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Biomedical subjects

J M Manning

Publications and source records attributed to J M Manning.

At least 109 records · Page 6Linked to original sources
PubMed1987

Effects of methyl acetyl phosphate on hemoglobin S: a novel acetylating agent directed towards the DPG binding site.

Methyl acetyl phosphate (MAP) is an acetylating agent which prevents the polymerization of sickle cell hemoglobin (HbS). MAP can penetrate sickle erythrocytes and can acetylate intracellular HbS. Tryptic peptide mapping of modified HbS on reversed phase HPLC and amino acid analysis of isolated peptides revealed the acetylation sites to be Val-1 (beta), Lys-82 (beta), and Lys-144 (beta). MAP, therefore, is specifically directed towards the 2,3-DPG binding site of Hb.

Acetylation↗
PubMed1986

Enzymic cleavage of the blocked amino terminal residues of peptides.

The substrate specificity of an enzyme that removes some N-terminal blocked amino acids from blocked peptides has been further explored with several naturally-occurring peptides. Chloride ion is an effective modulator of enzyme activity. Although the relative efficiency of the enzyme varies considerably with different peptide substrates, in each case there was significant although less than quantitative release of the N-terminal blocked amino acid. The possible application of this enzyme to structural studies on polypeptides is evaluated.

Amino Acids↗
PubMed1986

Methyl acetyl phosphate: a novel acetylating agent. Its site-specific modification of human hemoglobin A.

A novel acetylating agent, methyl acetyl phosphate (MAP), has been designed to react with a nucleophile near an anion binding site of proteins. We examined the effect of MAP on hemoglobin (Hb), which has a well defined binding site for 2,3-diphosphoglycerate (DPG), to determine whether this reagent recognizes the DPG binding site. The progress of the reaction was monitored by ion-exchange high-performance liquid chromatography (HPLC) on a TSK CM-SW column. Modified Hb was initially chromatographed on CM-52 and then separated into its component chains. The alpha- and beta-chains from modified and unmodified Hb were digested by TPCK-trypsin. The peptide mixtures were chromatographed on Whatman ODS-3 reversed-phase HPLC columns and the peptide maps of modified and unmodified chains were compared. The peaks formed by the modification with MAP were further purified on YMC ODS-S5 columns and then subjected to amino acid analysis on a Dionex D-500 instrument after acid hydrolysis. We found that the newly formed peptides are beta T1 and beta T14 + 15 and that the loss of a peptide corresponding to beta T9 and beta T 10 + 11 is significant. No change in the alpha-chains was observed. The results suggest that MAP is indeed specific for the DPG binding site, as the above peptides contain the amino acid residues involved in the binding of DPG. We have assigned the acetylation sites as Val-1(beta), Lys-82(beta) and Lys-144(beta).

2,3-Diphosphoglycerate↗
PubMed1986

Studies on the Amadori rearrangement in a model system: chromatographic isolation of intermediates and product.

The pH profile of the reaction of glyceraldehyde with either valylhistidine or alanylhistidine exhibits an optimum near pH 6.5. One of the intermediates in the reaction, the Schiff base (aldimine), can be readily detected on an amino acid analyzer. The product of the reaction, the ketoamine formed after Amadori rearrangement of the aldimine, has been isolated by chromatography on Dowex 50. Its structure has been established by elemental analysis, amino acid analysis, and the relative amounts of carbonyl and histidine moieties. These chromatographic systems should facilitate studies on the mechanism of this reaction as it relates to peptides and proteins.

Dipeptides↗
PubMed1986

Site-specific modification of hemoglobin by methyl acetyl phosphate.

Methyl acetyl phosphate, which was originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase [R. Kluger and W.-C. Tsui (1980) J. Org. Chem. 45, 2723], also has an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin. Three residues in or near this cleft between the beta-chains are acetylated by this reagent, i.e., Val-1, Lys-82, and Lys-144. There is no detectable acetylation of any of the amino groups of the alpha-chain. These results indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin.

Acetylation↗
PubMed1986

Enhanced survival of sickle erythrocytes upon treatment with glyceraldehyde.

Glyceraldehyde has been demonstrated to be an antisickling agent in vitro. In the present investigation, chromium-51 red cell studies were used to investigate the life span in vivo of sickle erythrocytes after treatment with glyceraldehyde in vitro. The mean survival (T1/2) of control cells was 5.8 +/- 1.6 days, whereas cells treated with 10 mmol/L or 20 mmol/L glyceraldehyde survived 9.0 +/- 1.4 (P less than .05) and 11.3 +/- 0.8 (P less than .002) days, respectively. The extent of modification by glyceraldehyde was 0.4 to 1.0 lysine residue per hemoglobin tetramer. These studies demonstrate not only a prolongation of the life span of sickle erythrocytes by treatment with glyceraldehyde but also the absence of any deleterious effects that would be revealed by this study.

Adult↗
PubMed1985

Selectivity in the modification of the alpha-amino groups of hemoglobin on reductive alkylation with aliphatic carbonyl compounds. Influence of derivatization on the polymerization of hemoglobin S.

The reactivity of the alpha-amino groups of the alpha- and beta-chains of hemoglobn toward reductive alkylation using limiting concentrations of the aliphatic carbonyl compounds, acetaldehyde (ethylation), glyoxylic acid (carboxymethylation), glycolaldehyde (hydroxyethylation), glyceraldehyde (dihydroxypropylation), and dihydroxyacetone (dihydroxyisopropylation) has been investigated. Hemoglobin A reductively ethylated at the alpha-amino groups eluted on CM-52 ahead of unmodified hemoglobin A, and hemoglobin A reductively ethylated at the epsilon-amino groups. This observation is similar to that seen on hydroxyethylation and dihydroxypropylation of the alpha-amino group of hemoglobin A. The presence of the alpha-hydroxyl or the carboxyl group in the carbonyl component used in the reductive alkylation influences considerably the selectivity pattern during the derivatization. The alpha-amino groups of the alpha- and beta-chains are modified to nearly the same degree during reductive hydroxyethylation as well as during reductive dihydroxypropylation. Reductive ethylation (aldehyde lacking the alpha-hydroxyl group) exhibited a slight preferential reaction at Val-1(beta). The presence of a negatively charged carboxyl group in the carbonyl component, i.e. glyoxylic acid, made this preferential reaction at Val-1(beta) even more pronounced. When the reductive alkylation is carried out with dihydroxyacetone (a ketone instead of an aldehyde), the dihydroxyisopropylation occurred at a slower rate and exclusively at Val-1(beta). The ethylation, hydroxyethylation, carboxymethylation, and dihydroxypropylation of the alpha-amino groups of hemoglobin S increased its solubility from the value of 16 g/dl for the unmodified protein to about 25 g/dl for the modified protein. Thus, the alkyl chains on the alpha-amino groups on the polymerization have a strong inhibitory influence. In order to determine the influence of the alkyl chains at the alpha-amino groups of alpha- and beta-chains on polymerization, hybrid hemoglobin S tetramers with hydroxyethylation either at Val-1(alpha) or at Val-1(beta) have been prepared. The solubility of each hybrid is about 26 g/dl. Thus, the hydroxyethyl group either on the alpha- or the beta-chain appears to interfere with the polymerization of deoxygenated HbS to the same degree. The inhibitory influence of the hydroxyethyl chain at Val-1(alpha) on the polymerization, compared with the lack of such an influence when this alpha-amino group is modified by cyanate, suggests that a carbamoyl group on Val-1(alpha) can be accommodated in the intermolecular contact region involving this segment of the molecule without seriously perturbing the mo

Alkylation↗
PubMed1985

Acylpeptide hydrolase activity from erythrocytes.

Acylpeptide hydrolase, which cleaves the NH2-terminal acetylated or formylated amino acid from a blocked peptide, has been purified to apparent homogeneity from human erythrocytes. The enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. Zinc inhibits to the same extent the hydrolysis of both the most efficient and the least efficient substrates. This enzyme may play a pivotal role in the processing of polypeptide chains during biosynthesis.

Alanine↗
PubMed1985

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase.

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.

Bacillus↗
PubMed1984

Enzyme-activated inhibition of bacterial D-amino acid transaminase by beta-cyano-D-alanine.

beta-Cyano-D-alanine is an efficient suicide substrate (Ki = 10 microM) of D-amino acid transaminase. This apparent inactivation is temperature dependent: it is irreversible at 10 degrees C or below and becomes progressively reversible at higher temperatures. Since at higher temperatures the apparent reactivation process predominates over the inactivation reaction, the reactivation process is considered to be endothermic. The nature of this reversibility suggests the formation of a heat labile bond between the inhibitor molecule and a nucleophilic group on the enzyme.

Alanine↗
PubMed1984

Inhibition of deoxyhemoglobin S polymerization by glyceraldehyde.

Glyceraldehyde reacts with hemoglobin S in the intact erythrocyte to reduce the degree of polymerization, thereby inhibiting sickling of the erythrocyte. Only five of the 24 amino groups per alpha beta dimer react with glyceraldehyde; the adducts are present as ketoamine structures, formed by Amadori rearrangement of the initial Schiff base adducts on the protein. The reactive amino groups are the epsilon-amino group of Lys-16 of the alpha-chain, and the alpha-amino group of Val-1 as well as the epsilon-amino groups Lys-82, Lys-59, and Lys-120 of the beta-chain. Hybrid tetramers were prepared with the modification only on Lys-16 of the alpha-chain or on the reactive lysine residues of the beta-chain. The former derivative gels at a much higher hemoglobin concentration (23 g/dl) than either the latter derivative (16 g/dl) or unmodified deoxyhemoglobin S (15 g/dl). Thus, the modification at Lys-16 of the alpha-chain is a major factor in the inhibition of sickling by glyceraldehyde.

Anemia, Sickle Cell↗
PubMed1984

The mechanism of action of two anti-sickling agents: sodium cyanate and glyceraldehyde.

Two compounds that inhibit the sickling of erythrocytes in vitro are sodium cyanate and glyceraldehyde. The former compound reacts selectivity with the NH2-terminus of the alpha-chain of hemoglobin S and thereby leads to an increased oxygen affinity of the protein and inhibition of erythrocyte sickling. The toxicity associated with oral administration of sodium cyanate precludes its use in the treatment of sickle cell anemia; administration by extracorporeal routes is still under consideration. The compound glyceraldehyde also inhibits the sickling of erythrocytes in vitro but does so by a different mechanism than sodium cyanate; it interferes directly with the gelation of deoxyhemoglobin S. Glyceraldehyde also displays selectivity; only five of a total 24 amino groups per alpha beta dimer of hemoglobin S are reactive. Preclinical studies on this compound as a potential treatment for sickle cell anemia are in progress.

Antisickling Agents↗
PubMed1983

Specific modification of the carboxyl groups of hemoglobin S.

The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23 degrees C for 1 h resulted in the modification of, on an average, three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7, indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the P50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide beta T5, i.e. the segment representing amino acid residues 41 to 59 of beta-chain, accounted for nearly 75% of the label associated with the globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized beta T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl group of Glu-43(beta), identifying it as the most reactive carboxyl group in hemoglobins. The results suggest that modification of the carboxyl group of hemoglobins S, presumably the gamma-carboxyl of Glu-43(beta), reduces the propensity of deoxyhemoglobin S to polymerize.

2,3-Diphosphoglycerate↗
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↗
PubMed1983

Reaction of glycolaldehyde with proteins: latent crosslinking potential of alpha-hydroxyaldehydes.

The Schiff base adducts of glyceraldehyde with hemoglobin undergo Amadori rearrangement to form stable ketoamine structures; this reaction is similar to the nonenzymic glucosylation of proteins. In the present studies the analogous rearrangement of the Schiff base adducts of glycolaldehyde with proteins has been demonstrated. However, the Amadori rearrangement of the Schiff base adduct produces a new aldehyde function, an aldoamine, which is generated in situ and is capable of forming Schiff base linkages with another amino group, leading to covalent crosslinking of proteins. Sodium dodecyl sulfate gel electrophoresis of the glycoaldehyde-RNase A adduct showed the presence of dimers, trimers, and tetramers of RNase A, demonstrating the crosslinking potential of this alpha-hydroxyaldehyde. The crosslinked products exhibited an absorption band with a maximum around 325 nm and fluorescence around 400 nm when excited at 325 nm. The crosslinking reaction, the formation of a 325-nm absorption band, and the development of fluorescence were prevented when the incubation was carried out in the presence of sodium cyanoborohydride. This finding indicates that the Amadori rearrangement that generates a new carbonyl function is a crucial step in this covalent crosslinking. Glycolaldehyde could be a bifunctional reagent of unique utility because its crosslinking potential is latent, expressed only upon completion of the primary reaction.

Acetaldehyde↗
PubMed1983

Influence of trifluoroacetic acid on retention times of histidine-containing tryptic peptides in reverse phase HPLC.

The tryptic peptides of the aminoethylated alpha- and beta-chain of hemoglobin have been separated on a Partisil-10 ODS-2 column with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The elution profile of the tryptic peptides of the chains obtained with this acetonitrile trifluoroacetate system has been compared with that obtained using phosphoric acid as the ion-pairing reagent. This comparison demonstrated that trifluoroacetate influences the retention times of the histidine-containing tryptic peptides much more than it affects those peptides that do not contain histidine residues. This behavior has been rationalized on the basis of ion-pairing of trifluoroacetate with the histidine residues of the tryptic peptides.

Chromatography, High Pressure Liquid↗