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H E Witkowska

Publications and source records attributed to H E Witkowska.

35 records · Page 2Linked to original sources
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↗
PubMed1993

Hemoglobin D Ibadan-beta zero thalassemia: detection by neonatal screening and confirmation by electrospray-ionization mass spectrometry.

We describe an infant with hemoglobin D Ibadan-beta zero thalassemia whose hemoglobinopathy was initially detected by neonatal screening. This previously undescribed condition was confirmed by family studies and by globin chain analysis by mass spectrometric techniques. The case illustrates the importance to neonatal screening programs of confirmatory testing and of linkage with reference laboratories capable of globin chain analysis. Hematologic studies at 36 months of age suggested that the presence of hemoglobin D Ibadan had no deleterious effect on this child with heterozygous beta zero thalassemia.

Globins↗
PubMed1993

Collisionally activated dissociation and tandem mass spectrometry of intact hemoglobin beta-chain variant proteins with electrospray ionization.

Electrospray ionization collisionally activated dissociation (CAD) mass spectra of multiply charged human hemoglobin beta-chain variant proteins (146 amino acid residues, 15.9 kDa), generated in the atmospheric pressure/vacuum interface and in the collision quadrupole of a triple-quadrupole mass spectrometer, are shown and compared. Several series of structurally informative singly and multiply charged b- and y-mode product ions are observed, with cleavage of the Thr 50-Pro 51 CO-NH bond to produce the complementary y96 and b50 sequence ions as the most favored fragmentation pathway. The eight different beta-globin variants studied differ by a single amino acid substitution and can be differentiated from the observed m/z shifts of the assigned product ions. The overall fragmentation patterns for the variant polypeptides are very similar, with the exception of the Willamette form, in which Arg is substituted for Pro- 51, and multiply charged y96 product ions are not observed. Circular dichroism spectra of normal beta A and beta Willamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.

Adult↗
PubMed1993

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

Amino Acid Sequence↗
PubMed1993

Expediting rare variant hemoglobin characterization by combined HPLC/electrospray mass spectrometry.

Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ESMS) and the method should be almost universally applicable. We have eliminated preparative scale HPLC of globin chains and semi-preparative HPLC of proteolytic digests which had been used prior to mass spectrometry. Use of microbore HPLC columns reduced the time required for analysis substantially and solvent usage by 100x. Molecular masses of intact globins and masses and sequence information of tryptic peptides could be obtained without collecting and separately analyzing chromatographic fractions. As an example of the use of these methods, we report the characterization of an unknown hemoglobinopathy case that was finally authenticated as Hb P-Galveston [beta 117(G19)His-->Arg], using the following sequence of analyses: 1) ESMS of complete hemolysate, 2) analytical HPLC of globin chains, 3) combined microbore HPLC/ESMS of globin chains to determine their molecular masses, 4) cysteine derivatization and tryptic digestion of mixture of all globins, followed by microbore separation of the peptides, molecular mass determination, and generation of fragmentation patterns allowing confirmation of amino acid sequences. This four-part strategy should allow characterization of almost all variant Hbs. Exceptions would be mutations in regions of globin chains which give rise to small (< four residues) tryptic peptides, either normal or produced by addition of new tryptic sites and mutations that introduce only minute difference in molecular weight (MW) of tryptic peptides. Since only 10% of each separated peptides is mass analyzed, 90% is available for collection and further structural identification (e.g. by tandem MS or Edman sequencing) if the identity is still in doubt.

Adult↗
PubMed1992

Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.

Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue (Ser-101) assisted by a histidine, tentatively identified as His-237. To determine whether in the folded protein His-237 is close enough to accept a proton from the Ser-101 hydroxyl, we have made use of a Ser101Cys mutant which retains up to 90% of catalytic activity. Unlike the wild-type enzyme, the S101C thioesterase is inhibited with stoichiometric amounts of the bifunctional alkylating reagent 1,3-dibromopropanone. To facilitate identification of the alkylated residue(s), the keto group introduced into the dibromopropanone-modified S101C mutant was radiolabeled by reduction with sodium [3H] borohydride. The protein was then digested and the radiolabeled peptides analyzed by amino acid sequencing and mass spectrometry. The experimental data unambiguously showed that dibromopropanone cross-linked the active site Cys-101 with His-237, demonstrating that these residues are positioned within 5 A of each other. These data strongly support the hypothesis that in the wild-type thioesterase His-237 accepts a proton from Ser-101, thus increasing its nucleophilic character and improving the catalytic efficiency of the enzyme. The possibility that exchange of cysteine and serine active site residues has occurred in the evolution of thioesterases is discussed.

Amino Acid Sequence↗
PubMed1991

Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins.

A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.

Amino Acid Sequence↗
PubMed1991

Laboratory diagnosis of hemoglobinopathies.

Diagnostic tests for most common hemoglobinopathies and recent advances in structural analysis of variant hemoglobins are reviewed. Routine and newly introduced methods that apply to the diagnosis of sickle cell anemia, thalassemia and the hemoglobin E disorders are presented. A brief description of the clinical course for each of these disorders is given, and potential pitfalls in diagnosis are discussed. Application of high-performance liquid chromatography and various mass spectrometric techniques (electrospray ionization mass spectrometry, liquid secondary ion mass spectrometry, and tandem mass spectrometry) for evaluation of hemoglobinopathy is presented.

Clinical Laboratory Techniques↗
PubMed1991

Hypoxia-induced in vivo sickling of transgenic mouse red cells.

To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions.

Animals↗
PubMed1990

The carboxyl-terminal region of thioesterase II participates in the interaction with fatty acid synthase. Use of electrospray ionization mass spectrometry to identify a carboxyl-terminally truncated form of the enzyme.

Medium-chain S-acyl fatty acid synthase thioester hydrolase (thioesterase II), a discrete 263-residue serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway in certain specialized tissues by hydrolyzing the thioester bond linking the growing acyl chain to the 4'-phosphopantetheine of the fatty acid synthase. Modification of one thioesterase II cysteine thiol with thionitrobenzoate inhibited interaction with the S-acyl-fatty acid synthase substrate but not with acyl-CoA model substrates. The identity of the sensitive cysteine residue was determined by treatment of the thionitrobenzoyl enzyme with cyanide and cleavage at the amino-terminal side of the S-cyanocysteinyl residue. Two small cleavage products were isolated; their molecular masses (889 and 675 Da) and amino acid compositions indicated that both originated from cleavage at Cys256. A new technique of electrospray ionization mass spectrometry was utilized to confirm that the heterogeneity displayed by the products of S-cyanocysteinyl cleavage resulted from the presence, in the purified preparations, of both full-length and a truncated form of the enzyme missing the carboxyl-terminal Leu-Thr peptide. The proportion of full-length polypeptide present appeared to correlate with the activity of the enzyme toward its natural substrate. The results of modification of Cys256 by thionitrobenzoate and removal of residues 262 and 263 by endogenous proteases indicate that integrity of the carboxyl-terminal region is important for interaction with its acyl-fatty acid synthase substrate.

Acyl Coenzyme A↗
PubMed1990

Tandem mass spectrometry in the clinical analysis of variant hemoglobins.

A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.

Hemoglobins, Abnormal↗
PubMed1989

Synthesis of derivatives of phosphonic glutathione analogs.

Five analogs of S-t-butyl glutathione containing phosphonic analogs of glycine and glutamic acid were obtained by standard procedures of MA activation in solution. Simultaneous deprotection of phosphonic, carboxylic and amino groups was achieved in the silylation reaction.

Chemical Phenomena↗
PubMedDate not supplied

Characterization of bacterially expressed rat estrogen receptor beta ligand binding domain by mass spectrometry: structural comparison with estrogen receptor alpha.

Functional rat estrogen receptor beta ligand binding domain (rER beta LBD, aa 210-485) and human estrogen receptor alpha ligand binding domain (hER alpha LBD, aa 301-553) were expressed in Escherichia coli. Hormone binding assays revealed that both ER beta and ER alpha LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd approximately 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16 alpha-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ER alpha and ER beta LBDs to ICI 164384 and 4-hydroxytamoxifen were observed, 16 alpha-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ER alpha and ER beta selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rER beta LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rER beta cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformational state. Concurrent analysis of hER alpha LBD showed that under the same experimental conditions, the two equivalent ER alpha cysteines were not alkylated (alpha C381 and alpha C447). These data support close structural relationship between the E2-bound ER alpha LBD and ER beta LBD proteins.

Alkylation↗