The time course of miniature endplate currents and its modification by receptor blockade and ethanol

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.     

Atypical miniature end plate currents in neuromuscular synapses of the rat under normal conditions, after acetylcholinesterase inhibition and cooling

In the end-plates of rat diaphragm among atypical miniature end-plate currents (MEPCs) 2.9% were giant and 5.1% were slowly rising. The frequency of the giant MEPCs was decreased when temperature was lowered and increased when acetylcholinesterase (AChE) was inhibited; the latter effect was reversed if d-tubocurarine was added. Frequency of the slowly rising MEPCs changed insignificantly by all conditions. It is suggested that a highly temperature-dependent presynaptic mechanism of giant MEPC generation does exist which is activated by acetylcholine (ACh). Data about changes in the time course of the slowly rising MEPCs by AChE inhibition and lowering of temperature make it possible to suggest that the slowly rising MEPCs may be accounted for either slow release of ACh quanta or release of quanta on large distances from synaptic cleft and postsynaptic cholinoreceptors. The latter is possible if ACh quanta are released from synaptic Schwann cell to periaxonial space.     

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.     

Electrogenic Protonation of the Secondary Quinone Acceptor Q<Subscript>B</Subscript> in Spinach Photosystem II Complexes Incorporated into Lipid Vesicles

The generation of transmembrane electric potential difference (delta psi) in quinone acceptor complex of proteoliposomes containing core complexes of photosystem II from spinach was studied using for the measurements a direct electrometric technique. Besides the fast increase in the membrane potential associated with the electron transfer between the redox-active tyrosine 161 residue (Y(Z)) in D1 polypeptide and the primary quinone acceptor Q(A), an additional electrogenic phase with tau approximately 0.85 msec at pH 7.3 and the maximal relative amplitude of approximately 11% of the Y(Z)ox Q(A)- phase was observed after the second light flash. The sensitivity of this phase to diuron (an inhibitor of electron transfer between Q(A) and the secondary quinone acceptor Q(B)), the dependence of its amplitude on the light flash parity, and also a decrease in its rate constant with increase in pH indicated that it was due to dismutation of Q(A)- and Q(B)- with the subsequent protonation of a doubly reduced plastoquinone molecule: Q(A)- Q(B)- + 2H+ --> Q(A)Q(B)H2.     

Determining K+ channel activation curves from K+ channel currents

Potassium ion channels are generally believed to have current-voltage (IV) relations which are linearly related to driving force ( V - E(K)), where V is membrane potential and E(K) is the potassium ion equilibrium potential. Consequently, activation curves for K+ channels have often been measured by normalizing voltage-clamp families of macroscopic K+ currents with (V - E(K)), where V is the potential of each successive step in the voltage clamp sequence. However, the IV relation for many types of K+ channels actually has a non-linear dependence upon driving force which is well described by the Goldman-Hodgkin-Katz relation. When the GHK dependence on (V - E(K)) is used in the normalization procedure, a very different voltage dependence of the activation curve is obtained which may more accurately reflect this feature of channel gating. Novel insights into the voltage dependence of the rapidly inactivating I(A) channels Kv1.4 and Kv4.2 have been obtained when this procedure was applied to recently published results.     

Evaluation of characteristic parameters for the neurotransmitter release mechanisms at the neuromuscular junction

The process of neurotransmitter release at the neuromuscular junction needs to be represented appropriately in modeling of the synaptic chemical transmission as a reaction-diffusion system. The release mechanisms of the expanding pore and the acceleration are analyzed by the computer simulation with respect to the effects of the characteristic parameters in the mechanisms on spontaneous generation of the miniature endplate current (MEPC), leading to the following evaluation. In the expanding pore mechanism the expanding rate of the pore more than 10 nm ms(-1) and the diffusion coefficient of acetylcholine in the synaptic cleft (D(c)) of about 1.0 x 10(-6) cm2 s(-1) yield the maximum amplitude, the rise time and the decay time constant of the MEPC in agreement with the empirical data. In the active release mechanism the 10-fold acceleration of the natural diffusion and a similar value of D(c) are required to suit for the empirical MEPC.     

The relationship between O2 saturation mixed venous blood and pressure in the trunk of the pulmonary artery for healthy people and patients with heart defects

Investigated the relationship between pulmonary artery pressure (P(LA)) and the oxygen saturation of mixed venous blood (S(V)) in 12 group's of surveyed individuals (1750 men and 1026 women). We have identified a function (P(LA)) between P(LA) = f(S(V)), and a function (S(V)) S(V) = f(P(LA)) was estimated for each group based on direct measurements of P(LA) and S(V). We found, that factors were subordinated to the dependences for a P(LA) = f(S(V)), P(LA) = a x (S(V))(-b), where b = = -0.2284a + 0.6564 men - and b = -0.285a + 1.2947 in women and the other for -S(V) = f(P(LA)), S(V) = c x (P(LA))(-d) where d = -0.251311n(c) + 1.0212; (R2 = 0.8993) men and d = -1.96451n(c) + 2.852; (R2 = 0.9674) women. Each group occupies a position on the curves represented by equations. The subjects with a diagnosis of functional murmur in the heart and patients with congenital stenos is of the aortic valve form a group, provisionally designated as "group norms", which is characterized by its dependence P(LA) = f(S(V)), and -S(V) = f(P(LA)). The men in "group norms" additionally include patients with coronary heart disease. The equation - CO = Cons.O2/(KEK(S(A) - (c x (P(LA))(-d). It relates the P(LA), caused by different reasons, with the corresponding saturation of mixed venous blood, and when the saturation of mixed venous blood is also caused by various factors, set the corresponding P(LA). Interdependent changes in physiological parameters of blood circulation and gas exchange in humans is established equilibrium between systemic and pulmonary circulation.     

Calibration of multiple poliovirus molecular clocks covering an extended evolutionary range

We have calibrated five different molecular clocks for circulating poliovirus based upon the rates of fixation of total substitutions (K(t)), synonymous substitutions (K(s)), synonymous transitions (A(s)), synonymous transversions (B(s)), and nonsynonymous substitutions (K(a)) into the P1/capsid region (2,643 nucleotides). Rates were determined over a 10-year period by analysis of sequences of 31 wild poliovirus type 1 isolates representing a well-defined phylogeny derived from a common imported ancestor. Similar rates were obtained by linear regression, the maximum likelihood/single-rate dated-tip method, and Bayesian inference. The very rapid K(t) [(1.03 +/- 0.10) x 10(-2) substitutions/site/year] and K(s) [(1.00 +/- 0.08) x 10(-2)] clocks were driven primarily by the A(s) clock [(0.96 +/- 0.09) x 10(-2)], the B(s) clock was approximately 10-fold slower [(0.10 +/- 0.03) x 10(-2)], and the more stochastic K(a) clock was approximately 30-fold slower [(0.03 +/- 0.01) x 10(-2)]. Nonsynonymous substitutions at all P1/capsid sites, including the neutralizing antigenic sites, appeared to be constrained by purifying selection. Simulation of the evolution of third-codon positions suggested that saturation of synonymous transitions would be evident at 10 years and complete at approximately 65 years of independent transmission. Saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete at 100 years. The rapid evolution of the K(t), K(s), and A(s) clocks can be used to estimate the dates of divergence of closely related viruses, whereas the slower B(s) and K(a) clocks may be used to explore deeper evolutionary relationships within and across poliovirus genotypes.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Miniature excitatory synaptic ion currents in the earthworm Lumbricus terrestris body wall muscles

The miniature excitatory postsynaptic currents (MEPCs) of the muscle cells of the earthworm Lumbricus terrestris were recorded by glass microelectrodes. In a single synaptic zone, three types of MEPC were recorded: a fast single-exponential type that decayed with tau =0.9 ms, a slow single-exponential with tau = 9.2 ms and a two-exponential MEPC with tau = 1.3 and 8.5 ms, respectively. The muscle cells of earthworms contain populations of yet-unidentified ionic channels that might be different from the common nicotinic and muscarinic groups of acetylcholine receptors, since these MEPCs are not sensitive to d-tubocurarine, atropine, benzohexonium or proserine. Alternatively, besides ACh receptors, the membrane may contain receptors for another yet-unidentified excitatory transmitter.     

Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Characteristics of postsynaptic potentials and ionic currents in synapses of fast and slow rat muscle fibers

Miniature end-plate currents and potentials (MEPPs and MEPCs, respectively) were recorded in fast and slow rat muscle fibers by extracellular focal recording and voltage clamp techniques. The rise time and the half-decay time of these potentials and currents were 1.3–1.4 times greater in slow fibers than in fast. A similar difference, but lesser in degree, also was observed after inhibition of acetylcholinesterase. Decline of the end-plate currents remained, generally speaking, exponential and its rate depended on the clamped voltage. The percentage distribution of fibers of different types by duration of MEPP and MEPC in fast and slow muscles correlated with the percentage distribution of fibers identified in these muscles on the basis of other parameters. Factors determining the time course of the responses (acetylcholinesterase activity, length of diffusion pathways, differences in passive electrical properties of the membrane), and their importance for synapses of different types, are discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 627–636, November–December, 1980.     

Effect of temperature on alpha olefin sulfonate induced softening of gelatin hydrogels

Dynamic light scattering (DLS) and oscillatory rheology experiments were performed to study temperature dependence (T=10-25 degrees C) of the interactions in hydrogels of gelatin with AOS (alpha olefin sulfonate, anionic surfactant) for surfactant concentrations in the range 25-100 mM, chosen larger than cmc (approximately 8mM). The network mesh size (xi) values deduced from fastmode diffusivity (D(f)) data obtained from dynamic structure factor measurements, S(q, t) approximately exp(-D(f)q(2)t) (for t相似文献   

Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with catechins (catechin (CA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG)) and related compounds (5-methoxyresorcinol (MR), 4-methylcatechol (MC), and n-propyl gallate (PG)) was performed in ethanol at 35 degrees C. MR, MC, and PG are considered to be a model of resorcinol (A)-, catechol (B)-, and gallate (G)-rings in catechins, respectively. The overall rate constants, k(Q) (= k(q) + k(r), physical quenching + chemical reaction), for the reaction of catechins with (1)O(2) increased in the order of PG < MR < MC < CA < EC < EGC < ECG < EGCG. In a comparison of the rate constants, the relationship between quenching rates and chemical structures is discussed. The catechins which have lower peak oxidation potentials, E(P), show higher reactivities. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by catechins. The k(Q) values of EGCG (1.47 x 10(8) M(-1) s(-1)) and ECG (7.81 x 10(7)) were found to be larger than those of lipids (1.3 x 10(5)-1.9 x 10(5) M(-1) s(-1)), amino acids (<3.7 x 10(7)), and DNA (5.1 x 10(5)). Further, these values are similar to those (1.15 x 10(8)-2.06 x 10(8) M(-1) s(-1)) of alpha- and gamma-tocopherol, ubiquinol-10, and gamma-tocopherol hydroquinone (plastoquinol model). The result suggests that catechins may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

Relation between subsynaptic receptor blockade and response to quantal transmitter at the mouse neuromuscular junction

When a quantum of transmitter is released into a synaptic cleft, the magnitude of the subsynaptic response depends upon how much transmitter becomes bound to receptors. Theoretical considerations lead to the conclusion that if receptor density is normally high enough that most of the quantal transmitter is captured, subsynaptic quantal responses may be insensitive to receptor blockade. The effectiveness of receptor blockers in depressing the subsynaptic response should be diminished by interference with processes that normally dispose of transmitter, but increased if receptor density is reduced. In conformity with equations derived from a simple mathematical model, the apparent potency of (+)- tubocurarine (dTC) to depress the peak height of miniature end-plate currents (MEPCs) in mouse diaphragm was substantially reduced by poisoning of acetylcholinesterase (AChE) and increased by partial blockade of receptors by immunoglobulin G from patients with myasthenia gravis or alpha-bungarotoxin. We calculated from the data that normally capture of quantal acetylcholine (ACh) by receptors is approximately 75% of what it would be if there were no loss of ACh by hydrolysis or diffusion of ACh form the synaptic cleft. This fraction is increased to approximately 90% by poisoning of AChE. Conversely, it normally requires blockade of approximately 80% of receptors-and after AChE poisoning, approximately 90% of receptors-to reduce ACh capture (and MEPC height) by 50%. The apparent potency of dTC to alter MEPC time- course (after AChE poisoning) and to depress responses to superperfused carbachol was much greater than its apparent potency to depress MEPC height, but corresponded closely with the potency of dTC to block receptors as calculated from the action of dTC on MEPC height. These results indicate that the amplitude of the response to nerve-applied acetylcholine does not give a direct measure of receptor blockade; it is, in general, to be expected that an alteration of subsynaptic receptor density may not be equally manifest in responses to exogenous and endogenous neurotransmitter.     

Effect of Temperature on Rates of Alternative and Cytochrome Pathway Respiration and Their Relationship with the Redox Poise of the Quinone Pool

We investigated the effect of short-term changes in temperature on alternative (Alt) and cytochrome (Cyt) pathway respiration, both in intact tissues and isolated mitochondria of 14-d-old cotyledons of soybean (Glycine max L. cv Stevens). We also established the extent to which temperature alters the interaction between the oxidizing pathways and the level of ubiquinone (UQ) reduction (UQ(r)/UQ(t)). No difference was found between the temperature coefficient of respiration (Q(10); proportional change per 10 degrees C) of Alt and Cyt pathway respiration in cotyledon slices (Q(10) = 1.92 and 1.86, respectively). In isolated mitochondria, the Q(10) of the fully activated Alt pathway (Q(10) = 2.24-2.61) was always equal to, or higher than, that of Cyt c oxidase (COX) alone (Q(10) = 2.08) and the complete Cyt pathway (Q(10) = 2.40-2.55). This was true regardless of substrate or whether ADP was present. There was little difference in the Q(10) of the Cyt pathway with or without ADP; however, the Q(10) of COX was substantially lower in the presence of an uncoupler (Q(10) = 1.61) than its absence (Q(10) = 2.08). The kinetics of Alt and Cyt pathway activity in relation to UQ(r)/UQ(t) were not affected by temperature. For a given UQ(r)/UQ(t) value, the proportion of maximum flux taking place was similar at all temperatures for both pathways (+/-ADP). However, the Q(10) of the Alt and the Cyt pathways (+ADP) increased with increasing UQ(r)/UQ(t). We conclude that the Alt pathway is not less temperature sensitive than the Cyt pathway or COX per se and that changes in the degree of control exerted by individual steps in the respiratory apparatus could result in changes in the Q(10) of mitochondrial O(2) uptake.     

Physical properties, crystallization, and spherulite growth of linear and 3-arm poly(L-lactide)s

The physical properties, crystallization, and spherulite growth behavior and mechanism of linear and 3-arm poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] have been investigated using absolute molecular weight as a molecular index. The branching reduces the chain mobility of PLLA and must be excluded from the crystalline regions. The former factor gives the higher glass transition temperature (T(g)) and starting temperature for thermal degradation (T(d,S)) of 3-arm PLLA compared with those of linear PLLA. On the other hand, both the former and the latter factors lead to the higher cold crystallization temperature (T(cc)), the longer induction period for spherulite growth (t(i)), the lower melting temperature (T(m)), crystallinitiy (X(c)), and radius growth rate of the spherulties (G) for the 3-arm PLLA compared with those for the linear PLLA. The G of 3-arm PLLA showed the vague dependence on number-average molecular weight (M(n)), probably because the branching effect was balanced with the molecular weight effect. At the M(n) exceeding critical values, the linear and 3-arm PLLA crystallize in regime II or regime III kinetics, depending on crystallization temperature (T(c)). In contrast, at the M(n) below critical values, the linear and 3-arm PLLA crystallize according solely to regime III and regime II kinetics, respectively, for all the T(c).     

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.