The binding of cupric ions to bovine pancreatic ribonuclease studied with diligand metal-ion buffers

A procedure has been developed for the use of metal-ion buffers that depends on the formation of 2:1 complexes between suitable chelators and metal ions. beta-Alanine has been used as the chelator for Cu(2+) ions in a study of Cu(2+) binding by bovine pancreatic ribonuclease by the equilibrium-dialysis technique at pH7.0, 6.1 and 5.2. The results indicated the presence of two avid binding sites, the more avid group being implicated in the inhibition of enzyme activity by Cu(2+) ions.The binding constants of the more avid site were 2.97x10(7), 7.97x10(5) and 1.25x10(4) at pH7.0, 6.1 and 5.2 respectively, and the binding constants of the less avid site were 5.27x10(6) and 1.71x10(5) at pH7.0 and 6.1 respectively.The data show that the Cu(2+) is chelated to the protein through at least two ligand groups on the ribonuclease molecule.     

Transition-state structure for a conformation change of ribonuclease

The rate constants k₁₂ⁿ for isomerization of the E₁H isomer (pK_a 8 in H₂O) of ribonuclease-A to the E₂H isomer (pK_a = 6.1 in H₂O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k₁₂ⁿ = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)²sec⁻¹ at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization.     

Reversible disruption of thymic function by steroid treatment

The effect of steroid treatment on the thymic output of T cells was examined in an avian model. Recent thymic emigrants in chickens transiently express the chicken T cell Ag 1 thymocyte marker, and thymic function can be monitored indirectly by measuring the levels of TCR gene rearrangement excision circles in peripheral T cells. Both parameters were used to show that intensive steroid treatment induces thymic involution and a profound reduction in the supply of naive T cells to the periphery. Conversely, resident T cells in the peripheral lymphocyte pool were relatively spared. Thymopoiesis immediately recovered following cessation of steroid treatment, concurrent with restoration of the thymic output of newly formed T cells. Repopulation of the peripheral T cell pool recapitulated the ontogenetic pattern of gamma delta T cell replenishment before alpha beta T cell reseeding, thereby indicating the complete recovery of thymic function after a course of steroid treatment.     

Chromatin conformation: a systematic analysis of helical parameters from hydrodynamic data

Chromatin has a “bead-and-bridge” appearance when viewed by electron microscopy. We have used quasielastic light scattering and sedimentation velocity techniques to study the hydrodynamic properties of chicken erythrocyte chromatin multimers in an attempt to determine the superstructure in solution. The functional dependence of the friction factor on the number of core particles in the multimer was analyzed by the Garcia de la Torre-Bloomfield formalism for a rigid array of odd-sized beads. The hydrodynamic parameters of the monomeric and dimeric subunit components, i.e., bead size and separation, form the basis of a systematic determination of the superstructure. These calculations support a helical conformation for chromatin multimers containing up to twenty repeat units. It is also shown that an “equivalent” helix can be obtained if the bead separation distance is not constrained to that determined for the dimer.     

Modification of pig heart lipoamide dehydrogenase by cupric ions.

The insertion of a second disulfide bridge into native pig heart lipoamide dehydrogenase, requires two Cu-2+ ions for each catalytic center inactivated under anaerobic conditions. During inactivation, both metal atoms become reducible by their juxtaposition to the two participating cysteine residues and may be removed as the Cu+-chelates of neocuproine and bathocuproinesulfonate, leaving an additional disulfide bridge on the protein. Inactivation does not require the presence of oxygen, but when substoichiometric levels of copper are used under aerobic conditions the slow regeneration of Cu-2+ becomes rate-limiting. The course of aerobic inactivation is markedly biphasic at 0 degrees using 2 Cu-2+/FAD, with 30% of the total change completed rapidly, followed by a much slower phase. Both the extent of the fast phase and the rate of the second phase are enhanced by increasing levels of Cu-2+, but are relatively unaffected when the Cu-2+/FAD ratio is maintained at 2 and the protein concentration is varied. The enzyme affords several binding sites for Cu-2+ at pH 7.8, and it is suggested that competition between these sites during the initial statistical distribution of metal ions may explain this biphasic behavior.     

Stimulation of carotenogenesis in Blakeslea trispora by cupric ions

Growth of mated Blakeslea trispora in the presence of trace amounts of cupric ions resulted in increases in (a) the amount and rate of initiation of carotenogenesis, (b) the utilization of glucose and Pi and (c) growth. It also caused an increase in trisporic acid synthesis and a two-fold increase in mevalonate kinase activity.     

Strain-specific prion-protein conformation determined by metal ions

In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.     

Reversible inhibition of lymphocyte proliferation by rubidium ions

Rb⁺ ions, in concentrations which are known to inhibit the Na⁺, K⁺-ATPase, displayed a marked suppressive effect on cell proliferation and differentiation as induced by a variety of B and T cell mitogens. This effect, which was noted at non-toxic concentrations of the cation, could be totally reverted by recultivation of cells in RbCl-free medium. These findings imply a close correlation between the Na⁺, K⁺-ATPase and the receptor regulating cell activation, as has previously been suggested. However, we do not favour the hypothesis of Na⁺, K⁺-ATPase being the mitogen receptor in lymphocyte triggering, since impairment of cell transformation probably only reflects a non-specific inhibition of cation dependent metabolic processes, such as amino-acid and carbohydrate uptake in the cells.     

Cyclic conformation of parallel polypeptide chains closed by cross bridges

The method of possible conformations calculations for cyclic structures, formed by two (or more) identical parallel polypeptide chains, closed by cross bridges has been elaborated. The algorithm is necessary for rigorous conformational analysis of cyclic regions in immunoglobulin, fibronectin and myosin.     

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.     

Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.     

Reversible assembly of helical filaments by de novo designed minimalist peptides

We have designed a series of 15 short, helical de novo peptides consisting of lysine, isoleucine, and alanine. We have termed this the KIA series. These peptides differ only in their hydrophobic interface, and thus their self-association is largely a consequence of hydrophobic interactions. One of these peptides, KIA13, forms insoluble helical fibers at specific NaCl concentrations. We have used CD spectroscopy, turbidity assays, and in situ tapping mode atomic force microscopy to characterize the reversible assembly pathway for this peptide. It is unfolded at low NaCl concentration, and forms helical, soluble fibers resembling a coiled-coil conformation at intermediate NaCl concentrations, and rope-like insoluble fibers at high NaCl concentrations. Reducing the NaCl concentration completely reverses this process. Another peptide from the KIA series specifically inhibits the formation of the insoluble KIA13 fibers, and reverses the process to some extent. This work sheds light onto protein fibrillogenesis and offers intriguing possibilities for the use of these types of peptides in drug delivery and biomaterials applications.