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E N Moudrianakis

Publications and source records attributed to E N Moudrianakis.

At least 37 records · Page 2Linked to original sources
PubMed1990

Spectropolarimetric analysis of the core histone octamer and its subunits.

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.

Ammonium Sulfate↗
PubMed1990

Effect of aggregation of histone octamers in high-salt solutions on circular dichroism spectra.

The circular dichroism (CD) of freshly prepared chicken erythrocyte core histones has been reexamined in high concentrations of ammonium sulfate and sodium chloride, conditions which cause drastic changes in the solubility and aggregative properties of these proteins. After sample clarification by ultracentrifugation, no significant net changes are detected in the secondary structure of the core histones in the range of 2.0-2.5 M ammonium sulfate. There is also no significant difference between the CD spectra of histone solutions in 2 M sodium chloride and clarified solutions of histones in high concentrations of ammonium sulfate. It was observed that sample clarification by ultracentrifugation immediately prior to taking CD spectra was necessary for signal stabilization, especially under conditions which begin to favor crystallization of the histones.

Ammonium Sulfate↗
PubMed1988

H2a-specific proteolysis as a unique probe in the analysis of the histone octamer.

We have utilized the H2a-specific protease as a unique probe to investigate the nature of the interactions between the protein subunits which form the core histone octamer. Upon incubation in high ionic strength media this protease, normally found tightly associated with isolated calf thymus chromatin, releases the 15 COOH-terminal amino acids of histone H2a by specifically cleaving the H2a polypeptide between Val114 and Leu115, yielding cleaved H2a (cH2a) and a free pentadecapeptide (Eickbush, T. H., Watson, D. K., and Moudrianakis, E. N. (1976) Cell 9, 785-792). We find that removal of this pentadecapeptide results in a marked dissociation of the octamer into its H2a:H2b dimer and H3:H4 tetramer subunits. Reconstitution experiments indicate that cH2a is capable of forming a dimer with H2b, but this cH2a:H2b dimer has a substantially lower affinity for the H3:H4 tetramer than native H2a:H2b dimer. Kinetic studies of H2a cleavage in high ionic strength solutions demonstrate that H2a molecules in the octamer are relatively resistant to proteolytic attack compared to H2a molecules in the dimer. The extent of this resistance, in response to various experimental parameters, is directly correlated to the strength of interaction between the H2a:H2b dimer and H3:H4 tetramer subunits. These reconstitution and kinetic experiments suggest that the histone domains proximal to the H2a cleavage site have an important function in maintaining the association of the histone octamer subunits.

Animals↗
PubMed1988

Regulation of H2a-specific proteolysis by the histone H3:H4 tetramer.

We have studied the limited cleavage of H2a in the H2a:H2b histone dimer by the H2a-specific protease under physiological conditions (neutral pH, 0.1 M NaCl) using a variety of histone-DNA reconstitutes as substrates and/or regulators of the partially purified enzyme. Under these conditions the protease cleaves H2a in "native" dimer-DNA reconstitutes but not in "native" octamer-DNA reconstitutes. Treatment of the enzyme with saturating amounts of H3:H4 tetramer-DNA prior to addition of dimer-DNA substrate results in complete inhibition of H2a-specific proteolysis. Sucrose gradient sedimentation experiments indicate that the protease binds reversibly to tetramer-DNA and that this leads to the reversible inhibition of enzymatic activity. Using three different tetramer-DNA complexes, we found native tetramer-DNA to be a more effective inhibitor than either trypsin-treated tetramer-DNA or acetylated tetramer-DNA. We conclude that under physiological conditions, the H2a-specific protease binds primarily to the highly basic amino-terminal domain of the H3:H4 tetramer, and this binding lowers the effective concentration of enzyme available to cleave H2a. Although no cleaved H2a is produced when protease is mixed with native octamer-DNA, incubation of the enzyme with acetylated octamer-DNA results in H2a-specific proteolysis. This is the first demonstration that the H2a-specific protease activity can be modulated by a physiologically relevant process (e.g. histone acetylation). We propose that the sequestered protease may be functionally regulated in vivo through reversible post-translational modifications to the NH2-terminal domains of the histone H3:H4 tetramer.

Acetylation↗
PubMed1986

Thiol reactivity of histone H3 in soluble and DNA-associated histone complexes: evidence for allosteric and torsional regulation.

The reactivity of chick erythrocyte and calf thymus histone H3 thiol groups toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been investigated both in the soluble, DNA-free state and in various nucleohistone complexes. We have found that the thiol reactivity of both tetramers and octamers decreases continuously as the ionic strength of the assay is increased, up to and beyond 2.0 M NaCl. Upon association of dimers with tetramers, there is loss of labeling by DTNB at one site, suggesting the existence of allosteric regulation [see also Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346] of dimer-tetramer interfaces emanating from within the tetramer complex. Comparison of the thiol reactivities of chick and calf tetramers indicates that the thiol groups at amino acid positions 96 and 110 are not chemically equivalent. When the histones are associated with DNA, in either reconstituted complexes, core particles, or long soluble chromatin, the thiol reactivity is greatly diminished, and this "DNA effect" overwhelms any influence of dimers. However, if single-strand nicks are introduced into the DNA backbone of core particles and other chromatin-like complexes by the action of DNase I, the influence of the DNA double helix upon thiol reactivity is reduced, and the effect of dimers can be detected once again. We can therefore conclude that the DNA effect derives from intranucleosomal torsional strain of the continuum of the double helix in equilibrium with coupled protein conformational changes. These observations support the concept that the octamer complex is a dynamic tripartite structure whose properties can be modulated through its interactions with DNA and by changes occurring in the dimer-tetramer interfaces.

Allosteric Regulation↗
PubMed1986

Effects of permeant buffers on the initiation of photosynchronous phosphorylation and postillumination phosphorylation in chloroplasts.

Under canonical chemiosmotic formulations, the development of a delocalized transmembrane proton gradient should precede and, in the absence of a membrane potential, should account for all the capacity of an energy transducing system to synthesize ATP. Furthermore, any agents, such as permeant proton-absorbing buffers, that slow down the kinetics of the development of this gradient should, consequently, delay ATP synthesis. We have studied the very early (0 through 1000 ms) steps of photosynthetic ATP synthesis utilizing real-time, rapid flow-quench techniques. We have investigated the effect(s) that permeant buffers exert on this process where these buffers show no uncoupling effects, and the transmembrane potential has been collapsed by valinomycin and K+. Experimentally this system was dissected into two ATP synthesizing components, as follows: synthesis of ATP strictly concomitant with light influx and unaffected by the addition of permeant buffers. We refer to this as photosynchronous phosphorylation and synthesis of ATP monitored after the light was extinguished and which was greatly diminished by the addition of proton-absorbing permeant buffers, thus exhibiting the characteristics of conventional postillumination phosphorylation, and we suggest that it represents part of capacitance phosphorylation. The potential for capacitance phosphorylation initiates very rapidly under light and gradually builds up to steady-state level, and it is governed by canonical chemiosmotic principles. We estimate that its contribution to overall ATP yield is minimal during the first few cycles of the system and that it increases gradually towards steady state when it contributes to the majority of ATP synthesized. Neither a delocalized transmembrane proton gradient nor a strictly localized intramembrane proton pathway can account for these observations so we have proposed that a gating mechanism exists which delivers intramembrane protons initially directly to the ATP synthetase complex but subsequently to the lumen as well, and thus, allows the lumen to act as a capacitor during the steady state. This study can reconcile the findings of Ort et al. (Ort, D. R., Dilley, R. A., and Good, N. E. (1976) Biochim. Biophys. Acta 449, 108-124) with the contrasting findings of Vinkler et al. (Vinkler, C., Avron, M., and Boyer, P. D. (1980) J. Biol. Chem. 255, 2263-2266) through the opposite effects which osmotic strength and KCl concentration exert on the two ATP synthetic phases (during and after illumination) of the rapid flash technique used in those studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1985

Millisecond kinetics of ATP synthesis driven by externally imposed electrochemical potentials in chloroplasts.

We have used rapid mixing and quenching techniques to measure the initial ATP synthesis rates and the duration of the ATP synthetic capacity derived from artificially imposed proton gradients and valinomycin-mediated K+ diffusion potentials in chloroplasts. The initial rate of ATP synthesis driven by a K+ diffusion potential was 10-fold slower than that driven by an acid-base transition of equivalent electrochemical potential. Total yields of ATP resulting from a K+ concentration shift were only slightly affected by the absence of Cl-, indicating that Cl- permeability does not significantly reduce the K+ diffusion potential. The ATP synthetic capacity decayed with a half-life of 0.2 s in the case of a K+ diffusion potential and a half-life of 1.0 s in the case of an acid-base shift. In both cases, ATP, added at the time of the pH or [KCl] shift, slowed the decay of the ATP synthesis rates, indicating that the coupling factor controls a channel for proton efflux, as proposed earlier (Portis, A.R., and McCarty, R.E. (1974) J. Biol. Chem. 249, 6250-6254). Because the proton gradient has a longer half-life than the K+ diffusion potential, when combinations of the two are employed to drive ATP synthesis, the proton gradient will make a larger contribution to the initial rate and total yield than that predicted from a strictly linear proportionality of the initial magnitudes of the two gradients.

Acid-Base Equilibrium↗
PubMed1985

Crystallographic structure of the octameric histone core of the nucleosome at a resolution of 3.3 A.

The structure of the (H2A-H2B-H3-H4)2 histone octamer has been determined by means of x-ray crystallographic techniques at a resolution of 3.3 angstroms. The octamer is a prolate ellipsoid 110 angstroms long and 65 to 70 angstroms in diameter, and its general shape is that of a rugby ball. The size and shape are radically different from those determined in earlier studies. The most striking feature of the histone octamer is its tripartite organization, that is, a central (H3-H4)2 tetramer flanked by two H2A-H2B dimers. The DNA helix, placed around the octamer in a path suggested by the features on the surface of the protein, appears like a spring holding the H2A-H2B dimers at either end of the (H3-H4)2 tetramer.

Animals↗
PubMed1984

Effects of ionic strength and state of assembly on kinetics of hydrogen exchange of calf thymus histones.

The kinetics of hydrogen exchange of calf thymus histone H2A-H2B dimers and (H3-H4)2 tetramers at pH 7 have been examined at low (0.16 M NaCl) and high (2 M NaCl) ionic strengths and after incorporation into (H2A-H2B-H3-H4)2 octamers. The similarity of the results for both species is noteworthy. Approximately 60% of the backbone amide protons are detectable in both low and high salt, and at least three kinetic phases can be distinguished. Increasing the ionic strength from 0.16 to 2 M accelerates exchange of some of the rapidly exchanging protons in both dimers and tetramers, while slowing exchange of others. Exchange of the more slowly exchanging protons is virtually unaffected. Incorporation of dimers into octamers accelerates exchange of approximately 40 protons to such an extent that they can no longer be detected. The effects of assembly upon the tetramer are qualitatively similar. These results indicate that both high ionic strengths and assembly destabilize some regions of the structure while stabilizing others. For both dimers and tetramers, the effects of ionic strength are dramatic, while those of assembly are more subtle. Higher resolution studies aimed at identifying the responsive protons would be of interest.

Animals↗
PubMed1984

Response of the adenosine triphosphatase activity of the soluble latent F1 enzyme from beef heart mitochondria to changes in Mg2+ and H+ concentrations.

The coupling factor of oxidative phosphorylation from beef heart mitochondria obtained as a "latent F1," exhibits negligible levels of ATPase activity, contains stoichiometric amounts of the specific F1 inhibitor protein, and is stable to incubation at low temperature (Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975) Biochemistry 14, 1727-1735). Incubation of the latent F1 enzyme at 60 degrees C activates its ATPase activity. We show in this paper that regulation of the interaction of the inhibitor protein with the latent F1 enzyme can be accomplished under more physiological conditions. At 37 degrees C, variations in the proton concentration led to changes in the degree and extent of activation of the enzyme, with maximal activation rates occurring after preincubation at pH 9.6. The energy for the pH 9.6-induced activation process (12.1 kcal/mol) was similar to that reported for the dissociation of the inhibitor protein from the membrane-bound F1 enzyme in energized mitochondria (Gomez-Fernandez, J. C., and Harris, D.A. (1978) Biochem. J. 176, 967-973). The rates of activation were higher in the presence of 5 mM ATP and inhibited by the presence of Mg2+, suggesting the existence of a specific binding site for Mg2+ between the inhibitor subunit and the F1 enzyme. A model is presented in which the activation of the latent F1 enzyme is brought about by a rapid titration of positively charged amino acid residues on the inhibitor subunit, followed by a slow release of a tightly bound Mg2+ atom. This model predicts that the initial event leading to the appearance of ATP synthesis is the deprotonation of the inhibitor subunit and that the onset of ATPase activity in mitochondria is due to sequestering of the available free Mg2+.

Adenosine Triphosphate↗
PubMed1984

Crystals of the octameric histone core of the nucleosome.

The undegraded core histone octamer has been crystallized in a form suitable for x-ray analysis. The hexagonal bipyramidal crystals reproducibly grow larger than 1.0 by 0.6 millimeter, X-ray reflections are observed from Bragg planes with spacings larger than 3.5 angstroms. The crystals have the symmetry of the space group P3l21 or its enantiomorph. There appears to be one histone octamer per asymmetric unit.

Animals↗
PubMed1984

Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria.

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used (U. K. Laemmli (1970) Nature (London) 277, 680-685). The determination of the apparent molecular weights by analysis of Ferguson plots (K. A. Ferguson (1964) Metabolism 13, 985-1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the beta subunit. A dramatic increase in the apparent molecular weight of the delta subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the alpha and the beta subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the beta subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the delta subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the delta subunit required for binding of the coupling factor to the mitochondrial membrane.

Animals↗
PubMed1983

The effect of permeant buffers on initial ATP synthesis by chloroplasts using rapid mix-quench techniques.

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KCl are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KCl pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post-illumination ATP synthesis are driven by distinct components of the proton motive potential.

Adenosine Triphosphate↗
PubMed1983

Minor histone 2A variants and ubiquinated forms in the native H2A:H2B dimer.

Histone octamers from calf thymus were separated into (H3:H4)2 tetramers and H2A:H2B dimers by chromatography through Sephadex G100. The tetramers and dimers were analyzed for variants, ubiquitin adducts, and proteolyzed forms. The minor histone variants H2A.X and H2A.Z were found to be associated with histone H2B as H2A.X:H2B and H2A.Z:H2B dimers, respectively. Ubiquitin adducts of the H2A's and H2B were also present in H2A:H2B dimers.

Animals↗
PubMed1983

Initial products of photophosphorylation with AMP and [32P]Pi.

The role of AMP in photophosphorylation was studied using rapid mixing acid quench techniques. Fragmented spinach chloroplast membranes or subchloroplast particles were illuminated and rapidly mixed with [32P]orthophosphate and AMP at pH 7 for 10 ms to 60 s after which time perchloric acid was added to quench the reaction. ATP was found to be the primary and predominant nucleotide labeled. It was found that after illumination, an adenylate kinase-like activity carried out an AMP-dependent conversion of labeled ATP to labeled ADP which was inhibited by the presence of ADP. This reaction was characterized as being similar to chloroplast adenylate kinase in Mg2+ dependency and in sensitivity to phlorizin and tentoxin and distinct from chloroplast coupling factor 1. The small amounts of adenylate kinase activity present in fragmented well washed chloroplast membranes were found to be sufficient to carry out this rapid reaction. These results necessitated a reinterpretation of the earlier findings of Tiefert and Moudrianakis (Tiefert, M.A., and Moudrianakis, E.N. (1979) J. Biol. Chem. 254, 9500-9508) and no longer support the role of ADP as a phosphorylated intermediate in ATP synthesis.

Adenosine Diphosphate↗
PubMed1982

Crystallization of the tetramer of histones H3 and H4.

Crystals of the histone tetramer (H3-H4)2 from calf thymus have been grown. The crystals yield x-ray diffraction patterns with Bragg spacings as small as 3.5 angstroms. Crystals grown from two types of preparations have the symmetry of the space group P61 (or P65). The best crystals were grown from histones that had the amino terminal arms removed by mild trypsinization.

Crystallization↗
PubMed1982

Histone-dependent reconstitution and nucleosomal localization of a nonhistone chromosomal protein: the H2A-specific protease.

We have described earlier a chromatin-bound protease with unique specificity for histone H2A [Eickbush, T. H., Watson, D. K., & Moudrianakis, E. N. (1976) Cell (Cambridge, Mass.) 9, 785--792]. In the present study, we explore the nature of interactions that form and stabilize the enzyme-chromatin system by using the activity of the protease to monitor its binding to DNA and DNA-histone complexes. During salt extraction of chromatin, the protease is released at an ionic strength between that required for the extraction of the slightly lysine-rich histones (H2A and H2B) and the arginine-rich histones (H3 and H4). The reassociation of this nonhistone protein to DNA has an absolute requirement for the H3--H4 tetramer and is only enhanced by the H2A--H2B dimer in the presence of the tetramer. We believe that the binding of the enzyme onto DNA requires some histone-elicited compaction of the helix. We have also examined the distribution of this enzyme within the chromatin fiber by isolating pools of monomer nucleosomes from micrococcal nuclease digests of 0.6 M NaCl extracted chromatin and from reconstituted DNA-protein complexes. The H2A-protease is found with these monomer nucleosome pools, and no activity can be detected in the low molecular weight products released during the digestion. Thus, by virtue of its extraction characteristics from chromatin and its association with isolated nucleosomes, this nonhistone protein exhibits properties hitherto assigned only to the inner histones.

Animals↗