Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc 1 Man 7-9 GlcNAc 2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc 1 Man 7-9 GlcNAc 2 , determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K b ) and changes in enthalpy of binding (⌬H b°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 ؋ 10 4 M ؊1 at 279 K is obtained when glucose occurs in ␣-1,3 linkage to Man␣Me as in Glc␣1-3Man␣Me. The binding constant increases by 25-fold from di-to trisaccharide and doubles from tri-to tetrasaccharide, demonstrating that the entire Glc␣1-3Man␣1-2Man␣1-2Man␣Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed. Calreticulin (CRT),1 along with calnexin, serves as a molecular chaperone in the endoplasmic reticulum (ER) of eukaryotic cells. Although calreticulin is a soluble, luminal protein, calnexin is a type I membrane protein (1, 2). Segments of these proteins share amino acid identity ranging from 42 to 78% (3). Calreticulin is a highly conserved ubiquitous protein (M r 46,000) and has been implicated in Ca 2ϩ storage and intracellular Ca 2ϩ signaling in the sarcoplasmic and endoplasmic reticula (4, 5). CRT has been divided into three regions: the N-terminal, the C-terminal, and the central P-domain, which consists of short sequence motifs repeated three times in tandem. The N-terminal domain is highly conserved among CRTs from different species and potentially mediates interactions between CRT and the ER folding catalysts, protein disulfide isomerase and ERp57 (6, 7). Recent studies show that the P-domain, previously thought to be involved in oligosaccharide binding, interacts directly with ERp57 (8 -10). The C-domain is characterized by a high content of acidic residues (4, 11), which is consistent with the location of a low affinity (K d ϭ ϳ1-2 mM), high capacity (ϳ25-50 mol) calcium-binding site (12) and contains the ER retrieval sequence.The ER plays an essential role in the folding and maturation of newly synthesized proteins in the secretory pathway. ER quality control operates at various levels; one of the most comm...

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An observed higher degree of autolysis in irradiated (20–200 Krads) wheat is apparently due to increased susceptibility of proteins to protease action. Total amino acid profiles of wheat and of isolated gluten reveal no appreciable changes on irradiation up to 1 Mrad. However, there is an overall increase in free amino acid levels in wheat irradiated at 1 Mrad. Lysine availability in wheat is not affected by radiation treatment. Studies on radiosensitivities of wheat proteins show a shift in molecular weight distribution to lower values.

Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT1B receptor) and complex lesions (SERT, 5-HT1B, 5-HT2B receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT2B receptor). Selective inhibitors of individual signaling elements – SERT, 5-HT2A, 5HT2B, and combined 5-HT2A/B receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine in vivo system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH.