Rapid-equilibrium rate equations for the enzymatic catalysis of A+B=P+Q over a range of pH

This article shows how pKs for the enzymatic site and enzyme-substrate complexes can be obtained from kinetic experiments on the reaction A+B=P+Q, with and without the consumption of hydrogen ions. The rapid-equilibrium rate equation makes it possible to obtain the pKs and chemical equilibrium constants involved in the mechanism, the apparent equilibrium constant K' for the catalyzed reaction, and the number of hydrogen ions consumed in the rate-determining reaction. Experimentally-determined Michaelis constants can be adjusted for the pKs of the substrates A, B, P, and Q so that it is easier to obtain the pKs of E, EA, EB, EAB, EQ, and EPQ, and the chemical equilibrium constants. Reaction rates are discussed for the forward reaction ordered A+B=ordered P+Q with zero, one, or two hydrogen ions consumed in the rate-determining reaction and for random A+B=ordered P+Q with zero, one, or two hydrogen ions consumed in the rate-determining reaction. When hydrogen ions are consumed in the rate-determining reaction, there is a new factor 10(n)(pH) in the rate equation, where n is the number of hydrogen ions consumed in the rate-determining reaction for the forward reaction. The integer n can be obtained from rate measurements over a range of pH, but it cannot be determined from thermodynamic measurements.     

Analysis of Ter-Ter enzyme kinetic mechanisms by computer simulation of isotope exchange at chemical equilibrium: development and application of ISOTER, a personal-computer-based program

A convenient, personal-computer-based program has been developed that allows simulation of isotopic exchange kinetics at chemical equilibrium catalyzed by a three reactant-three product (TerTer) enzyme system: A + B + C integral of P + Q + R. This program, ISOTER, utilizes a rapid algebraic method to calculate the exchange rate between any reactant-product pair as a function of the substrate concentration and avoids altogether the necessity of deriving an explicit (but cumbersome and impractical) equation for exchange rate. ISOTER was used to generate model saturation patterns for 16 different TerTer kinetic mechanisms, varying different combinations of reactant-product pairs in constant ratio at equilibrium: [all substrates], [A, P], [B, Q], and [C, R], while holding the nonvaried components constant. These model studies indicate that virtually every one of these mechanisms can be distinguished from the others. In addition, ISOTER has been used to fit multiple sets of experimental data for Escherichia coli glutamine synthetase, which produced a set of rate constants consistent with the previously proposed "preferred order random" kinetic mechanism.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Interactions between the donor and acceptor sides in bacterial reaction centers

The apparent equilibrium constant K'(2) for electron transfer between the primary (Q(A)) and secondary (Q(B)) quinone acceptors of the reaction center was measured in chromatophores of Rhodobacter capsulatus. In the presence of the oxidized primary donor P(+), we obtained a value of K'(2)(P(+)) approximately 100 at pH 7.2, based on the rates of recombination from P(+)Q(A-) and P(+)Q(B-). K'(2) was also measured in the presence of reduced P, from the damping of semiquinone oscillations during a series of single turnover flashes. A 5-fold smaller value, K'(2)(P) approximately 20, was found. Additional information on the interactions between the donor and acceptor sides was obtained by measuring the shift of the midpoint potential of P caused by the presence of Q(B-) or Q(A-)S (where S indicates the presence of the inhibitor stigmatellin). A stabilization of the oxidized state P(+) was observed in both instances, by 10 mV for Q(B-) and 30 mV for Q(A-)S. The larger stabilization of P(+)Q(A-)S with respect to P(+)Q(B-) does not account for the effect of P(+)/P on K'(2). Analysis of these results indicates that the interactions between P(+)/P and Q(A)/Q(A)(-) are markedly modified depending on the occupancy of the Q(B) pocket by ubiquinone or by stigmatellin. We propose that the large value of K'(2)(P(+)) results essentially from a conformational destabilization of the P(+)Q(A-) state, that is relieved when the proximal site of the Q(B) pocket is occupied by stigmatellin.     

Modulation of the free energy of the primary quinone acceptor (QA) in reaction centers from Rhodobacter sphaeroides: contributions from the protein and protein-lipid(cardiolipin) interactions

The redox midpoint potential (E(m)) of Q(A), the primary quinone of bacterial reaction centers, is substantially modulated by the protein environment. Quite subtle mutations in the Q(A) binding site, e.g., at residues M218, M252 and M265, cause significant increases in the equilibrium constant for electron transfer to Q(B), which indicate relative lowering of the E(m) of Q(A). However, reports of functional linkage between the Q(A) and Q(B) sites make it difficult to partition such effects between Q(A) and Q(B) from purely relative changes. We report here measurements on the yield of delayed fluorescence emission from the primary donor (P) accompanying the thermally activated charge recombination of P(+)Q(A)(-) to form the excited singlet state of the primary donor, P*. The results show that for mutations of the Q(A) site residues, Met(M218) and Ile(M265), essentially all the substantial thermodynamic effect is localized at Q(A), with no evidence for a significant effect of these residues on the properties of Q(B) or the mutual influence (linkage) of Q(A) and Q(B). We also report a significant lowering of the E(m) of Q(A) by the native lipid, cardiolipin, which brings the E(m) in isolated reaction centers more in line with that seen in native membrane vesicles (chromatophores). Possible origins of this effect are discussed in the context of the Q(A) binding site structure.     

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.     

Equilibrium and kinetic parameters for the binding of inhibitors to the QB pocket in bacterial chromatophores: dependence on the state of QA

The equilibrium and kinetic parameters for the binding of various inhibitors to the Q(B) pocket of the bacterial reaction center were investigated in chromatophores from Rhodobacter capsulatus and Rhodobacter sphaeroides. By monitoring the near-IR absorption changes specific to Q(A)(-) and Q(B)(-), we measured the fraction of inhibited centers in the dark and the kinetics and extent of inhibitor displacement after one flash due to the formation of the Q(A)Q(B)(-) state. The inhibitor release rate was much faster for triazines and o-phenanthroline (t(1/2) in the 50 ms to 1 s range) than for stigmatellin (t(1/2) approximately 20 s). For inhibitors with a rapid release rate, the fast phase of P(+) decay observed in the absence of secondary donor reflects the competition between P(+)Q(A)(-) recombination and inhibitor release: it is thus faster than the P(+)Q(A)(-) recombination, and its relative extent is smaller than the fraction of initially inhibited centers. At appropriate inhibitor concentrations, one can have almost total binding in the dark and almost total inhibitor displacement after one flash. Under such conditions, a pair of closely spaced flashes resets the two-electron gate in a single state (Q(A)Q(B)(-)), irrespective of the initial state. The apparent dissociation constant of terbutryn was significantly increased (by a factor of 4-7) in the presence of Q(A)(-), in agreement with the conclusion of Wraight and co-workers [Stein, R. R., et al. (1984) J. Cell. Biochem. 24, 243-259]. We suggest that this effect is essentially due to a tighter binding of ubiquinone in the Q(A)(-) state.     

Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.     

Secondary quinone in photosystem II of Thermosynechococcus elongatus: semiquinone-iron EPR signals and temperature dependence of electron transfer

The secondary quinone acceptor, Q(B), has been studied in photosystem II (PSII) isolated from Thermosynechococcus (T.) elongatus. Thermoluminescence indicated that Q(B) was present in this preparation. An EPR signal observed at low temperature at g = 1.9 was attributed to Fe2+ Q(B)- on the basis of the characteristic period-of-two variations in its intensity depending on the number of laser flashes given at 20 degrees C. When samples showing the Fe2+ Q(B)- signal were illuminated at 77 K, an EPR signal at g = 1.66 appeared with an amplitude proportional to that of the Fe2+ Q(B)- signal. This signal is attributed to the Q(A)- Fe2+ Q(B)- state. While these attributions have been made previously in PSII from other origins, they have remained relatively tentative since the characteristic period-of-two oscillations of Q(B) had not previously been observed. The flash experiments indicated that more than one exchangeable plastoquinone is associated with the isolated PSII. The g = 1.66 signal from the Q(A)- Fe2+ Q(B)- state was used to study the temperature dependence of electron transfer between the two quinones. Electron transfer occurred in half of the centers (after 30 s incubation) at -28 degrees C for Q(A)- to Q(B) but at -58 degrees C for Q(A)- to Q(B)-. This marked difference for the two electron transfer reactions indicates different types of rate-limiting reactions. In the better studied but homologous system, the purple bacterial reaction center, the Q(A)- to Q(B) step is limited by a gating process, while the Q(A)- to Q(B)- step is limited by protonation events. Similar reactions in PSII could give rise to the observed temperature dependence.     

A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

Homology modeling of gastric H,K-ATPase based on the E2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E2 form-specific salt bridge between Glu820 (M6) and Lys791 (M5). In the E820Q mutant this salt bridge is no longer possible, and the head group of Lys791, together with a water molecule, fills the position of the K+ ion and apparently mimics the K+-filled cation binding pocket. This gives an explanation for the K+-independent ATPase activity and dephosphorylation step of the E820Q mutant (Swarts, H. G. P., Hermsen, H. P. H., Koenderink, J. B., Schuurmans Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. (1998) EMBO J. 17, 3029-3035) and, indirectly, for its E1 preference. The model is strongly supported by a series of reported mutagenesis studies on charged and polar amino acid residues in the membrane domain. To further test this model, Lys791 was mutated alone and in combination with other crucial residues. In the K791A mutant, the K+ affinity was markedly reduced without altering the E2 preference of the enzyme. The K791A mutation prevented, in contrast to the K791R mutation, the spontaneous dephosphorylation of the E820Q mutant as well as its conformational equilibrium change toward E1. This indicates that the salt bridge is essential for high-affinity K+ binding and the E2 preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K+-insensitive activity and an E1 preference. In addition, the study gives a molecular explanation for the electroneutrality of H,K-ATPases.     

Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).     

Effectors of Escherichia coli aspartate transcarbamoylase differentially perturb aspartate binding rather than the T-R transition

New systematic methods developed for equilibrium isotope exchange kinetics have been used to analyze the effects of activator ATP and inhibitor CTP with Escherichia coli aspartate transcarbamoylase. This indepth approach requires (a) variation of [modifier] with fixed subsaturating levels of substrates, and (b) variation of at least three combinations of reactant-product pairs in constant ratio at equilibrium: [A,B,P,Q], [A,P], and [B,Q] with the co-substrates held constant, in the presence and absence of added modifier. Both ATP and CTP had much stronger effects on the [14C]Asp in equilibrium C-Asp exchange rate than on [32P]C-P in equilibrium Pi. The bisubstrate analog N-phosphonacetyl-L-aspartate activated, then inhibited, Asp in equilibrium C-Asp more strongly than C-P in equilibrium Pi. N-Phosphonacetyl-L-aspartate gave complete (100%) inhibition, whereas CTP inhibition of either exchange was only partial. Substrate saturation curves in the presence and absence of effectors indicate that ATP and CTP perturb the observed values of Rmax and S0.5 in different fashions without appreciably changing the observed Hill number. Computer simulations indicate that the primary site of ATP and CTP action is the association rate for Asp, not the allosteric T-R transition. This finding is substantiated by previous studies in which modified aspartate transcarbamoylase had lost cooperative Asp binding without loss of sensitivity to effectors, or in which sensitivity to one effector could be deleted selectively. The present results, with newly devised computer simulation and analysis methods, illustrate the usefulness of equilibrium isotope exchange kinetic probes for providing unique insights to enzyme regulatory mechanisms, to define exactly which steps are altered in a given kinetic mechanism.     

The position of QB in the photosynthetic reaction center depends on pH: a theoretical analysis of the proton uptake upon QB reduction

Electrostatics-based calculations have been performed to examine the proton uptake upon reduction of the terminal electron acceptor Q(B) in the photosynthetic reaction center of Rhodobacter sphaeroides as a function of pH and the associated conformational equilibrium. Two crystal structures of the reaction center were considered: one structure was determined in the dark and the other under illumination. In the two structures, the Q(B) was found in two different positions, proximal or distal to the nonheme iron. Because Q(B) was found mainly in the distal position in the dark and only in the proximal position under illumination, the two positions have been attributed mostly to the oxidized and the reduced forms of Q(B), respectively. We calculated the proton uptake upon Q(B) reduction by four different models. In the first model, Q(B) is allowed to equilibrate between the two positions with either oxidation state. This equilibrium was allowed to vary with pH. In the other three models the distribution of Q(B) between the proximal position and the distal position was pH-independent, with Q(B) occupying only the distal position or only the proximal position or populating the two positions with a fixed ratio. Only the first model, which includes the pH-dependent conformational equilibrium, reproduces both the experimentally measured pH dependence of the proton uptake and the crystallographically observed conformational equilibrium at pH 8. From this model, we find that Q(B) occupies only the distal position below pH 6.5 and only the proximal position above pH 9.0 in both oxidation states. Between these pH values both positions are partially occupied. The reduced Q(B) has a higher occupancy in the proximal position than the oxidized Q(B). In summary, the present results indicate that the conformational equilibrium of Q(B) depends not only on the redox state of Q(B), but also on the pH value of the solution.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes determination of the equilibrium between Q(-)AQB and QAQ(-)B

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q(A)-Q(B) and Q(A) Q(-B). A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of α = [Q(A)-Q(B)I/(IQ(A)-Q(Bl)+ [Q(A)Q(-B)I) = 0.065 +/- 0.005 (T= 21°C, pH 8).     

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.     

中国栎属九种植物的核型分析

报道了中国栎属 ( Quercus L inn.) 9种植物的核型。结果如下 :帽斗栎 ( Q.guyavaelofia) :2 n=2 4 =2 2 m+2 sm,属于“1A”类型 ;富宁栎 ( Q.setulosa) :2 n=2 4 =2 0 m+4sm,属于“2 A”类型 ;炭栎 ( Q.utilis) :2 n=2 4 =2 4 m,属于“2 A”类型 ;乌冈栎 ( Q.phillyraeoides) :2 n=2 4 =2 0 m+4sm,属于“2 B”类型 ;匙叶栎 ( Q.dolicholepis) :2 n=2 4 =2 0 m+4sm,属于“2 B”类型 ;岩栎( Q.acrodonta) :2 n=2 4 =2 2 m+2 sm,属于“2 B”类型 ;麻栗坡栎 ( Q.marlipoensis) :2 n=2 4 =2 4 m,属于“1B”类型 ;锥连栎 ( Q.franchetii) :2 n=2 4 =2 2 m +2 sm ,属于“1B”类型 ;麻栎 ( Q.acutissima) :2 n=2 4 =2 0 m+4sm,属于“2 B”类型 .其中除麻栎外 ,另 8种的核型系首次报道。研究结果表明 ,上述核型在种间较相似 ,但以组为单位进行比较时 ,则有一定的价值 ,有可能为揭示栎属的系统演化提供新的证据。     

Influence of insulin sensitivity and the TaqIB cholesteryl ester transfer protein gene polymorphism on plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities and their response to hyperinsulinemia in non-diabetic men.

Lecithin:cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP), and lipoprotein lipases are involved in high density lipoprotein (HDL) metabolism. We evaluated the influence of insulin sensitivity and of the TaqIB CETP gene polymorphism (B1B2) on plasma LCAT, CETP, and PLTP activities (measured with exogenous substrates) and their responses to hyperinsulinemia. Thirty-two non-diabetic men without hyperlipidemia were divided in quartiles of high (Q(1)) to low (Q(4)) insulin sensitivity. Plasma total cholesterol, very low + low density lipoprotein cholesterol, triglycerides, and apolipoprotein (apo) B were higher in Q(4) compared to Q(1) (P < 0.05 for all), whereas HDL cholesterol and apoA-I were lowest in Q(4) (P < 0.05 for both). Plasma LCAT activity was higher in Q(4) than in Q(1) (P < 0. 05) and PLTP activity was higher in Q(4) than in Q(2) (P < 0.05). Insulin sensitivity did not influence plasma CETP activity. Postheparin plasma lipoprotein lipase activity was highest and hepatic lipase activity was lowest in Q(1). Insulin infusion decreased PLTP activity (P < 0.05), irrespective of the degree of insulin sensitivity. The CETP genotype exerted no consistent effects on baseline plasma lipoproteins and LCAT, CETP, and PLTP activities. The decrease in plasma PLTP activity after insulin was larger in B1B1 than in B2B2 homozygotes (P < 0.05). These data suggest that insulin sensitivity influences plasma LCAT, PLTP, lipoprotein lipase, and hepatic lipase activities in men. As PLTP, LCAT, and hepatic lipase may enhance reverse cholesterol transport, it is tempting to speculate that high levels of these factors in association with insulin resistance could be involved in an antiatherogenic mechanism. A possible relationship between the CETP genotype and PLTP lowering by insulin warrants further study.     

Elevated P/Q type (alpha1A) and L2 type (alpha1D) Purkinje cell voltage-gated calcium channels in the cerebella of seizure prone gerbils

Differences in expression of N-methyl-D-aspartate (NMDA) receptor and voltage gated Ca2+ channels (VGCC) in the gerbil cerebellum were investigated to identify routes of Ca2+ influx that may be involved in Purkinje cell damage. Immunodensities of NR1 and NR2A/B were the same in seizure resistant (SR) and seizure sensitive (SS) gerbils. However, both P/Q type (alpha1A) and L2 type (alpha1D) VGCC levels were higher in the Purkinje cells of SS gerbils than in those of SR gerbils, whereas N type (alpha1B) and L1 type (alpha1C) VGCC levels were similar in the two groups. Our findings suggest that increases in P/Q type (alpha1A) and L2 type (alpha1D) VGCC are implicated in the degeneration of Purkinje cells in SS gerbils.     

Low-temperature interquinone electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis: characterization of Q(B)- states by high-frequency electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.