Haemoglobin Avicenna (beta 47 (CD6) Asp replaced by Ala). A new abnormal haemoglobin.

In a survey for abnormal haemoglobin variants in voluntary blood donors in Iran, a new variant was found in a young male who presented no clinical symptoms. It had the same electrophoretic mobility as haemoglobin D in alkaline buffers. Separation of the constituent polypeptide chains in acid urea buffer revealed it to be different from haemoglobin D previously found among Iranians. Analysis of its structure demonstrated a substitution to alanine (beta 47 Asp replaced by Ala) in the same residue as involved in haemoglobin G-Copenhagen (beta 47 Asp replaced by Asn).     

Haemoglobin Titusville: alpha94 Asp replaced by Asn. A new haemoglobin with a lowered affinity for oxygen.

A new haemoglobin variant with a decreased oxygen affinity is described, in which the substitution, alpha 94 (G1) Asp replaced by Asn, affects the alpha1beta2 contact alpha1G1-beta2G4. The relevance of this variant to our understanding of the importance of the hydrogen bond between alpha1G1 and beta2G4 in Perutz's model of oxyhaemoglobin A is discussed.     

Haemoglobin North Shore, beta134 Val replaced by Glu, a new unstable haemoglobin.

A new mildly unstable haemoglobin, Hb North Shore (beta134 Val replaced by Glu) is described. It was found in a 4th generation Australian of Anglo-Celtic origin with a mild microcytic anaemia. The charge change involved is partially obscured as evidenced by the electrophoretic mobility of the native haemoglobin. The structural studies illustrate the usefulness of Staphylococcus aureus strain V8 protease as a specific cleavage enzyme at glutamyl residues.     

The oxygen affinity of haemoglobin Tak, a variant with an elongated beta chain.

The oxygen affinity was investigated of purified Hb Tak, a human haemoglobin variant with elongated beta-chains. A very low P50 value was found which was not influenced by the addition of 2,3 diphosphoglycerate. The n value was 1, indicating non-cooperativity. The oxygen equilibrium curve of the whole blood haemolysate containing Hbs A and Tak was close to that of Hb A at the top of the curve, while the bottom of the curve greatly deviated from the latter, indicative of small if any interaction between Hb A and Tak during oxygenation.     

Haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) structure and function.

The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.     

A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor.

Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells. Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades. The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Recently, the feedback inhibitor suppressor of cytokine signaling-3 (SOCS-3), also referred to as cytokine-inducible SH2-containing protein 3 (CIS-3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR-associated Janus kinase 2 (Jak2) [Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I. & Yoshimura, A. (2000) J. Biol. Chem 275, 29338-29347]. In this study tyrosine 401 was identified as a binding site for SOCS-3 on the EpoR. Here we show that human SOCS-3 binds to pY401 with a Kd of 9.5 microm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS-3 but with a ninefold higher affinity than we found for the previously reported motif pY401. In addition, SOCS-3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS-3 binding. Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS-3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS-3 on the EpoR.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.     

Molecular modeling and prediction of binding mode and relative binding affinity of Art-Qui-OH with P. falciparum Histo-Aspartic Protease (HAP)

The relative binding affinity in terms of ΔΔG _bind-cald value of the antimalarial compound artemisinin-quinine hybrid is primarily derived and is discussed in this article with reference to the ΔG _bind-cald values of two known inhibitors Pepstatin-A and KNI-10006 complexed with HAP enzyme. The ΔG _bind-cald value for KNI-10006 and Pepstatin-A is -14.10 kcal/mol and -13.09 kcal/mol respectively. The MM-GB/SA scoring results in the relative binding energy (ΔΔG _bind-cald) of the hybrid molecule with respect to Pepstatin-A as 2.43 kcal/mol and 3.44 kcal/mol against KNI-10006. The overall binding mode of Art-Qui-OH resembles that of Pepstatin-A binding in HAP active site. We suggest here that the ΔΔG _bind-cald value & proposed binding mode of the Art-Qui-OH for HAP enzyme should be considered for further structure-based drug design effort.     

3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.     

A new haemoglobin variant: haemoglobin Anantharaj alpha 11 (A9) lysine replaced by glutamic acid.

Four heterozygotes for a fast alpha-chain variant in a Thai family were detected on starch gel electrophoresis during a survey study on iron deficiency anaemia in a rural area not far from Bangkok. They were healthy and had normal haematological profiles except for the presence of around 44% abnormal pigment, quantitated by cellulose acetate electrophoresis. The structural characterization of the variant by globin chain separation, peptide mapping, and amino acid analyses of the abnormal peptides indicated that lysine residue 11 (A9) of alpha-chain was replaced by glutamic acid. This mutation has not been previously described and it is proposed that it be called Haemoglobin Anantharaj.     

Blood oxypurines and erythrocyte 2, 3-diphosphoglycerate levels at high altitude hypoxia

Effect of acute high altitude hypoxia on the levels of plasma oxypurines and erythrocyte 2, 3-diphosphoglycerate (2, 3-DPG) was investigated. Exposure to simulated altitude at 4000, 5000, and 6000 m rapidly increased plasma oxypurines as well as erythrocyte 2, 3-DPG in human and mice. A close correlation of the concentration of oxypurines and 2, 3-DPG in human with the oxygen pressure, suggesting that the levels of these metabolites may be indicative of tissue hypoxia.     

The C2A-C2B linker defines the high affinity Ca(2+) binding mode of rabphilin-3A

The Ca(2+) binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca(2+) binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca(2+)-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca(2+) ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca(2+) binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca(2+)-bound state of the C2B domain. In addition, this Ca(2+) binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca(2+) binding mode not only shows how a C2 domain increases its intrinsic Ca(2+) affinity, but also provides the structural base for an atypical protein-Ca(2+)-phospholipid binding mode of rabphilin-3A.