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The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.

Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor-alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought.

Protein kinase C theta (PKCtheta) is a novel Ca(2+)-independent PKC isoform, which is selectively expressed in skeletal muscle and hematopoietic cells, especially T cells. In T cells, it colocalizes with the T cell antigen receptor (TCR).CD3 complex in antigen-stimulated T cells and is involved in the transcriptional activation of the interleukin-2 gene. In the present study, we report that PKCtheta is tyrosine phosphorylated in Jurkat T cells upon TCR.CD3 activation. The Src family protein-tyrosine kinase, Lck, was critical in TCR-induced tyrosine phosphorylation of PKCtheta. Lck phosphorylated and was associated with the regulatory domain of PKCtheta both in vitro and in intact cells. This association was constitutive, but it was enhanced by T cell activation, with both Src-homology 2 and Src-homology 3 domains of Lck contributing to it. Tyrosine 90 (Tyr-90) in the regulatory domain of PKCtheta was identified as the major phosphorylation site by Lck. A constitutively active mutant of PKCtheta (A148E) could enhance proliferation of Jurkat T cells and synergized with ionomycin to induce nuclear factor of T cells activity. However, mutation of Tyr-90 into phenylalanine markedly reduced (or abolished) these activities. These results suggest that Lck plays an important role in tyrosine phosphorylation of PKCtheta, which may in turn modulate the physiological functions of PKCtheta during TCR-induced T cell activation.

Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. Poly-glxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Oxidations of hemoglobin in oxygen-saturated and deoxygenated media by 10 structurally variant alkyl nitrites have been examined in kinetic detail. Pronounced structural influences on rate constants, whose values span a range of 80 in oxidations of both oxy- and deoxyhemoglobin, have been observed. tert-Butyl nitrite provides the slowest oxidation rate that for deoxyhemoglobin terminates after only half of the available iron(II) heme units have been oxidized. Activation parameters have been determined for oxidations of oxyhemoglobin and deoxyhemoglobin by ethyl, isopropyl, and neopentyl nitrites from kinetic evaluation of these reactions as a function of temperature. The differences in free energies of activation (delta G) between hemoglobin R and T states range from 1.8 to 2.9 kcal/mol for the three alkyl nitrites examined. The composite data portray alkyl nitrite oxidations as inner sphere electron transfer processes whose kinetic characteristics reflect the ligand binding properties of hemoglobin. A sulfhydryl-induced alkyl nitrite oxidation of oxyhemoglobin that is most pronounced in oxygen-saturated media has been observed, and its cause has been traced to nitrosyl exchange of alkyl nitrites with the beta-93 cysteine sulfhydryl group of hemoglobin.

In anaerobic phosphate buffer, pH 6.3-7.5, deoxyhemerythrin is oxidized to semi-methemerythrin (semi-met) by excess sodium nitrite. This oxidation is quantitative as judged by EPR spectroscopy. Further oxidation to methemerythrin is not detected. The absorbance changes of hemerythrin during the oxidation are biphasic. The rate of the faster first phase is linearly dependent on [H+] and [NO2-] suggesting that the oxidant is nitrous acid rather than nitrite. During the slower second phase, the characteristic EPR spectrum of semi-methemerythrin appears. The first phase can be interpreted by a scheme in which nitrous acid transforms deoxyhemerythrin (FeIIFeII) to the semi-met nitrosyl adduct (FeIIFeIIINO) and hydroxide. Independent experiments confirm that the combination of semi-met plus NO produces an EPR-silent adduct. The rates of the absorbance changes for the second phase are nearly independent of nitrite concentration and pH in the range 6.3-7.5. This slower phase involves the transformation of the EPR-silent intermediate to the semi-met nitrite adduct (FeIIFeIIINO2-) and is consistent with rate-limiting dissociation of nitric oxide followed by rapid attachment of nitrite. Nitrite appears to be a unique oxidant of deoxyhemerythrin in that when employed in excess, the final, stable product is semi-met- rather than methemerythrin. The lack of reactivity of ethyl nitrite with deoxyhemerythrin suggests that HONO oxidizes deoxyhemerythrin via an “inner-sphere” process in contrast to oxidants such as Fe(CN)6(3-). A proposed generalization is that excesses of “inner-sphere” oxidants convert deoxy to (semi-met)R, which is stabilized with respect to (semi-met)R, which is stabilized with respect to (semi-met)0 and met because the oxidant and/or a product of the oxidant can bind to the iron site.

An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-fcomplex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-fcomplex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 °C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr= 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr= 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr= 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr= 17,000 protein.

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for a-thrombin in solution. γ-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd= 5.7 × 10−10M) and stimulating the saturated rate of protein C activation by factor Xa (kcat= 149 min−1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody α-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.receptor alpha beta complex and signal transduction.

The stoichiometry of CO ligation to the dimer heme protein Rhodospirillum molischianum cytochrome c' is determined. We have recently measured the enthalpy change of CO ligation to this molecule by the van't Hoff method and found the value of -10.7 +/- 1.2 kcal/mol CO (aqueous) (Doyle, M. L., Weber, P. C., and Gill, S. J. (1985) Biochemistry 24, 1987-1991). In the present paper the enthalpy change of CO ligation, measured directly by titration calorimetry, is found to be -9.5 +/- 0.2 kcal/mol heme. Since the van't Hoff method gives the heat value in units/mole of CO and the calorimetric method gives the heat value in units/mole of heme, the stoichiometry of the reaction is given by the ratio of the two values and found to be 0.9 +/- 0.1, or within experimental error, one CO molecule bound per heme.