Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

Contact inhibition within hemoglobin S polymer by thiol reagents.

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.

Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties.

Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.

Effects of the alpha 20 mutation on the polymerization of Hb S.

The contribution of the alpha 20 residues in intermolecular contacts present in hemoglobin S fibers was investigated with mixtures of Hb Le Lamentin alpha 2(20)His----Gln beta 2A and of hemoglobin S alpha 2A beta 2(6)Glu----Val and with artificial hybrids alpha 2(20)His----Gln beta 2(6)Glu----Val. This study showed an increased solubility and delay time of polymerization of Hb S in solution only when the mutation at the alpha 20 residue is cis to the beta 6 Val contact. No modification of the polymerization process occurs when the mutation is trans to this beta 6 Val contact. This result is in agreement with the crystal model of Wishner and Love, who showed that one of the two alpha 20 residues of the Hb S tetramer was involved in an axial contact between hemoglobin S molecules in the crystals of Hb S ( Wishner , B.C., Ward, K.B., Lattman , E.E. and Love, W.E. (1975) J. Mol. Biol. 98, 179-194). The present observation is a new illustration of the validity of the crystal model for the structure of the fibers based on pairs of double filaments.

Changes of polymerization and conformation of hemoglobin S induced by thiol reagents.

Thiol reagents, covalently bound to cysteine beta 93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2'-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.

Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.

Kinetics of polymerization of hemoglobin S modified by thiol reagents and by oxidation.

The effects of four thiol reagents on the kinetics of polymerization of hemoglobin S have been studied in high phosphate buffer (1.8 M), in the presence (3 mM) or absence of sodium dithionite, depending on the reduction of mixed disulfides of Hb in the presence of this reducing agent. The effect of oxidized forms (methemoglobin) of HbS on the kinetics of aggregation of deoxyHbS was also studied because of the presence of 33% metHbS when HbS was modified by 4-aminophenyl disulfide. In the presence of sodium dithionite, the delay times prior to polymerization of deoxyHbS modified by N-ethylmaleimide, iodoacetamide and 4-aminophenyl disulfide were, respectively, 1.5-, 1.35- and 1.15-times longer than that of native deoxyHbS. The results indicate that the radicals bound to the cysteine beta 93 residue inhibit the contacts in the polymer formation to various extents but do not modify the size of the nuclei.

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.

Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

Structural modeling of the human erythrocyte bisphosphoglycerate mutase.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Reactivity of 42 disulfides with thiol group of human haemoglobin and human serum albumin.

The reactivities of disulfides of different compound families towards thiol groups of human haemoglobin and human serum albumin were determined at physiological pH 7.4 by anion-exchange liquid chromatography. The apparent second-order kinetic rate constants, K1, were calculated for the reaction of these disulfides with each protein. The results show that the studied heterocyclic disulfides are the most reactive compounds with both proteins. The lipophilic properties of these disulfides were evaluated by reversed-phase high performance liquid chromatography, using the percentage of acetonitrile (PAC) required for eluting each compound of the chromatographic column in a water-acetonitrile gradient. The structure-reactivity correlations between log K1 and log PAC are stated for each protein and compared. They fit a parabolic curve which permits one to define a lipophilic domain corresponding to a quantitative reaction of disulfides towards these proteins. The studied disulfides present a similar optimum of reactivity for both proteins.

Filterability of sickle cells as a function of pO2: role of physico-chemical factors.

A rigidity index (RI) related to red blood cell deformability was measured by using the hemorheometre. The RI for 13 patients homozygous for sickle cell disease was 109 +/- 44 at 37 degrees C and at atmospheric pO2. The filtration time curve as a function of pO2 is biphasic for sickle cell suspensions. The pO2 at which filtration time is maximum, pO2max., correlated with the rigidity index measured at atmospheric pO2. This pO2max. value was very sensitive to small changes in physico-chemical parameters such as osmolality, pH, temperature, hematocrit, and cell density. Conditions which reduced the Hb S polymerization induced a leftward shift of pO2max.. The experimental curves are in agreement with theoretical models based on the presence of two abnormal cell types: filtrable "slow cells" and infiltrable "sickled cells".

[Evaluation of three commercial kits for the estimation of total glycolysed hemoglobin: Hb Al (author's transl)]. / Evaluation de trois trousses commerciales pour le dosage de l'hémoglobine glycosylée totale : Hb Al.

It has been demonstrated by numerous workers that variations in levels of glycolysed hemoglobins Hb Al (Alb, Alb, Alc) are an excellent means of control of the quality of diabetes control. The estimation of the main glycolysed hemoglobin (Hb Alc) is very difficult, and various chromatographic methods have been developed to permit rapid estimation of all these glycolysed hemoglobins. Our work consisted of testing the three microcolumn kits on the french market: Isolab, Helena and Biorad. The great thermo-dependency of the first two kits did not permit us to obtain results in correlation with those of our reference technic derived from Trivelli's method. On the other hand, the levels of Hb Al obtained with the Biorad kit were perfectly correlated with the levels of Hb Alc. The average normal of Hb Al was 6,45 +/- 0,66 p. cent, CV 10,2 p. cent; the average value of Hblc was 5,4 +/- 0,4 p. cent. CV 7,4 p. cent. The average levels of Hb A obtained in diabetic subjects was 12 p. cent, the levels of Hblc being 10 p. cent. Certain restrictions in the use of these microcolumns were demonstrated: presence of Hb F or abnormal Hb, hyperlipemia and presence of chylomicrons.