glutamate (K_i = 3–5 μM); (d) the ADP transport, indirectly (K_i ≈ 10 μM).

6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH⁻ carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.

7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.

8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.

9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.

Thermal responses of the fresh water turtle Mauremys caspica to step-function changes in the ambient temperature

1. 1.|The turtle Mauremys caspica cools significantly faster than it heats in air. The heating/cooling ratio is 0.49.

2. 2.|The variation of body temperature in relation to time-course in response to a step-function change of environmental temperature, fitted to a second-order system improves that of a first-order system.

3. 3.|The gradient between ambient temperature (T_a) and equilibrium body temperature (T_b) increases significantly and progressively when ambient temperature rises over 25°C.

4. 4.|At 40°C thermoregulatory hyperventilation was detected, implying an increase in air convection requirement (ventilation relative to O₂ consumption, ).

Polar bear thermoregulation: Effect of oil on the insulative properties of fur

1. 1.|Oil caused a substantial decrease in the insulative value of polar bear (Ursus maritimus) pelts measured in vitro.

2. 2.|Following oil contamination the calm air heat transfer coefficient increased by a factor of 2 to 5: the wind coefficient averaged 290% greater and the solar utilization increased by 55%.

3. 3.|Conductance through oil-covered furs remained high at winter temperatures (T_a = 0.6°C) but decreased with time at summer temperatures (T_a = 24.7°C).

4. 4.|The most viscous of the three oils tested had a more consistently negative effect on insulation.

Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

Thermal responses and temperature tolerance of a dessert ant-lion larva

1. 1.Cueta trivirgata larvae construct pits in the dry Kuiseb River bed in the Namib Desert.

2. 2.An art, Ocymyrmex robustior comprises 65.4% of the biomass of prey consumed by the ant-lions.

3. 3.O. robustior is active between surface temperatures of 27–68°C.

4. 4.Ant-lions tolerate high body temperatures (LD₅₀ = 53.4°C).

5. 5.By exploiting the pit microclimate and by digging below the surface during extreme thermal loads, ant-lions can capture prey at surface temperatures of 13–63°C.

6. 6.These behavioural and physiological adaptations enable ant-lions to maximize the duration of vigilance and hence prey capture success.