Firing statistics of inhibitory neuron with delayed feedback. I. Output ISI probability density

Activity of inhibitory neuron with delayed feedback is considered in the framework of point stochastic processes. The neuron receives excitatory input impulses from a Poisson stream, and inhibitory impulses from the feedback line with a delay. We investigate here, how does the presence of inhibitory feedback affect the output firing statistics. Using binding neuron (BN) as a model, we derive analytically the exact expressions for the output interspike intervals (ISI) probability density, mean output ISI and coefficient of variation as functions of model's parameters for the case of threshold 2. Using the leaky integrate-and-fire (LIF) model, as well as the BN model with higher thresholds, these statistical quantities are found numerically. In contrast to the previously studied situation of no feedback, the ISI probability densities found here both for BN and LIF neuron become bimodal and have discontinuity of jump type. Nevertheless, the presence of inhibitory delayed feedback was not found to affect substantially the output ISI coefficient of variation. The ISI coefficient of variation found ranges between 0.5 and 1. It is concluded that introduction of delayed inhibitory feedback can radically change neuronal output firing statistics. This statistics is as well distinct from what was found previously (Vidybida and Kravchuk, 2009) by a similar method for excitatory neuron with delayed feedback.     

Selectivity of chemoreceptor neuron

Discriminating ability (selectivity) of chemoreceptor neuron is compared with that of its receptor proteins. The process of neuronal triggering is expected to be cooperative and threshold-type in a sense that the neuron can fire if and only if the number of its receptor proteins, which are bound with odor molecules, is above a definite threshold. Both deterministic and stochastic pictures are considered. The stochastic case is treated based on birth and death stochastic process and first passage technique. In both pictures, it is shown that a chemoreceptor neuron can have much a higher selectivity than its individual receptor proteins, provided the chemical stimuli are presented at low concentrations, and the threshold is high enough. This is in agreement with a preliminary estimate based on simplified probabilistic reasoning (Vidybida, A.K., 1999. Cooperative mechanism for improving the discriminating ability in the chemoreceptive neuron. Binomial case. Biol. Cybern. 81, 469-473). The mechanism of selectivity improvement is similar to that described before in cooperative chemical systems. A possibility for this mechanism to be valid at higher stages of processing of chemical signals, as well as in other sensory systems is discussed.     

Sensitivity to initial conditions in the route-finder model

A model of stimulus generalization [Staddon, J.E.R., Reid, A.K., 1990. On the dynamics of generalization. Psychol. Rev. 97, 576-578] was extended by Reid and Staddon [Reid, A.K., Staddon, J.E.R., 1998. A dynamic route finder for the cognitive map. Psychol. Rev. 105, 585-601] to account for the route-finder issue in the Tolman-Guthrie discussion about cognitive maps. This deterministic model has been described as sensitive to initial conditions in terms of the parameters of a diffusion equation [Reid, A.K., Staddon, J.E.R., 1998. A dynamic route finder for the cognitive map. Psychol. Rev. 105, 585-601]. A simulation study was carried out to test this assertion, evaluating whether the pattern of variation in the model output resembled a chaotic pattern. Results indicate that the model is sensitive to initial conditions, suggesting that the spatial navigation task addressed by this model involves non-linear relationships and complexity beyond the apparent simplicity of the stimulus generalization process.     

Event-driven simulation of cerebellar granule cells

Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model.     

Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

The complex formed between adenovirus DNA-binding protein (AdDBP) and poly(rA) was investigated with circular dichroism spectroscopy. The binding process was studied at a variety of salt concentrations, and the titration curves were analyzed according to the contiguous cooperative binding model given by McGhee and von Hippel [McGhee, J.D., & von Hippel, P.H. (1974) J. Mol. Biol. 86, 469-489]. The cooperativity factor omega of the binding process is low (omega approximately 20-30) and independent of the salt concentration. This in contrast to the binding constant K for which a moderately strong salt dependence is observed: delta log (K omega)/delta log [NaCl] = -3.1. The size of the binding site was consistently calculated to be about 13. We also studied the C-terminal 39-kDa fragment which is sufficient for DNA replication in vitro. Complex formation between this fragment of AdDBP and poly(rA) appeared to be characterized by spectroscopic and binding properties similar to those of the intact protein. Only, the binding constant in 50 mM NaCl is a factor of 5 lower.     

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.     

Alternating zinc fingers in the human male-associated protein ZFY: HX3H and HX4H motifs encode a local structural switch

The two-finger repeat in the human male-associated protein ZFY provides a model for comparative 2D-NMR studies of classical and variant Zn fingers. This repeat is defined in part by an alternation in spacing between consensus (HX3H) and variant (HX4H) histidine spacings. To investigate the effects of a "switch" between alternative histidine spacings, we have designed an HX3H analogue of a representative HX4H domain of known structure [ZFY-6; Kochoyan, M., Havel, T., Nguyen, D. T., Dahl, C. E., Keutmann, H. T., & Weiss, M. A. (1991) Biochemistry 30, 3371-3386]. The HX3H analogue (designated ZFY-switch) forms a tetrahedral Co2+ complex whose thermodynamic stability is similar to that of the parent peptide. 2D-NMR studies demonstrate that ZFY-switch and ZFY-6, although similar in overall structure, exhibit significant local changes near the site of deletion. Whereas the HX4H site in the native finger forms a nonstandard loop, the HX3H site in ZFY-switch folds as a 3(10) extension of the C-terminal alpha-helix, as observed in the NMR solution structure of a consensus HX3H domain [Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A., & Wright, P. E. (1989) Science 245, 635-637] and in the crystal structure of a representative Zn finger-DNA complex [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. We propose that variant histidine spacings (HX3H and HX4H) encode a local switch between alternative surface architectures with implications for models of protein-DNA recognition.     

Tryptophan residues of creatine kinase: a fluorescence study

Spectroscopic studies of rabbit skeletal muscle creatine kinase (CPK) and its complexes with adenosine phosphates have long suggested the occurrence of a tryptophan residue at or near the coenzyme binding sites [K?gi, J. H. R., Li, T.-K., & Vallee, B. L. (1971) Biochemistry 10, 1007-1015; Price, N. C. (1972) FEBS Lett. 24, 21-23]. This conjecture was further supported by nuclear Overhauser effect (NOE) 1H NMR studies indicating through-space interactions between protons of the adenine ring of bound ADP and one or more aromatic side chains of the proteins [Vasák, M., Nagayama, K., Wüthrich, K., Mertens, M. L., & K?gi, J. H. R. (1979) Biochemistry 18, 5050-5055]. Further evidence for a tryptophan residue in the environment of the active site has now been obtained by fluorescence-quenching studies using iodide and acrylamide as external quenchers. Thus, while by the addition of iodide the tryptophan fluorescence of unliganded CPK is reduced to about 75% of the unquenched control, no such effect is manifested upon addition of this quencher to the CPK.ADP and CPK.ATP complexes. Similarly, the relative effectiveness of quenching of the CPK-coenzyme complexes by acrylamide is only about 60% of that measured in the unliganded enzyme. Both these data and the spectral characteristics of the quenched fluorescence suggest that coenzyme binding perturbs a tryptophan residue that is close to the active site and that is partially exposed to the solvent. The differential effectiveness of external quenchers on unliganded and liganded CPK allows the determination of the ligand binding equilibria by fluorescence-quenchability titration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 ?) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.     

Stochastic automaton models for interaction of excitatory and inhibitory impulse sequences in neurons

The mechanisms by which excitatory and inhibitory input impulse sequences interact in changing the spike probability in neurons are examined in the two mathematical neuron models; one is a real-time neuron model which is close to physiological reality, and the other a stochastic automaton model for the temporal pattern discrimination proposed in the previous paper (Tsukada et al., 1976), which is developed in this paper as neuron models for interaction of excitatory and inhibitory input impulse sequences. The interval distributions of the output spike train from these models tend to be multimodal and are compared with those used for experimental data, reported by Bishop et al. (1964) for geniculate neuron activity and Poisson process deleting model analyzed by Ten Hoopen et al. (1966). Special attention, moreover, should be paid to how different forms of inhibitory input are transformed into the output interval distributions through these neuron models. These results exhibit a clear correlation between inhibitory input form and output interval distribution. More detailed information on this mechanism is obtained from the computations of recurrence-time under the stationary condition to go from active state to itself for the first time, each of which is influenced by the inhibitory input forms. In addition to these facts, some resultant characteristics on interval histogram and serial correlation are discussed in relation to physiological data from the literature.     

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.     

Small conductance potassium channels cause an activity-dependent spike frequency adaptation and make the transfer function of neurons logarithmic

We made a computational model of a single neuron to study the effect of the small conductance (SK) Ca2+-dependent K+ channel on spike frequency adaptation. The model neuron comprised a Na+ conductance, a Ca2+ conductance, and two Ca2+-independent K+ conductances, as well as a small and a large (BK) Ca2+-activated K+ conductance, a Ca2+ pump, and mechanisms for Ca2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca2+ concentration ([Ca2+]i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca2+]i (. Pflügers Arch. 422:223-232; K?hler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709-1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechner's law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.     

Interaction of phalloidin with chemically modified actin

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67-72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.     

Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of streptomycin to the 50S subunit of Escherichia coli with phenyldiglyoxal

[3H]Dihydrostreptomycin was covalently linked to the 50S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the specific interaction of streptomycin with the ribosome. The binding primarily involved the ribosomal RNA and also a limited number of proteins, namely, L2, L6, and L17. This suggests that the binding domain for streptomycin is close to the peptidyl transferase center, in the valley between the central protuberance and the wider lateral protuberance of the 50S subunit. This domain faces the binding domain for streptomycin which we have previously characterized on the 30S subunit [Melan?on, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703]. Our results indicate that the 50S subunit is involved in the binding of streptomycin to the bacterial ribosome, in addition to the 30S subunit which is generally considered as the specific target of the antibiotic. They are consistent with the occurrence of a single binding site for streptomycin on the ribosome, comprised of regions of both subunits.     

The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.     

Analysis of the gastric H,K ATPase for ion pathways and inhibitor binding sites

New models of the gastric H,K ATPase in the E1K and E2P states are presented as the first structures of a K+ counter-transport P2-type ATPase exhibiting ion entry and exit paths. Homology modeling was first used to generate a starting conformation from the srCa ATPase E2P form (PDB code 1wpg) that contains bound MgADP. Energy minimization of the model showed a conserved adenosine site but nonconserved polyphosphate contacts compared to the srCa ATPase. Molecular dynamics was then employed to expand the luminal entry sufficiently to allow access of the rigid K+ competitive naphthyridine inhibitor, Byk99, to its binding site within the membrane domain. The new E2P model had increased separation between transmembrane segments M3 through M8, and addition of water in this space showed not only an inhibitor entry path to the luminal vestibule but also a channel leading to the ion binding site. Addition of K+ to the hydrated channel with molecular dynamics modeling of ion movement identified a pathway for K+ from the lumen to the ion binding site to give E2K. A K+ exit path to the cytoplasm operating during the normal catalytic cycle is also proposed on the basis of an E1K homology model derived from the E12Ca2+ form of the srCa ATPase (PDB code 1su4). Autodock analyses of the new E2P model now correctly discriminate between high- and low-affinity K+ competitive inhibitors. Finally, the expanded luminal vestibule of the E2P model explains high-affinity ouabain binding in a mutant of the H,K ATPase [Qiu et al. (2005) J. Biol. Chem. 280, 32349-32355].