Biosynthesis of cat hemoglobins A and B: Amino terminal acetylation of the β chain of HbB

Acetylation of the amino terminal serine of the β chains of cat HbB occurs during synthesis of hemoglobin in a mRNA dependent rabbit reticulocyte lysate protein synthesizing system in the presence of acetyl-CoA and cat reticulocyte mRNA. Both of the major cat hemoglobins, the nonacetylated HbA and acetylated HbB, are synthesized efficiently in the rabbit lysate system. The acetylation of HbB-β chains occurs during the biosynthesis of these proteins. Radioautography of tryptic peptide maps reveals that acetylation occurs specifically at the amino terminal serine of HbB-β globin, and not on HbA-β globin or on the α chain common to both hemoglobins A and B. Because of the similarity of the structures of HbA-β and HbB-β globin, it is suggested that the amino terminal residue determines whether the peptide chain is recognized for acetylation by a ribosomal acetyltransferase.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.     

Heterogeneity in the globin chain composition of a human minor fetal hemoglobin component

The first hemoglobin found to contain an acetyl blocking group was the minor human fetal hemoglobin, Hb FI, present as 10-15% of the total fetal hemoglobin in umbilical cord blood red cells. Acetylation occurs at the amino-terminal glycine of the gamma-globin chain. Assays for the acetyl group by two different methods gave values less than the 2 per tetramer expected for a fully acetylated hemoglobin. We have purified acetylated fetal hemoglobin FIc to homogeneity. The globin chain composition of Hb FIc has been examined by both globin chain separation on CM-cellulose and by tryptic peptide mapping by HPLC. The identities of the gamma globin chains and of the gamma T-1 peptides were confirmed by amino acid analysis. Globin chain separation profiles showed the presence of 22.3 +/- 7.0% of gamma 0 globin (of the total gamma globin) in Hb FIc. Accordingly, the tryptic peptide maps of Hb FIc tetramers also showed the presence of a similar amount of gamma 0T-1 peptide. The gamma 0T-1 peptide was not present in the maps of isolated gamma Ic globin. It is evident that column purified Hb FIc contains a certain percentage of non-acetylated gamma-globin chains, thus indicating a hybrid globin chain composition for this minor fetal hemoglobin component.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Interaction between the synthesis of alpha and beta globin.

We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the slender loris, Loris tardigradus.

alpha and beta chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the alpha chains and 2 in the beta chains were recognized.     

Amino acid sequences of the aplpha and beta chains of adult hemoglobin of the tupai, Tupaia glis.

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.     

Amino-acid sequence of the alpha and beta chains of adult hemoglobin of the harbor seal, Phoca vitulina

The complete amino-acid sequences of the alpha and beta chains of adult hemoglobin of harbor seal, Phoca vitulina that belong to carnivora were determined as follows. The alpha and beta chains isolated by chromatography on a CM-cellulose column were digested with trypsin after S-carboxymethylation. Amino-acid sequences of the tryptic peptides derived from both chains were analysed. Comparing the primary structures of the alpha and beta chains of the seal hemoglobin with those of human, dog, bear, badger and cat, 19, 12, 12, 11, and 16 substitutions, respectively, were recognized in the alpha chain, and 12, 10, 4, 6, and 19 (22) in the beta chain.     

Amino-acid sequences and functional differentiation of hemoglobins A and D from swift (Apus apus, Apodiformes)

The blood of the adult swift contains one major (HbA = alpha 2A beta 2) and two minor components (HbD = alpha 2D beta 2 and HbD'). The components were separated by FPLC with a TSK SP-5 PW-column in phosphate buffers, and were eluted with a linear NaCl gradient. HbD' could be detected only in freshly prepared hemolysates with the sensitive FPLC separation method. The globin chains were separated on a cation exchanger (CM-cellulose), the tryptic peptides by HPLC with a RP-2 LiChrosorb column. Their amino-acid sequences were determined by automatic Edman degradation with the film- or gas-phase method. For the alpha A-, alpha D- and beta-chains, peptide alignment was achieved by homologous comparison with the corresponding chains of the greylag goose (Anser anser). The structural significance of the substitutions was examined with the aid of molecular graphics. The oxygen-binding properties of the stripped hemolysate and of HbA and HbD and their dependence on pH, temperature and inositol polyphosphate are presented and discussed with reference to molecular structures and hypothermy that occurs during torpidity.     

Free alpha-like material from bovine pituitaries. Removal of its O-linked oligosaccharide permits combination with lutropin-beta

Further characterization of the free alpha subunit immunoreactive material, not combined with beta subunit in extracts of bovine pituitaries, shows that the only significant modifications, relative to alpha subunits themselves, are the oligosaccharide O-linked to threonine-43, and heterogeneity of the carboxyl terminus. Removal of the O-linked carbohydrate with a mixture of glycosidases from Streptococcus pneumoniae results in an alpha-like material capable of combining with lutropin beta subunit and, thus, the presence of the oligosaccharide is responsible for the inability of the free alpha-like material to combine with beta subunits. Amino acid compositions of tryptic peptides spanning the entire sequence indicate no change in amino acid sequence of the free alpha-like material as compared to lutropin alpha. Further, based on the similar behavior reverse phase high performance liquid chromatography of the tryptic peptides as compared to their lutropin alpha counterparts, it is concluded that no additional post-translational modifications are present. The N-linked oligosaccharides of the free alpha-like material most likely contain terminal O-sulfated N-acetylhexosamines (as do the asparagine-linked carbohydrates from the pituitary hormones) as indicated by the presence of 3 mol of sulfate/mol of free alpha-like material and the resistance of these oligosaccharides to enzymatic deglycosylation. The O-linked oligosaccharide does not contain sulfated residues.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Turnover of amino-terminal acetylated and non-acetylated hemoglobins in vivo

The significance of amino terminal acetylation in the turnover of hemoglobin was analyzed by measuring the synthesis and degradation of hemoglobins A and B in domestic cat blood. The two hemoglobins occur as mixtures in cat blood and are structurally very similar except for β-chain amino terminal acetylation found in HbB which is chiefly responsible for their difference in isoelectric pH. The hemoglobins were labeled by administering radioactive amino acids to anemic animals and their specific radioactivities were monitored at intervals for several days thereafter until the average life span of the erythrocytes was exceeded. The results showed that the turnover of hemoglobin is unrelated to isoelectric pH or to amino-terminal acetylation of the protein.     

Structural adaptation of bird hemoglobins to high-altitude respiration and the primary sequences of black-headed gull (Larus ridibundus, Charadriiformes) alpha A- and beta/beta'-chains

Two hemoglobin components HbA (alpha A2 beta 2) and (alpha D2 beta 2) have been detected by analytical electrophoresis in the lysed erythrocytes of the adult Black-Headed Gull (Larus ridibundus). We report the complete primary structure of the alpha A- and beta-chains of the major hemoglobin component HbA. Following the chain separation and isolation of the tryptic peptides by RP-HPLC, the amino-acid sequence was established by automatic Edman degradation in spinning cup and gas-phase sequencers. The primary structures of alpha A- and beta-chains from the Black-Headed Gull HbA differ by 11 and by 6 amino-acid residues from the corresponding chains of Greylag Goose. These changes are randomly distributed over both alpha-helical and interhelical regions. The presence of beta/beta'-chains is indicated by the observation of Ile/Leu at position beta 78. An exchange at position beta 55 (D6)Leu-Asn which is known to be involved in the alpha 1 beta 1-interface with alpha 119(H2)Pro has been found. It is suggested that packing contacts in the alpha 1 beta 1-interface are important for high altitude respiration in birds.     

Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contact inhibition within hemoglobin S polymer by thiol reagents

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Gene expression in an interspecific hybrid: Analysis of hemoglobins in donkey,horse, and mule by peptide mapping

The fast and slow components of horse, donkey, and mule hemoglobin were analyzed by mapping of tryptic peptides. The two horse components share a common beta chain but differ in the alpha chain. Donkey hemoglobin differs from the slow horse component by replacement of histidine by asparagine in position 20 and replacement of a glycine residue by alanine in peptide V. Mapping of mule hemoglobins indicates that (1) the slow mule component contains the alpha and the beta chains of the slow horse component and the alpha and beta chains of the donkey, and (2) the fast mule component contains the alpha chain of the fast horse component and the beta chains of both the horse and the donkey. It is concluded that all six theoretically possible tetrameric combinations of the polypeptide chains encoded by the horse and donkey parental genes are formed in the mule.     

The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.     

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.