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H E Witkowska

Publications and source records attributed to H E Witkowska.

At least 19 recordsLinked to original sources
PubMed1999

Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.

Acetone↗
PubMed1998

Site-specific spin trapping of tyrosine radicals in the oxidation of metmyoglobin by hydrogen peroxide.

The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

Amino Acid Substitution↗
PubMed1998

Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia.

Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of delta-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease.

5-Aminolevulinate Synthetase↗
PubMed1997

Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.

To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, and betaS-globin were generated and bred with knockout mice that had deletions of the murine alpha- and beta-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease.

Anemia, Sickle Cell↗
PubMed1997

Characterization of adult alpha- and beta-globin genes in the zebrafish.

Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major alpha globins and two beta globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult alpha- and beta-globin cDNA clones that encode some of these globins. High levels of alpha- and beta-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the alpha- and beta-globin genes are tightly linked on the same chromosome and are arrayed in a 3'-5' to 5'-3' configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.

Amino Acid Sequence↗
PubMed1997

HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N-Baltimore] with opposing effects upon HbC crystallization.

Compound heterozygotes of variant haemoglobins (Hbs) with HbC, with or without novel phenotypic changes, have provided insight into the molecular basis of the interacting haemoglobins and information concerning the role of specific residues in the crystallization of oxy HbC. A high phosphate buffer system has proved useful for studying the effects of variant haemoglobins (naturally co-existing with HbC in the red cell) on the oxy HbC crystallization process and has led us to conclude that beta87 and beta73 are contact sites of the oxy HbC crystal. We now present investigations from two HbC compound heterozygotes which exhibit opposing effects upon HbC crystallization: HbC/Hb N-Baltimore (beta95 Lys-->Glu) and HbC/Hb Riyadh (beta120 Lys-->Asn). The latter inhibits the in vitro crystallization of HbC, explaining the lack of erythrocyte abnormalities (with the exception of microcytosis) in the doubly heterozygous infant. In contrast, Hb N-Baltimore accelerates the crystallization of HbC, contributing to multiple abnormalities in red cell morphology, albeit in the absence of morbidity. We conclude that (1) beta120 and beta95 are additional contact sites in the crystal, and (2) the HbC/Hb Riyadh haemoglobinopathy demonstrates that crystallization may not be required for the generation of the observed microcytosis and increased red cell density in HbC-containing red cells.

Adult↗
PubMed1997

Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro.

cDNAs of various lengths encoding the second domain of the multifunctional fatty acid synthase (FAS) have been expressed in Escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. This shortened transacylase, consisting of FAS residues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity. When expressed as a soluble enzyme in Spodoptera frugiperda cells, this transacylase has the same specific activity as the enzyme that has been refolded in vitro. The refolded transacylase consisting of FAS residues 488-809, but not the longer enzyme consisting of residues 428-815, can be crystallized readily. These results suggest that FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, are not required for catalysis of the transacylase reaction. This region of the FAS is less well conserved than the transacylase catalytic domain and may constitute an extended structural linker that facilitates the functional interaction between the transacylase and acyl carrier protein domains.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

Expression of soluble human beta-globin chains in bacteria and assembly in vitro with alpha-globin chains.

Authentic soluble human beta-globin chains were produced in Escherichia coli using an expression plasmid (pHE2beta) containing full-length cDNAs coding for human beta-globin chain and methionine aminopeptidase. Spectral properties of the purified beta-globin were identical to those of authentic beta-globin. Soluble beta-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric beta-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric beta-globin. Monomeric forms of beta-globin, like authentic native beta-globin, formed tetrameric hemoglobin (Hb) A (alpha2beta2) in vitro upon incubation with alpha-globin, while dimeric forms did not. When beta-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the beta-globin chain monomers assembled with alpha-globin and formed hemoglobin tetramers. alpha-Globin was more thermally unstable than beta-globin, while assembled tetramers promoted higher stability. Disulfide-bonded beta-globin dimers showed a slight increase in thermal stability compared with beta-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of alpha chains favors assembly with beta-globin, beta-beta dimers cannot bind alpha chains, and that Hb A tetramer formation results in the most thermally stable species.

Biopolymers↗
PubMed1996

Carboxymethylation of the human estrogen receptor ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows that cysteine 447 does not react with iodoacetic acid.

Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.

Alkylation↗
PubMed1996

Intact noncovalent dimer of estrogen receptor ligand-binding domain can be detected by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESMS) of the estrogen receptor ligand binding domain (ER LBD) in its estradiol-binding form was performed. A dimeric ER LBD was observed, with a greatly reduced capacity for protonation (major charge state for dimer +16 vs. +23 for a monomer). Peak broadening (probably due to heterogeneity resulting from salt and water adduct formation) adversely affected our ability to distinguish between multiple discreet dimeric species and thus prevented us from establishing an accurate average mass for the dimerized domain. A mixture of species with molecular masses between 57,240 Da and 57,900 Da was observed, which would compare to 57,274 Da, 57,546 Da, and 57,818 Da for the calculated masses of the dimer without estradiol, or with one or two bound ligand molecules, respectively. Hence, nonliganded ER LBD dimer appeared to constitute the major species. The presence of low levels of a singly liganded ER LBD dimer cannot be ruled out, but the data argue against the possibility of the ER LBD dimer carrying two molecules of estradiol. Allowing for current limitations in the technology, our data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range (4000 Da for singly charged ions) has potential utility for studying ligand-binding proteins. In particular, in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.

Binding Sites↗
PubMed1995

Defining of the minimal domain of protein 4.1 involved in spectrin-actin binding.

The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-amino acid alternative exon and a 59-amino acid constitutive exon. To characterize the minimal domain active for interactions with spectrin and actin, we functionally characterized recombinant 4.1 peptides containing the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59). Peptide 21.43 was shown fully functional in binary interactions with spectrin (by cosedimentation and coimmunoprecipitation experiments) and in ternary complex formation with spectrin and actin (by an in vitro gelation assay). Further truncation produced peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), shorter peptides with reduced ternary complex activity and altered kinetics (21.26 and 0.59), and inactive peptides (21.20 and 21.10). Binding studies and circular dichroism experiments suggested that residues 37-43 of the constitutive domain were directly involved in spectrin binding. These data indicate that 4.1-spectrin binary interaction requires the 21-amino acid alternative cassette plus the 43 N-terminal residues of the constitutive domain. Moreover, the existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-spectrin interactions, and a second by 4.1-actin interactions. The latter may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain.

Actins↗
PubMed1995

Intact protein electrospray ionization tandem mass spectrometry can be the sole technique used for confirming the structure of a variant hemoglobin.

A mutation within the human alpha-globin resulting in an 18 u mass increment was characterized by sequencing the intact M(r) 15000 protein in the gas phase by electrospray ionization tandem mass spectrometry. No separation of the variant protein from its normal counterpart and other components of the hemolysate was performed prior to mass spectrometric analysis. Collision-induced dissociation of the globin molecules carrying 18 protons was affected by their exposure to methane in the collision cell of a triple quadrupole mass spectrometer equipped with an electrospray ion source. Consideration of the major fragmentation ions (b758+, y"284+ and y"617+) narrowed down the mutation site to the last 28 amino acid residues within the protein sequence. Examination of low-abundance product ions allowed the unequivocal identification of the mutation as alpha 136 Leu --> Met, a hemoglobin previously named Hb Chicago.

Amino Acid Sequence↗
PubMed1994

Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis.

Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.

Amino Acid Sequence↗
PubMed1994

Reengineering the specificity of a serine active-site enzyme. Two active-site mutations convert a hydrolase to a transferase.

Two residues are known to play important catalytic roles in fatty acyl-thioester hydrolase, thioesterase II: Ser-101, the site of a covalent acyl-enzyme intermediate, and His-237 which is within hydrogen bonding distance of Ser-101 and facilitates catalysis by increasing the nucleophilicity of this residue. In this study we have examined the effect of mutations at these two residues on the ability of the enzyme to function as a hydrolase and, in the presence of a thiol acceptor, as an acyltransferase. In the hydrolase reaction kcat values for the wild-type, H237R, S101C, and S101C, H237R thioesterase enzymes were 0.11, < 0.002, 0.10, and < 0.002 s-1, respectively, and at steady state, the proportion of each enzyme present as the covalent acyl-enzyme intermediate was 11, 91, 71, and 100%, respectively. In the acyltransferase reaction no activity could be detected for the wild-type or H237R enzymes but the specific activities of the S101C and S101C/H237R thioesterases were 170 and 1300 nmol/min/mg of protein, respectively. From this data we conclude the following: the wild-type enzyme functions exclusively as a hydrolase. The H237R mutant acts ineffectively as a hydrolase primarily because the deacylation reaction is drastically retarded. The S101C enzyme functions well as a hydrolase, even though the rate of deacylation is adversely affected, and this enzyme can also perform as an acyltransferase. Mutation of both catalytic residues leads to a complete loss of hydrolase activity and the S101C,H237R mutant functions as an effective acyltransferase exhibiting kcat values higher then those of the wild-type enzyme acting as a hydrolase. This study reveals that, with only two amino acid replacements, an enzyme capable of functioning exclusively as a hydrolase can be converted into an equally active enzyme performing solely as an acyltransferase.

Acylation↗
PubMed1993

Compound heterozygosity for hemoglobin C and Korle-Bu: moderate microcytic hemolytic anemia and acceleration of crystal formation [corrected].

We report here that compound heterozygosity for hemoglobin Korle-Bu (HbKB) and HbC (beta 6 Glu-->Lys) is associated with moderate chronic hemolytic anemia with microcytosis. To understand the pathogenesis of this syndrome, we have studied the effect of Hb Korle-Bu (KB = beta 73 Asp-->Asn) on the crystallization of HbC. We have previously established that fetal Hb (HbF) inhibits the crystallization of HbC. In contrast, HbS accelerates crystallization affecting the pathogenesis of SC disease. We now report on in vitro crystallization of mixtures of HbKB, HbC, and various amounts of HbF and the native hemolysate of a child who is a compound heterozygote for HbKB and HbC. At 6 months of age, the propositus' hemolysate contained 55% HbKB, 39% HbC, and 6% HbF. Crystal formed within 2 minutes compared with 30 minutes for the mixture of 40% HbC:60% HbS and with 180 minutes for 40% HbC:60% HbA. The morphology of the crystals formed was cubic, in contrast with the tetragonal crystals observed in CC and SC disease. Early crystals did not exhibit "sharp edges" until 45 minutes. Purified HbKB formed aggregates but not crystals after 24 hours. Isopycnic gradients showed that the KB/C compound heterozygotes have red blood cell (RBC) densities intermediate between the AC and CC phenotype and similar to SC disease. The surface residue beta 73, known to participate in areas of interaction of the deoxy HbS polymer, can now be assigned to areas of contact in HbC containing crystals. The hemolysis observed in the HbKB/C compound heterozygote is likely to be secondary to the acceleration of Hb crystallization. The microcytosis and increased RBC density is clearly the consequence of the presence of HbC, but the basis of the increased RBC pathology compared with AC trait, despite the low proportion of HbC (35% to 40%), remains to be elucidated.

Adolescent↗
PubMed1993

A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders.

BACKGROUND: Fetal-globin (gamma-globin) chains inhibit the polymerization of hemoglobin S (sickle hemoglobin) and can functionally substitute for the beta-globin chains that are defective or absent in patients with the beta-thalassemias. Identifying safe mechanisms to stimulate fetal-hemoglobin production is therefore of great interest. Previous studies have shown that administering butyrate selectively stimulates the promoter of the human fetal-globin gene and leads to increases in gamma-globin--gene expression in the developing fetus, cultured cells, and animal models. METHODS: To determine whether butyrate can stimulate fetal-globin production in humans, we treated three patients (3 to 13 years old) with sickle cell anemia and three patients (7 to 27 years old) with beta-thalassemia syndromes with a short course of intravenous infusions of arginine butyrate. The drug was infused continuously for either two or three weeks; the initial dose was 500 mg per kilogram of body weight per day. Globin-chain ratios, proportions of reticulocytes producing hemoglobin F (F reticulocytes), and levels of gamma-globin messenger RNA (mRNA) were determined before and during treatment. RESULTS: In all six patients, fetal-globin synthesis increased by 6 to 45 percent above pretreatment levels (P < 0.01). The proportion of F reticulocytes increased about twofold, and the level of gamma-globin mRNA increased twofold to sixfold. The increase in gamma-globin synthesis led to improvement in the globin-chain ratios in the patients with thalassemia. The treatment of one patient was extended for seven weeks, and her hemoglobin level increased from 4.7 to 10.2 g per deciliter (2.9 to 6.3 mmol per liter). Side effects were minimal; one patient had a transient increase in serum aminotransferase concentrations. CONCLUSIONS: In patients with beta-hemoglobinopathies butyrate, a natural fatty acid, can significantly and rapidly increase fetal-globin production to levels that can ameliorate beta-globin disorders. Further trials of this class of compounds are warranted to determine long-term tolerance and efficacy in patients with sickle cell anemia or beta-thalassemia.

Adolescent↗