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.

Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.

2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]₄₄₅ is 3.0·10⁵ degree·cm⁻²·dmole⁻¹ heme a for membrane-bound oxidase compared to 1.6·10⁵ degree·cm⁻²·dmole⁻¹ heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.

3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.

4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c₁ or from cytochrome b₅₆₁ in either the oxidized or the reduced form.

5. 5. Cytochrome b₅₆₆ in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b₅₆₆ reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b₅₆₆ is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b₅₆₆ on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

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.

Redox state of the partially reduced cytochrome aa3-cyanide complex

The low-spin ferric cyanide complex of beef heart cytochrome aa₃ can be partially reduced by stoichiometric additions of ferrous cytochrome c or by similar additions of N,N,N′,N′-tetramethyl-p-phenylene diamine. In both cases the initial ratio of cytochrome c oxidized: cytochrome a reduced or Wurster's Blue: cytochrome a reduced approximates the value 2. It is concluded that the binding of a single HCN prevents the reduction of both cytochrome a₃ and its associated EPR-invisible Cu atom.     

Skin temperatures, thermosensory and pleasantness estimates in case of heterogeneity in the thermal environment

1. 1. Seven thermal conditions were imposed on male sitting subjects (slightly clothed: 0.6 clo).

2. 2. A thermal mannikin was also used to determine the exact operative temperature, T₀.

3. 3. Conditions were: uniform (UN: all parameters at 24.5°C, air velocity at 0.15 ms⁻¹), heated ceiling (HC at 45°C), heated floor (HF at 34°C), cold floor (CF at 14°C), two conditions of one cold wall at 6°C (CW1 and CW2 respectively with and without air temperature compensation) and increased air velocity (AV at 0.4 ms⁻¹).

4. 4. Local skin temperatures and answers to questionnaires were obtained.

5. 5. Skin temperature variations were affected by conditions and slight T₀ changes.

6. 6. Comfort judgments were fairly well related to T₀, especially when expressed as differences between actual non-uniform environment and the uniform one.

7. 7. It is concluded that, in case of non-uniform environments close to thermoneutral zone, thermal comfort or discomfort reflects the climate alterations better than the thermal sensation does.

Dihydroxamic acid complexes of iron (III): ligand pKa and coordinated water hydrolysis constants

A series of dihydroxamic acid ligands of the formula [RN(OH)C(O)]₂(CH₂)_n, (n = 2, 4, 6, 7, 8; R = CH₃, H) has been studied in 2.0 M aqueous sodium perchlorate at 25.0 °C. These ligands may be considered as synthetic analogs to the siderophore rhodotorulic acid. Acid dissociation constants (pK_a) have been determined for the ligands and for N-methylacetohydroxamic acid (NMHA). The pK_a1 and pK_a2 values are: n = 2, R = CH₃ (8.72, 9.37); N = 4, R = CH₃ (8.79, 9.37); N = 6, R = CH₃; N = 7, R = CH₃ (8.95, 9.47); N = 8, R = CH₃ (8.93, 9.45); N = 8, R = H (9.05, 9.58). Equilibrium constants for the hydrolysis of coordinated water (log K) have been estimated for the 1:1 feeric complexes of the ligands n = 2, 4, 8; R = CH₃. The N = 8 ligand forms a monomeric complex with Fe(III) while the n = 2 and 4 ligands form dimeric complexes. For hydrolysis of the n = 8 monomeric complex, log K₁ = −6.36 and log K₂ = −9.84. Analysis of the spectrophotometric data for the dimeric complexes indicates deprotonation of all four coordinated waters. The successive hydrolysis constants, log K_1–4, for the dimeric complexes are as follows: n = 2 (−6.37, −5.77, −10.73, −11.8); n = 4 (−5.54, −5.07, −11.57, −10.17). The log K₂ values for the dimers are unexpectedly high, higher in fact than log K₁, inconsistent with the formation of simple ternary hydroxo complexes. A scheme is proposed for the hydrolysis of the ferric dihydroxamate dimers, which includes the possible formation of μ-hydroxo and μ-oxo bridges.     

Inhibition of chitosan-immobilized urease by slow-binding inhibitors: Ni, F and acetohydroxamic acid

The inhibitions by Ni²⁺ and F⁻ ions and by acetohydroxamic acid of jack bean urease covalently immobilized on chitosan membrane was studied (pH 7.0, 25°C) and compared with those of the native enzyme. The reaction progress curves of the immobilized urease-catalyzed hydrolysis of urea were recorded in the absence and presence of the inhibitors. They revealed that the inhibitions are of the competitive slow-binding type similar to those of native urease. The immobilization weakened the inhibitory effect of the inhibitors on urease as measured by the inhibition constants K_i^*. The increase in their values: 17.9-fold for Ni²⁺, 26.5-fold for F⁻ and 1.7-fold for acetohydroxamic acid, was accounted for by environmental effects generated by heterogeneity of the urease–chitosan system: (1) mass transfer limitations imposed on substrate and reaction product in the external solution, and (2) the increase in local pH on the membrane produced by both the enzymatic reaction and the electric charge of the support. By relating the K_M/K_i^* ratio to the electrostatic potential of chitosan it was found that while the reduced Ni²⁺ inhibition is mainly brought about by the potential, inhibition by acetohydroxamic acid is independent of the potential, and the acid inhibits urease in its non-ionic form. The reduction in F⁻ inhibition was ascribed to the increased pH in the local environment of the immobilized enzyme.     

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.

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.     

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10⁴ M⁻¹ · s⁻¹. In the absence of cyanide, ferrocytochrome a₃ appears at a rate (k_obs) of 0.016 s⁻¹. Ferricytochrome a₃ maintains its 418 nm Soret maximum until reduced. The rate of a₃ reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a₃/cyanide complex occurs at the same rate as a₃ reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a₃ with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a₃ shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a₃. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (k_obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu_A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu_A at redox values more negative than +240 mV) followed by a slow reduction of Cu_B. Cu_B reduction is proposed to spin-uncouple cytochrome a₃ to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.     

Body temperature in arboreal and nonarboreal anuran amphibians: Differential effects of a small change in water vapor density

1. 1.|Body temperatures (T_b) and contaneous evaporative water loss rates (CWL) were measured in tree frogs (Hyla cinerea) and toads (Bufo valliceps) exposed to cyclical ramp changes in water vapor density (WVD) between 7.5 and 9.8 gm⁻³ (1 cycle h⁻¹ at an air temperature of 27.0°C.

2. 2.|CWL was 3.3 times greater in toads than in tree frogs.

3. 3.|T_b in toads cycled directly with WVd; WVD accounted for 98% of the variation in toad T_b.

4. 4.|T_b in tree frogs was independent of WVD, probably due to changes in skin resistance to water loss.

Shifts in thermoregulatory patterns with pregnancy in the poikilothermic mammal—The naked mole-rat (Heterocephalus glaber)

1. 1. The naked mole-rat (Heterocephalus glaber) is a poikilothermic mammal. During gestation metabolic shifts that differ from both mammalian and reptilian thermoregulatory patterns occurred.

2. 2. Body temperature was directly dependent on ambient temperature. At low ambient temperatures the temperature differential (T_b − T_a) was approximately 3°C, whereas at higher ambient temperatures the temperature differential diminished.

3. 3. In early pregnancy (prior to week 3) oxygen consumption at low ambient temperatures was greater than that of non-reproductive animals. A maximal metabolic rate (3.2 ± 1.0 ml O₂ . g⁻¹ . h⁻¹) occurred at an ambient temperature of 27°C. Thereafter the endothermic pattern of metabolism with increasing ambient temperatures was evident. Oxygen consumption decreased with increasing ambient temperature to minimal rates of 1.2 ± 0.1 ml O₂ . g⁻¹ . h⁻¹ over the ambient temperature range of 31–34°C.

4. 4. Oxygen consumption in late pregnancy (1.8 ± 0.1 ml O₂ . g⁻¹ . h⁻¹) was not correlated with ambient temperature over the entire ambient temperature range measured (24–36°C).

5. 5. Differences in thermoregulation in early and late pregnancy may be attributed to different rates of heat loss as a consequence of (a) changes in surface area and body mass or (b) vascular changes. Furthermore the thermoregulatory changes in late pregnancy may indicate that maximal overall metabolic capacity had been reached, for peak resting metabolism (expressed per animal rather than per gram body mass) in early pregnancy was similar to observed metabolism in late pregnancy.

6. 6. The dissociation of metabolism from both ambient temperature and body temperature in late pregnancy could confer an energetic advantage to this arid dwelling underground inhabitant; for it may enable the breeding female to partition a greater portion of available energy into reproduction.

Kinetics and equilibria of Ga(III)---thiocyanate complex formation. Mechanism of ligand substitution reactions of Ga(III) in aqueous solution

The kinetics and equilibria of complex formation by Ga(III) with NCS⁻ in aqueous solution have been measured over a range of acidities and temperatures, the contributing paths to the reaction resolved, and their rate constants and activation parameters determined. The hydrolysis equilibria required to carry out this resolution of kinetic behaviour have also been measured.

Unlike the other reported complexation reactions of Ga(III) in aqueous solution, the separate reaction pathways can be assigned with no ambiguity. At 25 °C and ionic strength 0.5 M, the observed forward rate constant for the complex formation is described by {k₁ + k₂K_1h/[H⁺] + k₃K_1hK_2h/[H⁺]²} M⁻¹ s⁻¹. For these conditions, the first and second successive hydrolysis constants of Ga(H₂O)₆³⁺ are given by pK_1h = 3.69 ± 0.01 and pK_2h = 3.74 ± 0.04. The rate constants corresponding to the reactions of the species Ga(H₂O)₆³⁺, Ga(H₂O)₅(OH)²⁺ and Ga(H₂O)₄(OH)₂⁺ with NCS⁻ are k₁ = 57 ± 4 M^{−1 −1}, k₂ = (1.08 ± 0.01) × 10⁵ M⁻¹ s⁻¹ and k₃ = 3 × 10⁶ M⁻¹ s⁻¹ respectively. The complexation equilibrium quotient [GaNCS²⁺]/([Ga³⁺][NCS⁻]) has been independently determined by spectrophotometric titration to be 20.8 ± 0.3 M⁻¹ at 25 °C and ionic strength 0.5 M.

These kinetic results lead to an interpretation of the data, and a reinterpretation of other data for aquo-Ga(III) complex formation kinetics from the literature which support the assignment of a dissociative interchange mechanism for these reactions rather than the associative activation mode sometimes proposed.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Comparative aspects of the thermal biology of the short-tailed gerbil, Desmodillus auricularis, and the bushveld gerbil, Tatera leucogaster

1. 1. The response of oxygen consumption (VO₂), thermal conductance (C_d and C_min, body temperature (T_b), and evaporative water loss (EWL) of Tatera leucogaster and Desmodillus auricularis were measured over the range of ambient temperatures (T_a) from 5–35°C.

2. 2. Basal metabolic rate (BMR) of T. leucogaster was 0.841 ± 0.049 ml O₂ g⁻¹ h⁻¹ and lower than predicted, while that of D. auricularis was similar to the expected value (1.220 ± 0.058 ml O₂ g⁻¹ h⁻¹). D. auricularis had a high, narrow thermoneutral zone (TNZ) typical of nocturnal, xerophilic, burrowing rodents.

3. 3. D. auricularis and T. leucogaster regulated T_b over the range T_a = 5–35°C and kept EWL and dry thermal conductance at a minimum below the TNZ. However, the EWL of T. leucogaster increased rapidly above T_a = 30°C.

4. 4. After comparison with data from other species, it was concluded that there is an optimum size for xeric, nocturnal, burrowing rodents.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

Transport of glutamate in rat-liver mitochondria

The transport of glutamate across the membrane of rat-liver mitochondria has been studied.

1. 1. Glutamate can be transported across the mitochondrial membrane in exchange for OH⁻ (or together with H⁺).

2. 2. Intramitochondrial glutamate is not extruded from the mitochondria by addition of aspartate when the mitochondria are preloaded with glutamate.

3. 3. N-Ethylmaleimide is a specific inhibitor of the movement of glutamate across the mitochondrial membrane.

Relative stability of chlorophyll complexes in vivo

The time course of aerobic photobleaching of various chlorophyll-protein complexes in vivo at high light intensities was studied with isolated Aspidistra elatior chloroplasts.

1. 1. C_a680 bleaching starts with the onset of irradiation and, initially, proceeds linearly with time. Washing the chloroplasts causes a nearly constant increase of the bleaching rate throughout the experiment.

2. 2. C_a670 does not appreciably, if at all, bleach initially; subsequently, bleaching proceeds linearly with time and at a slightly higher rate than that for C_a680. Washing makes C_a670 bleach concomitantly with the onset of illumination, and at a nearly constant rate.

3. 3. Bleaching at 665 nm is likely to start only after a relatively long period of illumination. Washing shows no effects during this period. Once bleaching has started, washing causes its rate to increase.

4. 4. No indication of the occurrence of “short-wave” chlorophyll a forms other than C_a670 and C_a665 was obtained.

5. 5. C_b bleaching starts concomitantly with illumination at a low rate. The rate increases more or less exponentially with time. Washing enhances bleaching in two steps.

6. 6. The importance of the results is discussed.