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Biomedical subjects

H F Bunn

Publications and source records attributed to H F Bunn.

At least 73 records · Page 4Linked to original sources
PubMed1988

Cotranslational amino-terminal processing of cytosolic proteins. Cell-free expression of site-directed mutants of human hemoglobin.

The diversity of protein structure is significantly enhanced by cotranslational and posttranslational modifications. Structural features at the amino terminus are especially important contributors to protein function and stability. Amino-terminal processing of cytosolic proteins includes the cleavage of the initiator methionine and N alpha-acetylation. To better understand the rules that govern these cotranslational events, site-directed mutants of the human beta-globin gene encoding for all 19 amino acid replacements of Val-beta 1 were expressed in a cell-free transcription and translation system. The initiator methionine was 100% cleaved when the side chain of the adjacent residue was relatively small (radius of gyration less than 1.29 A), whereas the initiator methionine was 100% retained when the side chain was relatively large. The extent of N alpha-acetylation ranged from 0 to 100% depending on the amino-terminal sequence. The experimental results in this cell-free system faithfully mimic what occurs in nature as judged by the structures of normal and variant human hemoglobins as well as a broad spectrum of other cytosolic proteins.

Amino Acids↗
PubMed1988

Non-enzymatic glycation of antithrombin III in vitro.

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.

Amino Acid Sequence↗
PubMed1988

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age.

The nonenzymatic glycation of glomerular basement membranes (GBMs) from 14 diabetic and 19 nondiabetic human subjects was determined after boronic acid affinity and high-performance cation-exchange chromatography of their NaB[3H]4-reduced ketoamine adducts. The glucitol-lysine (Glc-Lys) and the glucitol-hydroxylysine (Glc-Hyl) content of diabetic GBM was found to be about twofold higher than that of nondiabetic samples (P less than .001). The content of these glycated amino acids did not correlate with age over the range examined (20-91 yr) or with the length of disease in diabetic subjects (2-16 yr). However, analyses of Glc-Lys and Glc-Hyl in calf and adult bovine GBM and lens capsules indicated that the levels of these glycated amino acids were several times greater in basement membranes from older animals. We also observed that guanidine-insoluble collagen of bovine GBM is more extensively glycated (approximately 4-fold) than primarily noncollagenous proteins that are extracted by this reagent. In all of the basement membranes examined, the percentage of glycation of lysine was greater than of hydroxylysine. Characterization of the components released by alkaline hydrolysis indicated that O-glycosylated hydroxylysine residues are nonenzymatically N-glycated to the same extent as those without an enzymatically attached carbohydrate unit. Our study indicates that more than a hundred times as many hydroxylysine residues are enzymatically glycosylated in human and bovine GBM as those containing the nonenzymatically formed ketoamine adduct.

Adult↗
PubMed1987

The kinetics of assembly of normal and variant human oxyhemoglobins.

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.

Humans↗
PubMed1987

The regulated expression of erythropoietin by two human hepatoma cell lines.

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

Carcinoma, Hepatocellular↗
PubMed1987

Subunit assembly of hemoglobin: an important determinant of hematologic phenotype.

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.

Anemia, Sickle Cell↗
PubMed1987

Association of hemoglobin H disease with Hb J-Iran (beta 77 His----Asp): impact on subunit assembly.

A young Iranian female was found to be heterozygous for hemoglobin (Hb) J-Iran (beta 77 His----Asp) in combination with Hb H disease. The proportion of Hb J in the patient's hemolysate was surprisingly high: 65% Hb J, 30% Hb A. Thus, the interaction of a negatively charged beta subunit variant of Hb with alpha-thalassemia leads to a marked increase in the relative amount of the variant Hb within red cells. This observation provides further support for an electrostatic model of Hb subunit assembly.

Globins↗
PubMed1986

Regulation of erythrocyte cation and water content in sickle cell anemia.

The pathophysiological events in sickle cell disease are critically dependent on the intracellular concentration of hemoglobin S, which varies inversely with cell cation and water content. Erythrocytes of SS homozygotes exposed to oxygen or carbon monoxide decrease their potassium and water content through a pathway for potassium transport that is activated by both cell swelling and decrease in internal pH. This pathway is not inhibited by ouabain either with or without bumetanide. When SS erythrocytes were separated according to density, the pH- and volume-dependent potassium transport was greatest in the least dense fraction and was reduced in the densest cells. This pathway, which does not depend on polymerization of sickle hemoglobin, may be important in regulating the cation and water content of SS erythrocytes.

Anemia, Sickle Cell↗
PubMed1986

Electrostatic attraction governs the dimer assembly of human hemoglobin.

We have investigated the effect of surface charge on the rate of assembly of alpha beta dimers of human hemoglobin A: alpha + beta k a----alpha beta. Heme intact beta A subunits were compared with four mutant subunits which differ by integral units of charge: beta N(Lys-95----Glu) (2-); beta J(Gly-16----Asp) (1-); beta S(Glu-6----Val) (1+); beta C(Glu-6----Lys) (2+). Subunit competition experiments were performed as follows. Varying amounts of 3H-labeled alpha A subunits were added to a mixture containing equal amounts of beta A and beta X subunits so that alpha/(beta A + beta X) ranged from 0.05-1.0. The reconstituted 3H-labeled Hbs A and X were analyzed by ion-exchange high pressure liquid chromatography as well as by gel electrofocusing and fluorography. Under the solvent conditions employed (10 mM PO4(Na), pH 7.0, 0 degrees C) a predominant proportion of the beta subunits was monomeric. Therefore, the ratio of Hb X to Hb A formed from subunit reconstitution when alpha/(beta X + beta A) approached zero provides a direct measure of the relative rates of monomer combination: kXa/kAa. The experimental values of this ratio decreased monotonically with the overall charge of the variant beta subunit: beta N = 2.6; beta J = 1.5; beta S = 0.41; beta C = 0.13. In contrast surface charge had no significant effect on the rate of dissociation of the alpha beta dimer: alpha beta kd----alpha + beta. At pH 8.0, where the alpha chains lack a net surface charge, they combined equally well to beta A and beta C chains. These experiments are consistent with a two-step mechanism, alpha + beta in equilibrium (alpha...beta) in equilibrium alpha beta, where the oppositely charged monomers diffuse together under the influence of their mutual electrostatic interaction to form a nonspecifically bound encounter complex [alpha...beta] that undergoes a surface charge-independent rearrangement to form the stable dimer.

Chromatography, High Pressure Liquid↗
PubMed1986

Dissociation of dimers of human hemoglobins A and F into monomers.

Dissociation of alpha beta and alpha gamma dimers of human hemoglobins (Hb) A and F into monomers was studied by alpha chain exchange (Shaeffer, J. R., McDonald, M. J., Turci, S. M., Dinda, D. M., and Bunn, H. F. (1984) J. Biol. Chem. 259, 14544-14547). Unlabeled carbonmonoxy-Hb A was incubated with trace amounts of preparatively purified, native, 3H-alpha subunits in 10 mM sodium phosphate, pH 7.0, at 25 degrees C. At appropriate times, free alpha monomers were separated from Hb A tetramers by anion exchange high performance liquid chromatography. Transfer of radioactivity from the alpha chain pool into Hb A was measured, yielding a first order dimer dissociation rate constant, k2 = (3.2 +/- 0.3) X 10(-3) h-1. The Arrhenius plot of k2 was linear between 7 and 37 degrees C, yielding an enthalpy of activation of 23 kcal/alpha beta dimer. As the chloride concentration was raised from 0 to 0.2 M, the dissociation rate increased 3-fold; with higher salt concentrations, however, the rate gradually returned to baseline. This rate was not altered by raising the pH from 6.5 to 7.2, but as pH was further raised to 8.4, kappa 2 increased about 3-fold. Hb F, which has an increased stability at alkaline pH, dissociated into alpha and gamma monomers 3 times more slowly than Hb A. Moreover, the dimer-monomer dissociation of Hb F was characterized by a significantly reduced pH dependence. These results demonstrate that both alpha beta and alpha gamma dimers of Hb A and Hb F dissociate reversibly into monomers under physiologic conditions. The differential pH dependence for dimer dissociation between Hb A and Hb F suggests that specific amino acid replacement at the alpha 1 gamma 1 interface confers increased resistance to alkaline denaturation.

Chlorides↗
PubMed1986

"Replacement" of COOH-terminal truncation of v-fms with c-fms sequences markedly reduces transformation potential.

Protooncogenes when transduced by retroviruses may undergo structural modifications that render their gene products oncogenic. The c-fms gene encodes a transmembrane protein with tyrosine kinase activity that is very similar or identical to the receptor for the monocyte-macrophage colony-stimulating factor. Its transforming homologue (v-fms) in the Susan McDonough strain feline sarcoma virus causes fibrosarcomas in cats. Molecular cloning and sequence analysis of the cDNA that encodes the cytoplasmic domain of the human c-fms gene shows that the product of the transduced viral homologue, v-fms, is truncated at the COOH-terminal end. The COOH-terminal 40 amino acids of the c-fms gene product are replaced in the v-fms gene product by 11 amino acids encoded by the retroviral genome. Hybrid v-fms/c-fms genes, in which either the entire cytoplasmic domain or the COOH-terminal coding sequences of the v-fms gene were replaced by the corresponding segments of the c-fms gene, had a reduced ability to transform fibroblasts despite a high level of encoded protein on the cell surface. These data indicate that the COOH-terminal modifications contribute to the transforming potential of the v-fms viral oncogene product.

Amino Acid Sequence↗
PubMed1985

Expression of the human c-fms proto-oncogene in hematopoietic cells and its deletion in the 5q- syndrome.

The c-fms proto-oncogene was shown to be expressed in human bone marrow and in differentiated blood mononuclear cells, suggesting that its gene product plays a role in hematopoietic maturation. The c-fms mRNA was not detected in HL-60 cells, an established promyelocytic line, whereas c-fms expression appeared 48 hr after induction when most cells had differentiated into macrophages. An acquired deletion of chromosome 5 (5q-) in bone marrow cells is associated with abnormalities in blood cell production. The normal 5 and 5q- chromosomes were segregated by construction of cell hybrids between bone marrow and rodent cells. A selective system was used that requires retention of the structural gene for dihydrofolate reductase, located on human chromosome 5. Analysis of DNA from individual hybrid clones revealed that the 5q- deletion had removed the c-fms gene. We postulate that hemizygosity at the c-fms locus leads to abnormalities in hematopoietic maturation.

Anemia, Aplastic↗
PubMed1985

Amino-terminal processing of proteins: hemoglobin South Florida, a variant with retention of initiator methionine and N alpha-acetylation.

The hemoglobin variant South Florida has been shown by protein sequencing and fast-atom-bombardment mass spectroscopy to have a substitution of methionine for the NH2-terminal valine of the beta-globin chain. In addition, there was complete retention of the initiator methionine on the mutant polypeptide. Approximately 20% of the protein was acetylated at the NH2 terminus of the beta chain. A search of a comprehensive data bank of protein and gene sequences revealed 84 unrelated vertebrate proteins that have not undergone cleavage of leader sequences. A highly nonrandom distribution of residues at the NH2 termini of these proteins predicts removal of the initiator methionine as well as NH2-terminal acetylation. Proteins that undergo removal commonly have serine, alanine, glycine, or valine, as the NH2-terminal residues. The first three residues favor N alpha-acetylation. Proteins that retain the initiator methionine commonly have a charged residue or methionine at the second position. Information on Hb South Florida and other hemoglobins coupled with this survey of primary sequence provides insights into the NH2-terminal processing of proteins.

Acetylation↗
PubMed1985

Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

Erythrocytes from patients with homozygous hemoglobin C disease (CC cells) contain less K, Na, and water than do erythrocytes from normal subjects that contain only hemoglobin A (AA cells). In this paper, we provide evidence that the reduced K content and volume of CC cells are due to the activity in these but not in AA cells of a K transport system that is: (a) insensitive to ouabain and bumetanide, and (b) stimulated by increased cell volume, and dependent on internal pH (pHi). When the cation and water content of CC cells was increased (by making the membrane temporarily permeable to cations with nystatin) and the cells were then incubated in an isotonic medium containing 140 mM NaCl and 4 mM KCl, they lost K and shrunk back toward the original volume. This regulatory K and volume decrease was not inhibited by ouabain or bumetanide. When CC cells were incubated in a hypotonic medium, with ouabain and bumetanide, they also lost K and shrunk toward the original volume. This behavior was not observed in control AA cells. The ouabain- and bumetanide-resistant K efflux from CC cells was volume and pH dependent: K efflux from CC cells rose from 5-6 to 20-25 mmol/liter of cells X h, when cell volume was increased by increasing cell solute content (nystatin method) or by exposure to hypotonic media. In CC cells, the dependence of K efflux on pHo had a bell shape, with a maximal flux (20-25 mmol/liter of cells X h) at pHo 6.8-7.0. In contrast, the K efflux from control cells was minimal at pH 7.4 (1.2 mmol/liter of cells X h) and was slightly stimulated by both acid and alkaline pH. In order to study the effect of pHi and pHo on K efflux, CC cells were incubated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM) and acetazolamide (1 mM) at different pHi (6.7, 7.3, and 7.8), and resuspended in media with different pHo (6.75, 7.4, and 8): K efflux was stimulated by reducing pHi but was independent of pHo. The ouabain- and bumetanide-resistant K efflux from CC cells was not inhibited by some inhibitors of the Ca2+-activated K permeability. It seems likely that the genetically determined change in the primary structure of hemoglobin C directly or indirectly causes this modification in K transport. One possible mechanism could involve an electrostatic interaction between C hemoglobin and components of the erythrocyte membrane.

Biological Transport↗
PubMed1984

Dimer-monomer dissociation of human hemoglobin A.

Isolated 3H-labeled human hemoglobin alpha chains were incubated with unlabeled carbonmonoxyhemoglobin A for 72 h in 0.01 M potassium phosphate, pH 7.0, at 25 degrees C. Following separation of the free alpha chain monomers and Hb A (alpha 2 beta 2) tetramers by electrophoresis on cellulose acetate strips or by chromatography on DEAE-cellulose columns, analysis of radioactivity showed a slow transfer of 3H-labeled alpha chains into Hb A. Separation of the globin chains of the isolated [3H] Hb A on a CM-cellulose column in 8 M urea showed that the radioactivity was in structurally intact alpha chains. Gel filtration of a reaction mixture on Sephadex G-75 in the presence of 0.15 M NaCl showed that the 3H-labeled alpha-chains in Hb A were an integral part of, and not nonspecifically adsorbed to, the tetramer. These results indicate that the free 3H-labeled alpha chain monomers were incorporated into Hb A by exchange with pre-existing unlabeled alpha chain subunits according to the scheme, alpha 2 beta 2 in equilibrium 2 alpha beta in equilibrium 2 alpha + 2 beta. The rate constant for the dissociation of alpha beta dimers to alpha and beta monomers, the rate-limiting step in this exchange reaction, was 4.0 (+/- 0.7) X 10(-3) h-1.

Carboxyhemoglobin↗
PubMed1984

Nonenzymatic glycosylation of human serum albumin alters its conformation and function.

Approximately 10% of the albumin in normal human serum is modified by nonenzymatic glycosylation, primarily at the epsilon-amino group of lysine residue 525. Incubation of albumin with glucose under physiological conditions in vitro resulted in glycosylation of the same residue. After separation of glycosylated human serum albumin from the nonglycosylated form by boronate affinity chromatography, the fluorescence emission characteristics of the sole tryptophan residue (Trp 214) were monitored. The quantum yield of tryptophan fluorescence for both in vivo and in vitro glycosylated albumin was reduced 30% relative to nonglycosylated albumin, and the maximal wavelength of the fluorescence emission band was shifted to shorter wavelengths. These observations show that nonenzymatic glycosylation induces a conformational change in human serum albumin. Ligand binding properties of glycosylated and unmodified albumin were compared. Hemin affinity was unaltered by glycosylation of albumin in vivo, whereas the affinity of bilirubin for glycosylated albumin was about 50% its value for the nonglycosylated form. The affinity of the long chain fatty acid cis-parinaric acid for albumin glycosylated in vivo and in vitro was reduced approximately 20-fold relative to nonglycosylated albumin. These differences in affinity suggest that lysine 525 plays a key role in the binding of physiologically important ligands to albumin.

Adult↗