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.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Oxidation-reduction potentials of human fetal hemoglobin and gamma chains. Effects of blocking sulfhydryl groups.

The oxidation-reduction equilibrium of the gamma chains of human fetal hemoglobin (Hb F) has been studied and compared with that of the alpha and beta chains of human adult hemoglobin (Hb A). The effects of the sulfhydryl (--SH) reagents, iodoacetate, iodoacetamide, and p-mercuribenzoate (PMB), on the three kinds of chains and on Hb F have been compared. The midpoint potentials (E-m) of all three sorts of chains are lower than those of tetrameric hemoglobin A or F. The E-m values of alpha chains are the lowest, E-m = 0.049 volt at 6 degrees, and are unaffected by pH change or by PMB treatment, at least from pH 6 to 8. The E-m values of beta-SH chains are higher; E-m = 0.102 volt at pH 7, decreasing to 0.050 volt at pH 8, both at 6 degrees. These results agree with those of Banerjee and Cassoly ((1969) J. Mol. Biol. 42, 337-349). They reported no effect of PMB on beta chains, but we find that 2 eq of PMB/chain raise E--M to 0.139 volt at pH 7 at 6 degrees, chiefly as the result of reaction at beta-93, not at beta-112. Carboxymethylation at beta-93 has an insignificant effect compared with that of PMB. The oxidation-reduction potential of gamma chains is similar to that of beta chains. E-m = 0.098 volt at pH 7 at 6 degrees, decreasing to 0.064 at pH 8 and 0.010 at pH 9. The effects of --SH reagents, reacting at position gamma-93 (the only --SH group present in gamma chains), are essentially the same as those seen with beta chains. The oxidation-reduction potential of Hb F is almost identical with that of Hb A, except for being 0.008 volt lower at pH 6 at 6 degrees. This agrees with the results reported by Flohe and Uehleke ((1966) Life Sci. 5, 1041-1045). PMB or iodoacetamide treatment lowers E-m by 0.02 to 0.03 volt, depending on the pH, from 6 to 9, in much the same way as previously reported for Hb A(Brunori, M., Taylor, J.F., Antonini, E., Wyman, J., and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 2295-2300). The "residual oxidation Bohr effect" noted in Hb F can be attributed to the oxidation Bohr effect of the gamma chains. The apparent pK of the heme-linked water molecule was found at 25 degrees to be, for Hb F, 8.1; for gamma-SH chains, 7.85; for gamma-PMB chains, 8.35; and for gamma chains treated with iodoacetate, 7.80. Sedimentation coefficients, s-20, w, at a protein concentration of 5 mg/ml, were found to be, for fetal hemoglobin 4.09, for iodoacetamide-treated fetal hemoglobin 4.04, for PMB-treated fetal hemoglobin 3.41, for fetal gamma-SH chains 4.25, and for fetal gamma-PMB chains 3.08.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Patterns of hemoglobin assembly in reticulocytes of sickle cell trait individuals.

Venous blood was obtained from five sickle cell trait donors with relatively high hemoglobin S concentrations (40% of total hemoglobin) and five donors with unusually low hemoglobin S concentrations (25 to 30%). A fraction of cells with 15 to 20% reticulocytes was isolated from the blood and incubated with [3H]leucine in a medium supporting protein synthesis for various times from 1.25 to 60 min. Previous studies showed an imbalance in globin chain synthesis in reticulocytes of "low hemoglobin S" donors which suggested the presence of an alpha-thalassemia gene; reticulocytes of "high hemoglobin S" donors had balanced globin chain synthesis (DeSimone, J., Kleve, L., Longley, M.A., and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 59, 564-569). In the present study the soluble phase of the 3H-labeled reticulocytes was examined by electrophoresis on strips of cellulose acetate. The tetramer hemoglobins A and S were separated from each other and from a small pool of free, newly synthesized alpha and beta chains. Kinetics of labeling studies showed that the free alpha and beta chains were intermediates in tetramer hemoglobin assembly. The distribution of radioactivity between the alpha and beta chains of each of the electrophoretically isolated components were determined by separation of their globin chains on CM-cellulose columns. After 5 min of 3H-labeling of the reticulocytes from donors with 40% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the tetramer hemoglobins A and S ranged from 0.37 to 0.58. This ratio increased with longer labeling times. Almost all of the radioactivity of the free chain intermediates was in the alpha chain. These results confirmed the presence of a significant pool of newly synthesized alpha chains and a normal pattern of hemoglobin assembly in which initially unlabeled alpha chains combined with labeled beta chains when the cells were exposed to [3H]leucine. Conversely, in the reticulocytes of donors with 25 to 30% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the completed hemoglobins A and S ranged from 0.96 to 1.37 and remained unchanged throughout the 3H-labelling period. The radioactivity of the free alpha chain pool was substantially less that the total radioactivity of the betaA and betaS chain pools. These results confirmed the existence of a decreased pool size of soluble alpha chain intermediates and a pattern of hemoglobin assembly consistent with the presence of the alpha-thalassemia gene.     

The chemical heterogeneity of human hemoglobin F. Direct evidence for the existence of three types of gamma chain, the G gamma I, A gamma I, and A gamma T chains.

Direct evidence is presented for the existence of three types of gamma chain of human hemoglobin F. A modification of a CM-cellulose chromatographic method has allowed the incomplete separation of these gamma chains while high pressure liquid chromatography and fingerprint analyses of tryptic peptides of zones of the isolated gamma chains, and amino acid analyses of isolated peptides were used to identify the chains. These studies have shown that the presence of a glycyl residue in position 136 (G gamma chain) is directly related to that of an isoleucyl residue in position 75 (I gamma chain), thus indicating the existence of an G gamma I chain, and that the presence of an alanyl residue in position 136 (A gamma chain) can be related to that of an isoleucyl residue in position 75, thus suggesting the existence of an A gamma I chain. When the isoleucyl residue at positive 75 is replaced by a threonyl residue, invariably it is related to the alanyl substitution at position 136 (A gamma T chain). These data support indirect evidence from case analyses and family studies which were published before, and indicate that the T gamma chain is an allele of the A gamma which should be renamed the A gamma T chain.     

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 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.     

Assembly of gamma- with alpha-globin chains to form human fetal hemoglobin in vitro and in vivo

Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

Protein conformational heterogeneity as a binding catalyst: ESI-MS study of hemoglobin H formation

Our previous studies of hemoglobin tetramer assembly in vitro suggested that the initial step in the oligomerization process, which ultimately dictates the high fidelity of the heterotetramer (alpha*beta*)2 assembly, is the binding of a flexible heme-free beta-globin chain to a highly ordered heme-bound alpha*-globin. In this work, we extend these studies to investigate formation of the homotetrameric hemoglobin H, whose formation in vivo is a well-documented clinical consequence of significant overexpression of beta-globin in alpha-thalassemic disorders. Upon reconstitution of the isolated beta-globin with excess heme, the predominant species in the ESI mass spectrum corresponds to the homotetramer beta*4, alongside homodimeric species and monomeric beta-globin chains in both apo and holo forms. The assembly process of the hemoglobin H homotetramer apparently follows a scenario similar to that of a normal heterodimeric hemoglobin (alpha*beta*)2 species, with the asymmetric binding event between compact and flexible polypeptide chains being the initial step. The extreme importance of large-scale chain dynamics and conformational heterogeneity for the protein assembly process is highlighted by the inability of highly structured alpha-globins to undergo ordered oligomerization to form dimers and tetramers as opposed to indiscriminate aggregation.     

The distribution of fetal hemoglobin and the types of gamma chain in red cell fractions separated by gradient centrifugation from blood of patients with sickle cell anemia and other hemoglobinopathies

Isopycnic separations of red cells from cord bloods, and from patients with sickle cell anemia, different forms of HPFH, S-beta O-thalassemia, and a beta +-thalassemia homozygosity were made in order to evaluate the distribution of Hb F and the relative levels of G gamma and A gamma chains over the cell fractions. As expected, the cord blood data showed decreased levels of both Hb-F and G gamma chains in the top cell fractions since the beta leads to gamma and high G gamma: A gamma low G gamma: A gamma switches are operative around the time of birth. Complete cell fractionations were made on the blood of three SS patients with low G gamma values (40%) and three SS patients with high G gamma values (60%). The proportion of G gamma chain was constant in all cell fractions, while the Hb-F level was higher in cells with higher densities. The difference in the quantities of the three types of gamma chain in the fetal hemoglobins of two SS patients with an A gamma T heterozygosity, one having a low G gamma value and the other a high G gamma level, can be explained by assuming an alteration in a regulatory mechanism. Considerable variation both in the level of Hb F and in the percentage of G gamma chain was observed in two G gamma A gamma-HPFH heterozygotes with a relatively low G gamma percentage of 30%; an inverse relationship was present between the two parameters. Such a phenomenon was not evident for the G gamma A gamma-HPFH homozygote, and also did not exist in two additional G gamma A gamma-HPFH heterozygotes with an associated alpha-thalassemia-2 heterozygosity who had a similar amount of Hb F but with higher G gamma values of about 50%. The difference in G gamma values between these two categories of G gamma A gamma-HPFH could be due to a higher affinity of the G gamma chains over A gamma chains for a slightly decreased amount of alpha chains as in an alpha-thalassemia-2 heterozygosity. 2 heterozygosity. Although an increased synthesis of Hb-F with G gamma chains was again observed after in vitro incubation of reticulocytes with [35S]methionine none of the isolated cell fractions contained a Hb F with the alpha 2 G gamma 2 composition.     

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 alpha and beta chains of adult hemoglobins of the Grand Galago, Galago crassicaudatus

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.     

Human fetal hemoglobin synthesis and its NH2-terminal acetylation in a rabbit reticulocyte lysate translational system

The biosynthesis of the acetylated (Hb FIc) and the non-acetylated (Hb F0) human fetal hemoglobin components has been examined in a cell-free translational system. The poly(A)-RNA was isolated from umbilical cord blood samples and translated in the heterologous translational system derived from rabbit reticulocyte lysates in the presence of labeled amino acid(s) or acetyl-CoA. The amount of each hemoglobin or globin chain made in the system was determined by separating the synthesis products by cation-exchange chromatographic methods. The in vitro synthesis ratios were close to the FIc/Ftotal values of the respective hemolysates. The same conclusion could be reached by determining the specific activity ratios of Hb FIc/Hb F0. Co-migration of radioactivity peaks with absorbance peaks indicated the synthesis of that hemoglobin or globin chain. Confirmation of the synthesis of true gamma 0 and gamma Ic was accomplished by high-pressure liquid chromatographic separation of 3H-labeled tryptic peptides. Each peptide corresponded well with the radioactivity peak. Labeled acetyl-group incorporation into Hb FIc and gamma IcT-1 provided direct evidence for acetylation of gamma chains in Hb FIc. The data indicate that the mRNA itself dictates whether a protein is acetylated and, if so, to what extent. The control appears to be not unique to the human red cell system.     

Interaction of hemoglobin and its component alpha and beta chains with band 3 protein

The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.     

The XmnI site 5' to the G gamma-globin gene polymorphism and its relationship to %Hb F and %G gamma in normal Japanese and Korean adults.

The ratio of fetal hemoglobin to total hemoglobin (%Hb F), the ratio of G gamma to total gamma globin (%G gamma), and the polymorphism of the XmnI site at -158 base pairs from the cap site of the G gamma-globin gene were examined in normal unrelated Japanese (n = 113) and Korean (n = 44) adults. The frequency of the presence of the XmnI site was 0.15 in the Japanese and 0.16 in the Korean population. There were statistically significant differences in the %G gamma values of the Japanese between those +/+ and those +/- or -/- at the XmnI site (p less than 0.01). The Korean %G gamma values showed a statistically significant difference (p less than 0.01) between those +/- and those -/-. The presence of the XmnI site was significantly associated with the elevation of G gamma-globin chain synthesis, but this relationship was not necessarily absolute. The absence of the XmnI site in an adult with a gamma-globin gene triplication (G gamma AG gamma A gamma/G gamma A gamma) more or less reduced the level of G gamma-globin chain synthesis, but the presence of the XmnI site in an adult with a gamma-globin gene deletion (GA gamma/G gamma A gamma) had no effects on the proportion of the two gamma-globin chains.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.