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Viruses coopt host intracellular Ca2 signaling pathways to optimize timing and effectiveness of infection stages against barriers to invasion, pathogenesis, replication, and release. Virus-induced changes in free cytosolic Ca2 levels facilitate virus adsorption, uncoating, catalysis, toxin production, structural assembly and stabilization, trafficking, and fusion and budding. Ca2-associated alterations in virus status also selectively precipitate host cytopathologies through, among other events, retardation or induction of apoptosis, elevation of metabolic stress and reactive oxygen species production, and promotion of proinflammatory cytokine and chemokine synthesis and release. Viral particles and proteins tune spatiotemporal dynamics of host free cytosolic Ca2 concentrations by modulating Ca2 entry from the extracellular environment, upstream first or second messengers, ion- and ATP-dependent Ca2 pumps that sequester or extrude free cytosolic Ca2, store-operated Ca2 mobilization and leakage, and viral capsid/envelope and downstream host Ca2 binding proteins and sensors. Each of these major viral mechanisms, briefly reviewed in this article, presents a suitable drug target capable of mitigating the severity and incidence of viral infections. Given its pivotal role in cellular response regulation, bioenergetics, posttranslational protein and lipid modification and transport, homeostasis, cell motility and morphogenesis, and T lymphocyte proliferation, targeting virally stimulated inositol 1,4,5-trisphoshate (IP3)-mediated store-operated Ca2 release especially offers unique, predictable benefits for augmenting immunoprotection in vertebrate clinical populations. We appraise possibilities of modulating this system with experimental proteins that gate activation kinetics of endoplasmic-reticulum-localized Ca2-conducting IP3 receptors via allosteric protein-protein interactions. Such compounds are expected to be valuable in treating primary disease symptoms and sequelae, including virus-associated dementia.

The heat shock protein GroEL from Escherichia coli is a tetradecameric oligomer that facilitates the refolding of nonnative polypeptides in an ATP-hydrolysis dependent reaction. A mutant in GroEL was prepared in which lysine 3 was substituted with glutamate, which destabilizes the oligomeric structure of GroEL (Horovitz, A., Bochkareva, E.S., and Girshovich, A.S. (1993) J. Biol. Chem. 268, 9957-9959). The highly expressed and purified GroELK3E was judged to be monomeric by sedimentation equilibrium, yielding a molecular weight of 54,500, despite a weak tendency of the mutant to reversibly form higher order aggregates above 4 mg ml-1. The monomeric variant appears to be folded based on the far UV circular dichroism spectrum, which shows significant alpha-helical content, but with slight differences in conformation relative to wild-type GroEL. The increase in exposed hydrophobic surface of the monomer was probed with the dye 4,4'-bis-1-anilino-3-naphthalenesulfonate (bis-ANS). The fluorescence of bis-ANS increases approximately 150-fold in the presence of the mutant, and about 4 mol of bis-ANS bind per mol of monomer, with a binding constant of 1.6 microM. Adenosine nucleotide binding to monomeric GroELK3E resulted in considerable quenching of bis-ANS fluorescence, correlating with significant structural changes as seen in the far UV circular dichroism, and permitted the measurement of binding isotherms for ATP and ADP. Hyperbolic ATP binding isotherms yield a dissociation constant of 82 microM, about 4-fold weaker than the K0.5 for ATP seen in steady-state kinetics assays of the wild-type GroEL ATPase.A similar difference was seen for ADP binding. These results suggest that the mutation disrupts the native tetradecameric quaternary structure through conformational changes that may also weaken nucleotide binding. The monomeric mutant exhibited no chaperone activity as evidenced by a filure to inhibit or facilitate the refolding of chemically denatured enolase, an inability to refold denatured rhodanese above spontaneous levels, and a lack of binding to alpha-casein, a competitor in many chaperonin-promoted refolding reactions. Thus, the formation of assembly incompetent monomers by the lysine 3 to glutamate mutation results in a dramatic decrease in the affinity for nonnative polypeptide chains and suggests that the oligomeric nature of GroEL is crucial for its molecular chaperone function.

Electron microscopic studies employing negative staining, rotary shadowing, and unidirectional shadowing have revealed the subunit architecture of ornithine transcarbamoylase and arginase from Saccharomyces cerevisiae. These techniques have confirmed the quaternary structure of these enzymes, and have permitted an estimate of the shape and dimensions of each of the individual enzymes as well as those of the corresponding subunits to be determined. Both enzymes are trimers exhibiting 3-fold rotational symmetry with subunits which are oblate ellipsoids of revolution. The overall dimensions determined for ornithine transcarbamoylase are a height of 39 A and a width of 102 A. The consequent subunit dimensions are 59 X 59 X 39 A, yielding a subunit axial ratio of 1.51. Arginase has a height of 42 A and a width of 97 A. The subunit dimensions are 56 X 56 X 42 A, yielding a subunit axial ratio of 1.33. The hydrodynamic behavior of the two enzymes is fully consistent with this molecular architecture, suggesting that the structures proposed here are similar to those of the proteins in aqueous solution.

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.

Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (c) and six regulatory (r) polypeptide chains. Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains. Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts. Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state. In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme. When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp. Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-asparatate in weakening intersubunit interactions. Subunit exchange increased about 9-fold for the r142 Glu----Asp mutant and almost 20-fold for the r142 Glu----Ala mutant in the presence of the ligand.

It has been proposed that regulatory multienzyme complex formation between yeast ornithine transcarbamoylase (OTCase) and arginase is triggered by a conformational change promoted by the binding of ornithine to a regulatory site in OTCase (Wiame, J.-M. (1971) Curr. Top. Cell. Regul. 4, 1-38). To isolate the binding of ornithine to the proposed regulatory site, the active site was blocked with the high affinity (Ki = 13 +/- 1.4 nM) bisubstrate analogue, delta-N-phosphonacetyl-L-ornithine (PALO). The binding of PALO to the active site produces large changes in the absorption (delta A290-296 = 0.010/mg of enzyme) and in the fluorescence (25% quenching) of the protein. These changes both saturate at one PALO/polypeptide chain. The binding of PALO also changes the rate constant for diffusional acrylamide quenching by 43% and increases the midpoint for the thermal denaturation of the enzyme by 13 degrees C. Finally, PALO binding results in a +2.8% change in the sedimentation coefficient demonstrating that these spectral and energetic changes are associated with a gross structural change in the enzyme. In an effort to detect ligand binding to the proposed effector site on OTCase, ornithine was added to the enzyme saturated with PALO, and consequent conformational changes were tested for using methodologies identical to those which demonstrated active site ligand binding-promoted conformational changes. In no instance were any additional differences observed. Hence, strong support for isosteric effector binding-promoted conformational changes cannot be presented. We conclude that active site ligand binding events themselves are responsible for conformational changes which promote enzyme-enzyme association of OTCase with arginase.

A one-session T-maze training procedure for cockroaches in which animals were trained to turn right or left to avoid a shock was utilized to investigate the effects of dieldrin and chlordimeform (two neurotoxic pesticides) on learning and memory. These animals were trained and then tested for retention 5 h later. Three behavioral measures were recorded: choice behavior or direction turned, the time taken to proceed down the runway (runway time), and the time taken to proceed from the runway to either choice arm (choice point time). In control animals the number of correct choices, the runway time, and the choice point time increased with succeeding trials during training. Furthermore, control animals showed retention of correct choice behavior from training to testing.A nontoxic dose (no overt behavioral signs) of dieldrin was injected 2 h before training or 15 min after training. Pretraining injections of dieldrin eliminated correct-choice learning but did not alter the increase with training in runway or choice point times. Posttraining dieldrin administration did not interfere with retention of correct-choice behavior upon testing. These findings indicate that dieldrin induces behavioral alterations in a low acute dose and that detrimental effects are more likely with more complex behavior. Furthermore, behavioral disruption is more likely the closer exposure is to the initial learning of a task. Nontoxic doses of chlordimeform injected 1 h before training eliminated correct-choice learning and facilitated an increase in runway times during training.

R67 dihydrofolate reductase (DHFR) provides resistance to the antibacterial drug trimethoprim. This R-plasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of active, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025) indicates that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-directed mutagenesis of residues in the pore will produce four mutations/active site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the essential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDS-polyacrylamide gel electrophoresis and is monomeric under nondenaturing conditions as measured by sedimentation equilibrium experiments. The catalytic activity of quadruple R67 DHFR is decreased only slightly when compared with native R67 DHFR. Folding of quadruple R67 DHFR is completely reversible at pH 5. However, at pH 8, folding is not fully reversible; this is likely due to a competition between productive intramolecular versus nonproductive intermolecular domain association. The production of a fully active, monomeric R67 DHFR variant will enable the design of more meaningful site-directed mutants where single substitutions per active site pore can be generated.

The molecular chaperone GroEl from Escherichia coli is a member of the highly conserved Hsp60 family of proteins that facilitates protein folding. A central question regarding the mechanism of GroEL-assisted refolding of proteins concerns its broad substrate specificity. The nature of GroEL-polypeptide chain interaction was investigated by isothermal titration calorimetry using proteins that maintain a non-native conformation in neutral buffer solutions. A single molecule of an unfolded variant of subtilisin BPN' binds non-cooperatively to GroEL with micromolar affinity and a positive enthalpy change. Additional calorimetric titrations of this chain with GroEL show that the positive enthalpy change decreases with increasing temperature between 6 and 25 degrees C, yielding a delta CP of -0.85 kcal mol-1 degree-1. alpha-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-20 degrees C, yielding a delta CP of -0.44 kcal mol-1 degree-1. The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of these substrates with the GroEL chaperonin.

Ornithine transcarbamoylase (OTCase) has been purified in 100-mg quantities from a plasmid-containing, enzyme-overproducing strain of Saccharomyces cerevisiae. The specific activity of the homogeneous enzyme is 2.5-fold above that previously reported. The molecular weight and partial specific volume of OTCase were determined by sedimentation equilibrium in solutions containing H2O and D2O. Data from two rotor speeds were simultaneously fit using nonlinear least squares analysis with multiple independent variables giving a molecular weight of 110,000 +/- 2,200 and a partial specific volume of 0.732 +/- 0.006 ml g-1. The ultraviolet absorption spectrum of OTCase gives a specific absorbance at 280 nm of 0.36. This low value is consistent with a small number of aromatic residues. Amino acid analysis, fluorescence, and multicomponent analysis yield 1 tryptophan, 4 tyrosine, and 24 phenylalanine/polypeptide chain. From an analysis of the circular dichroic spectrum, it was determined that OTCase contained 22% alpha-helix, 43% beta-sheet, 8% beta-turn, and 27% random structure. The fluorescence of the single tryptophan/polypeptide chain has an emission maximum at 320 nm, indicating a hydrophobic environment.

Shifts in the sigmoidal kinetics of allosteric threonine deaminase promoted by isoleucine and valine binding control branched chain amino acid biosynthesis in Escherichia coli. A highly conserved alpha-helix in the C-terminal regulatory domain of the tetrameric enzyme was previously implicated in effector binding and feedback inhibition. Double (447, 451) and triple (447, 451, 454) alanine replacements for the conserved amino acids leucine 447, leucine 451, and leucine 454 in this region yield enzyme variants that show increased sigmoidality in steady-state kinetics, and which are less sensitive to the allosteric modifiers isoleucine and valine. Equilibrium binding studies using fluorescence, enzyme kinetic, and calorimetric approaches indicate that the enzyme variants possess reduced affinity for isoleucine and valine, and suggest that heterotropic ligands can bind to the same site to promote their different effects. The increase in sigmoidal kinetics for the mutants relative to wild-type threonine deaminase may be attributable to the elimination of L-threonine binding to the effector sites, which activates the wild-type enzyme. Enzyme kinetic data and isotherms for active site ligand binding to the mutants can be analyzed in terms of a simple two-state model to yield values for allosteric parameters that are consistent with previous estimates based on an expanded two-state model for homotropic cooperativity for threonine deaminase.

The use of reperfusion therapy in patients with ST elevation acute coronary syndromes had been established. However, reperfusion therapy is usually considered contra-indicated in those with ST depression, despite the knowledge that regional posterior infarction is typically indicated by ST depression maximal in leads V"1 to V"3 and nonregional subendocardial infarction is typically indicated by marked ST depression maximal in other leads. This study of patients with non-ST-elevation acute coronary syndromes investigates the quantitative relationship between presenting ST depression and final QRS changes in both of these subgroups. The final QRS score was significantly higher (2.44 points) than that of a control group with not ST depression, (1.55 points) in the group with maximal ST depression in V"1 to V"3 (P=0.04). However, in the entire population, there was a highly significant correlation (P=.003) between the sum of the presenting ST depression and the final QRS score. Trials of reperfusion therapy will be required to determine if such evolution to electrocardiogram documented acute myocardial infarction can be prevented in patient with marked ST depression acute coronary syndromes.