Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 μM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.     

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl(-) conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca(2+)-activated Cl(-) conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl(-)/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca(2+) signal in response to mucosal nucleotides that may contribute to the increased Cl(-)/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca(2+) signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.     

Predisposition to multifactorial pathology in residents of the city in the zone around the "Maiak" atomic industry enterprise

According to present achievements in radiation and molecular genetics it is possible to expect that due to chronic radiation exposure the relative increasing of genetic risk in following generations will be observed. It will be due, in the first place, to increasing a percent of multifactorial diseases (MFD). Most of geneticists refer bronchial asthma (BA) to multifactorial diseases. Genetics and epidemiological analysis of liability to BA in population of Ozyorsk situated in the control area of "Mayak" Production Association (PA "Mayak") was accomplished. Population risk, as probability for an individual in population to develop BA up to the end of life is 2.69%. It's higher than in Moscow (2.03%) (p < 0.05). The excess is due to significantly higher values in females (2.96% against 1.93%). Population risk to fall ill of BA in females of Ozyorsk (2.96%) is significantly higher than in males (2.4%). Prevalence of BA in Ozyorsk (0.60%) is significantly higher than in Moscow (0.47%) due to higher values in females (0.65% against 0.47%). The total risk to fall ill of BA amongst relatives is 5.4% that is twice higher than the population risk. The heritability of BA is 0.71 (in Moscow-0.70).     

A two-state cell damage model under hyperthermic conditions: theory and in vitro experiments

The ultimate goal of cancer treatment utilizing thermotherapy is to eradicate tumors and minimize damage to surrounding host tissues. To achieve this goal, it is important to develop an accurate cell damage model to characterize the population of cell death under various thermal conditions. The traditional Arrhenius model is often used to characterize the damaged cell population under the assumption that the rate of cell damage is proportional to exp(-EaRT), where Ea is the activation energy, R is the universal gas constant, and T is the absolute temperature. However, this model is unable to capture transition phenomena over the entire hyperthermia and ablation temperature range, particularly during the initial stage of heating. Inspired by classical statistical thermodynamic principles, we propose a general two-state model to characterize the entire cell population with two distinct and measurable subpopulations of cells, in which each cell is in one of the two microstates, viable (live) and damaged (dead), respectively. The resulting cell viability can be expressed as C(tau,T)=exp(-Phi(tau,T)kT)(1+exp(-Phi(tau,T)kT)), where k is a constant. The in vitro cell viability experiments revealed that the function Phi(tau,T) can be defined as a function that is linear in exposure time tau when the temperature T is fixed, and linear as well in terms of the reciprocal of temperature T when the variable tau is held as constant. To determine parameters in the function Phi(tau,T), we use in vitro cell viability data from the experiments conducted with human prostate cancerous (PC3) and normal (RWPE-1) cells exposed to thermotherapeutic protocols to correlate with the proposed cell damage model. Very good agreement between experimental data and the derived damage model is obtained. In addition, the new two-state model has the advantage that is less sensitive and more robust due to its well behaved model parameters.     

Coordinated production and utilization of FADH2 by NAD(P)H-flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase

4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH(2)) utilizing monooxygenase. Its cosubstrate, FADH(2), is supplied by HpaC, an NAD(P)H-flavin oxidoreductase. Because HpaB is the first enzyme for 4HPA metabolism, FADH(2) production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA. An important question is how FADH(2) is produced and used, as FADH(2) is unstable in the presence of free O(2). One solution is metabolic channeling by forming a transitory HpaB-HpaC complex. However, our in vivo and in vitro data failed to support the interaction. Further investigation pointed to an alternative scheme for HpaB to sequester FADH(2). The intracellular HpaB concentration was about 122 microM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH(2) (K(d) of 70 nM), suggesting that most FADH(2) is bound to HpaB in vivo. The HpaB-bound FADH(2) was either used to rapidly oxidize 4HPA or slowly oxidized by O(2) to FAD and H(2)O(2) in the absence of 4HPA. Thus, HpaB's high intracellular concentration, its high affinity for FADH(2), its property of protecting bound FADH(2) in the absence of 4HPA, and its ability to rapidly use FADH(2) to oxidize 4HPA when 4HPA is available can coordinate FADH(2) production and utilization by HpaB and HpaC in vivo. This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date.     

Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method.

Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

Photoreaction cycle of the light, oxygen, and voltage domain in FKF1 determined by low-temperature absorption spectroscopy

Flavin-binding Kelch repeat F-box (FKF1) protein plays important roles in the photoregulation of flowering in Arabidopsis. FKF1 has a light, oxygen, and voltage (LOV) sensing domain binding a flavin mononucleotide (FMN) as a chromophore noncovalently. Photoreaction of the FKF1-LOV polypeptide was studied by low-temperature absorption spectroscopy. Upon blue light irradiation, a ground state, D(450), is converted to S(390) known as a cysteinyl-flavin adduct intermediate in the photoreaction of phototropin. Below 150 K, bleaching of D(450) was much reduced and a new photoproduct, Z(370), appeared as well as S(390) formation. The calculated absorption spectrum for Z(370) is very similar to those of flavoproteins in an anion radical state. On the basis of the results that S(390) formation proceeds to Z(370) formation and that Z(370) formed at low temperatures reverts to D(450) upon temperature increase, Z(370) is concluded to be not an intermediate from D(450) to S(390). Z(370) is suggested to be formed from the biradical triplet-excited state after relaxing to the ground state with the FMN anion radical trapped at the low temperature, in which the SH of the cysteine is in the wrong position that is able to produce a radical pair but unable to form the cysteinyl-flavin adduct. The counter SH in the cationic radical state may revert to the ground state by extracting an electron from the unidentified amino acid residue. Interestingly, S(390) that has been thought to be irreversible to D(450) was revealed to revert to D(450) very slowly with a half-life time of 62.5 h in solution at 298 K. The photoreaction mechanism is discussed in reference to the calculated activation energy of the reaction processes.     

Discovery of the true peroxy intermediate in the catalytic cycle of terminal oxidases by real-time measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O(2) to the fully reduced enzyme is followed by the fast (5 micros) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O(2) by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe (3+)(d)-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P(M) intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b(558) in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b(595) are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 micros; it decays into oxoferryl species due to oxidation of low spin heme b(558) that is linked to significant charge translocation across the membrane.     

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres

A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.     

Inhibition of Cell-Free Protein Synthesis by Hydrostatic Pressure

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.     

Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.     

Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).     

The Zellweger syndrome: deficient conversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) and normal delta 4-desaturase activity in cultured skin fibroblasts

The metabolism of docosahexaenoic acid (22:6(n-3)) and adrenic acid (22:4(n-6)) was studied in cultured fibroblasts from patients with the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. It was shown that [4,5- 3H]22:6(n-3) is retroconverted to labelled eicosapentaenoic acid (20:5(n-3)) in normal and X-ALD fibroblasts, while this conversion is deficient in Zellweger fibroblasts. [U- 14C]Eicosapentaenoic acid (20:5(n-3)) is elongated to docosapentaenoic acid (22:5(n-3)) in all three cell lines. With [U- 14C]20:5(n-3) as the substrate, shorter fatty acids were not detected. With [4,5- 3H]22:6(n-3) as the substrate, labelled fatty acids were esterified in the phospholipid- and triacylglycerol-fraction to approximately the same extent in all three cell lines. [2- 14C]Adrenic acid (22:4(n-6)) was desaturated to 22:5(n-6) and elongated to 24:4(n-6) in all three cell lines and to the largest extent in the Zellweger fibroblasts. This agrees with the view that the delta 4-desaturase is not a peroxisomal enzyme. The observation that the retroconversion of 22:6(n-3) to 20:5(n-3) is deficient in Zellweger fibroblasts strongly suggest that the beta-oxidation step in the retroconversion is a peroxisomal function. Peroxisomal very-long-chain (lignoceroyl) CoA ligase is probably not required for the activation of 22:6(n-3), since the retroconversion to 20:5(n-3) is normal in X-ALD fibroblasts.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested.     

Metabolism of D- and L-glyceraldehyde in adipose tissue: a stereochemical probe for glycerokinase activity

Distributions of (14)C have been determined in free glycerol, in glycerol from triglycerides, in glucose from glycogen, and in lactate after incubation of d-glyceraldehyde-3-(14)C and l-glyceraldehyde-3-(14)C with rat adipose tissue. The distributions are interpreted in terms of presently accepted possible reactions for the initial metabolism of glyceraldehyde. Formation of glycerol-1-(14)C from d-glyceraldehyde-3-(14)C indicates that in adipose tissue glyceraldehyde is reduced to glycerol. Incorporation of (14)C from d-glyceraldehyde-3-(14)C into carbon 3 of the glycerol of triglyceride indicates that d-glyceraldehyde is either phosphorylated or oxidized to d-glyceric acid, or both, in its initial metabolism. Incorporation of (14)C from l-glyceraldehyde-3-(14)C into carbon 3 of glycerol indicates that l-glyceraldehyde is reduced to glycerol, which is phosphorylated and (or) converted to d-glyceric acid via l-glyceric acid. Some (14)C from l-glyceraldehyde-3-(14)C is incorporated into carbon 1 of glycerol of triglycerides and carbon 4 of glycogen; the explanation for this incorporation is uncertain.     

The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria

The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.