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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.

Abstract: The phenomenon of habituation may be interpreted as a process that has evolved for filtering out iterative stimuli of little present relevance. That habituation is seen in aneural as well as neural organisms throughout phylogeny with remarkably similar characteristics suggests that its role is an important one in animal survival. If habituation is to be viewed as a process to filter out iterative stimuli that have no significant consequences, then how is sensitization to be viewed? One way of viewing these two behavioral changes, i.e. habituation and sensitization, is that they are homeostatic processes which optimize an organism’s likelihood of detecting and assessing the significance of a stimulus in a new iterative series or a change in it. If one views the level of initial responsiveness to a new stimulus as a function of an organism’s threshold just prior to stimulus occurrence, then “high responders” (i.e. those who initially react more strongly) are assumed to have a lower threshold for detecting and assessing the significance of this stimulus than are the “low responders” (i.e. those who initially react more weakly). Thus, high-responders would initially receive more sensory input and progressively decrease their responsiveness to a non-threatening stimulus (habituation). Likewise, initial low-responders would receive less sensory input followed by a decreased threshold and an increased response to the next stimulus occurrence (sensitization). The level of responsiveness achieved in both habituaters and sensitizers, as an asymptote is approached, is a balance between being too sensitive to an unimportant stimulus (and possibly missing other significant stimuli) and being too insensitive, and missing a change in the relevance of the present stimulus. These response changes can be taken as indices of the organism’s mechanisms for achieving an appropriate threshold level to an iterative stimulus in order to accurately assess its present significance and then eventually to asymptote at an optimal stable response level. This approach toward an asymptote is a behavioral homeostatic process that reflects the accumulated significance of the iterative stimulus at each occurrence. The purpose of adding “behavioral” to the term “homeostasis” is to extend the usual meaning of the concept from primarily internal processes to also include (a) iterative external stimulation, (b) the organism’s initial threshold to the initial stimulus as well as (c) the behavior which results from it. Since we are discussing organisms that range from intact, single-celled protozoa to intact mammals, as well as surgically simplified preparations, the termsstimulus, response andbehavior will be used broadly. While other investigators have focused on specific cellular mechanisms underlying habituation and sensitization in a given organism, this paper focuses on the adaptive significance of these two behavioral processes viewed across phylogeny.

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.

Abstract: The phenomenon known as “learned helplessness” (LH) is seen broadly across the animal kingdom. Some of the basic characteristics of this behavior are: failure to escape shock when it is possible to do so following non-escapable shock; reversion to non-escape behavior even after successful escape; if the animal is given escape/avoidance training prior to being given inescapable shocks, the latter will not interfere with its ability to later show normal escape/avoidance behavior (generally described as an immunization effect); following inescapable shock training the animals often become “passive and still” when confronted with an escapable shock. These behaviors are seen in intact mammals, lower vertebrates, and invertebrates. In fact, the basic characteristics are even seen in a spinal rat and, with the exception of one characteristic not yet examined, in an isolated thoracic ganglion of an insect. The brain is evidently not essential either in mammals or in invertebrates for demonstrating this behavior. Not only can an insect ganglion show the behavioral characteristics of LH, but the neural information underlying the phenomenon of LH can be shown to transfer from one ganglion innervating one pair of legs to another ganglion innervating a different pair of legs. Thus, how CNS information underlying LH is coded and transferred from one site to another within the CNS can be examined in such a system. The LH model has provided valuable insights into the physiology of depression. This model suggests that human depression is caused by one’s lack of control over traumatic events. It is supported by a number of parallels between depression and LH behavior. Tricyclic antidepressants, MAO inhibitors, and ECT, which are effective in treating depression, also can prevent and reverse LH in mammals. It would be important to find out if they are also effective in invertebrate models. The fact that the characteristics of the behavior called LH are seen in invertebrates such as slugs, cockroaches, and locusts provokes other intriguing questions about the presence of cognition at these phylogenetic levels, as well as what animal or preparation constitutes an appropriate model for human depression.

The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) beta chain has recently been determined to 1.7 A resolution. To investigate whether this soluble beta chain (murine V beta 8.2J beta 2.1C beta 1) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGs in the absence of MHC class II molecules. Dissociation constants (KDs) were determined using two independent techniques: surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing V beta 8.2. In contrast, SEA, which does not stimulate V beta 8.2-bearing cells, does not bind the recombinant beta chain. Binding of the beta chain to SAGs was characterized by extremely fast dissociation rates (> 0.1 s-1), similar to those reported for certain leukocyte adhesion molecules. Whereas the beta chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 microM, the corresponding value for SEB was approximately 140 microM. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3- to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma. We interpret these results in terms of the ability of SEC to activate T cells independently of MHC, in contrast to SEB. We have also measured SE binding to the glycosylated form of the beta chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V beta 8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V beta specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated beta chain.

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.

High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation.

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.

The sigmoidal steady-state kinetics of biosynthetic threonine deaminase from Escherichia coliis allosterically controlled by isoleucine and valine, the end-products of branched-chain amino acid biosynthesis. A basis for the regulation of threonine deaminase by heterotropic effecters has been studied by capitalizing on the intrinsic fluorescence of the essential pyridoxal phosphate cofactor in this enzyme in kinetic and equilibrium binding studies with the substrate analog D-threonine. D-Threonine binds cooperatively to four sites on the free enzyme, with an average dissociation constant of 19.8 mM. However, in the presence of saturating valine, or isoleucine, the D-threonine binding isotherms are noncooperative and characterized by dissociation constants of 3.9 and 24.8 mM, respectively. The rate of association of D-threonine with threonine deaminase in the presence of the regulatory ligands was biphasic. Analysis of the data in terms of a two-step scheme whereby the internal aldimine Schiff base in the initial encounter complex undergoes transimination with D-threonine to form an external aldimine yielded estimates for overall binding constants that were in good agreement with those determined from equilibrium binding isotherms. These analyses indicate that the positive allosteric effector valine acts solely to alter the binding of D-threonine to the active sites of threonine deaminase by shifting the equilibrium between a low-affinity and high-affinity state, consistent with predictions from a simple two-state model. However, isoleucine has a compound effect on the enzyme. The negative allosteric ligand promotes decreases in the rate and equilibrium constants for encounter complex formation, consistent with its preferential binding to the low-affinity state of the enzyme. In addition, however, isoleucine promotes a decrease in the transimination rate and equilibrium constants. Since transimination is generally considered to be protein-catalyzed in pyridoxal phosphate requiring enzymes, it is proposed that isoleucine affects bath binding and catalysis in threonine deaminase, which provides a possible explanation for the inadequacy of a simple two-state model to describe the allosteric regulation of this enzyme.

Using a 9.4 T MRI instrument, we have obtained images of the mouse brain response to photic stimulation during a period between deep anesthesia and the early stages of arousal. The large image enhancements we observe (often >30%) are consistent with literature results extrapolated to 9.4 T. However, there are also two unusual aspects to our findings. (i) The visual area of the brain responds only to changes in stimulus intensity, suggesting that we directly detect operations of the M visual system pathway. Such a channel has been observed in mice by invasive electrophysiology, and described in detail for primates. (ii) Along with the typical positive response in the area of the occipital portion of the brain containing the visual cortex, another area displays decreased signal intensity upon stimulation.

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.

The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction. The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin. The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates. The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1). The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding. The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex. The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides. These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin. The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.

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 discussion presented deals with the question of the existence of a neural code for learning. What parameters of nerve impulses might be involved in encoding at least one kind of avoidance learning ?

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.