Biomedical subjects

W J Fantl

Publications and source records attributed to W J Fantl.

25 records · Page 2Linked to original sources

PubMed1990

Functional measurements on carboxymethylated hemoglobin, a potential blood substitute: effects of sodium chloride on pH values that influence the alkaline Bohr effect.

The leakage of chloride from electrodes during measurements of the alkaline Bohr effect of hemoglobin (by the proton release method) amounted to 1-5 mM concentration of the anion depending on the type of electrode employed. This concentration, together with the amount of chloride found to be intrinsically bound to hemoglobin (0.2-0.6 mM), could mask the contribution of chloride to various hemoglobin functions. In addition, the concentration of chloride was found to affect the pH of buffers as measured either with a pH meter or with the dye, cresol red. Thus, for 20 mM phosphate buffer, the pH was lowered almost 0.4 pH units in the presence of 0.30 M chloride. For Tris-acetate buffer, the same concentration of chloride led to an increase in pH of about 0.05 units.

Blood Substitutes↗

PubMed1989

Mutations of the platelet-derived growth factor receptor that cause a loss of ligand-induced conformational change, subtle changes in kinase activity, and impaired ability to stimulate DNA synthesis.

The wild-type and two mitogenic-defective mutants of the type beta receptor for platelet-derived growth factor (PDGF) were expressed in Chinese hamster ovary cells. In the first mutant, delta Ki, 82 of 104 amino acids in the kinase insert region were deleted. This mutant was recently reported to be defective in mediating DNA synthesis. In the second mutant, Y825F, tyrosine 825 was converted to phenylalanine by a point mutation. We report here that this mutant is also defective in mediating PDGF-stimulated DNA synthesis. Both mutants were capable of eliciting many of the early responses to PDGF, including receptor autophosphorylation. However, neither mutant was capable of undergoing PDGF-stimulated change in receptor conformation or of phosphorylating exogenous substrate in an in vitro assay. These data suggest that changes in receptor conformation and efficient utilization of specific tyrosine kinase substrates are important for the stimulation of cell proliferation of PDGF and that phosphorylation of tyrosine 825 may be involved in signal transduction.

Animals↗

PubMed1988

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation.

The effect of ligand binding on PDGF receptor conformation was examined using peptide antisera directed against specific receptor domains. Antiserum 83, which recognizes the receptor's carboxyl terminus, preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the receptor was induced by the three different forms of PDGF (AA and BB homodimers and AB heterodimer) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by AMP-PNP, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in CHO fibroblasts transfected with wild type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site, the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis, even though [125I]PDGF binding was normal. Ligand-induced receptor autophosphorylation was reduced but present in cells expressing receptors that lacked a major site of autophosphorylation and was present in cells expressing receptors that lacked the kinase insert domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin.

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding, Competitive↗

PubMed1987

Properties of carboxymethylated cross-linked hemoglobin A.

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1983

Selective carboxymethylation of the alpha-amino groups of hemoglobin. Effect on functional properties.

Hemoglobin A hybrids with carboxymethyl groups at the alpha-NH2 termini of the alpha-chains or the beta-chains or at the termini of both chains have been prepared by reductive alkylation of the protein with glyoxylate and NaCNBH3 under controlled conditions. A hemoglobin derivative, which was selectively carboxymethylated at the NH2-terminal residues, was separated into its alpha- and beta-chains. These derivatized chains were recombined to yield alpha 2 Cm beta 2 Cm or were combined with unmodified beta- or alpha-chains, respectively, and purified to yield alpha 2 Cm beta 2 or alpha 2 beta 2 Cm. These hybrid tetramers retained their cooperativity and function (average n = 2.4). The hybrid alpha 2 Cm beta 2 had a lower oxygen affinity (p50 = 12 mm) than unmodified hemoglobin (p50 = 7 mm) and was reactive with 2,3-diphosphoglycerate (p50 = 48 mm). The oxygen affinity of the derivative alpha 2 beta 2 Cm was lower (p50 = 17 mm) than unmodified hemoglobin and was affected only slightly by 2,3-diphosphoglycerate (p50 = 25 mm). The tetramer carboxymethylated at all 4 NH2-terminal residues, alpha 2 Cm beta 2 Cm, exhibited a very low intrinsic oxygen affinity (p50 = 37 mm) that was further lowered to a limiting value of 50 mm by saturating amounts of 2,3-diphosphoglycerate. Each carboxymethyl tetramer, except alpha 2 Cm beta 2 Cm, was reactive with chloride to lead to a lower oxygen affinity. These carboxymethylated hybrids (Hb-NH-CH2COO-) may provide a useful model system for studies on the binding of anions to hemoglobin or on the interaction of CO2 with hemoglobin to form the carbamate Hb-NH-COO-.

2,3-Diphosphoglycerate↗

PubMed1981

Acetaldehyde adducts with hemoglobin.

Clinical studies on the minor hemoglobins (hemoglobin A1a-c) have suggested that a novel adduct may form in people abusing alcohol. Such patients were found to have an elevated concentration of minor hemoglobins, but normal or subnormal amounts of glycosylated hemoglobin (hemoglobin A1c) as determined by radioimmunoassay, Acetaldehyde, a reactive metabolite of ethanol, was postulated to form adducts with hemoglobin A that change its chromatographic properties. At physiological concentrations, acetaldehyde was found to form adducts with hemoglobin that were stable to extensive dialysis for several days. The amount of hemoglobin adducts formed were a function of the concentration and number of exposures to acetaldehyde. The reaction of acetaldehyde with hemoglobin A produced chromatographic variants, some of which migrated in the hemoglobin A1a-c region. Analysis of stable acetaldehyde-hemoglobin adducts demonstrated that valine, lysine, and tyrosine residues of globin were sites of reaction. The acetaldehyde-modified amino acid residues appear to exist in interconvertible conformations, only one of which is reducible by sodium borohydride. The amount of these adducts was found to be significantly elevated in hemoglobin from alcoholics as compared with normal volunteers.

Acetaldehyde↗