Binding of CO to mutant alpha chains of hemoglobin M Iwate; evidence for distal imidazole ligation.

The optical contribution of the beta chains to the spectrum of hemoglobin M Iwate (alpha87his leads to tyr)2beta2a was subtracted with the aid of a computer so that the spectrum of ferric alpha chains was obtained. Tyrosinate binding to the heme is suggested from spectral resemblance to ferric heme phenolate in dimethyl sulfoxide. The slow reduction of the abnormal ferric alpha chains in hemoglobin M Iwate by dithionite was studied spectrophotometrically both in the presence and absence of CO. The rate of reduction was found to be dependent on the state of ligation of the normal beta chains. The CO-ligated form of the reduced alpha chains bears strong spectral resemblance to the CO-ligated form of the reduced beta chains suggesting similar structures for the heme-ligand complex. A model compound with similar optical properties to the CO-ligated protein can be prepared in dimethyl sulfoxide from hemin chloride, imidazole, and CO using chromous acetate as the heme reductant. Substitution of phenolate for imidazole produces a spectral entity so different from that observed in the protein as to rule out tyrosinate ligation to ferrous heme of the alpha chains when CO is bound.     

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

Esterification of the propionate groups promotes alpha/beta hemoglobin chain homogeneity of CN-hemin binding

This study examines the post-translational role of peripheral propionate groups in the incorporation of the Fe-protoporphryin IX heme into nascent alpha- and beta-globin chains. Human apohemoglobin (a heme-free alpha/beta dimer) in 0.05 M potassium phosphate buffer, pH 7, at 20 degrees C was titrated with either CN-protohemin (native heme with two peripheral propionate groups), or CN-dimethylester hemin (a modified heme with two methyl ester groups in place of the propionate groups). Soret spectrophotometric CN-hemin titrations confirmed that a spectral shift resulted upon binding of protohemin, but no spectral shift occurred upon binding the dimethylester derivative. Recent studies have correlated a Soret spectral shift with the preferential heme binding to the alpha subunit of apohemoglobin. The absence of a Soret wavelength shift (in conjunction with molecular modeling) presented here suggested that the modification of heme propionate groups prevented the formation of an alpha-heme/beta-globin intermediate, a requisite step in the normal assembly of functional hemoglobin.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Functional characterization of human erythrocyte spectrin alpha and beta chains: association with actin and erythrocyte protein 4.1

Human erythrocyte spectrin alpha and beta chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of alpha-beta heterodimers with sedimentation characteristics identical with native alpha-beta dimers. The binding of 125I-labeled band 4.1 to alpha and beta chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. It was found that both alpha and beta chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified beta chains but not alpha chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified alpha chains, beta chains, and recombinant alpha-beta heterodimers to F-actin was measured in the presence of band 4.1. We found that alpha or beta chains separately exhibited no band 4.1 dependent association with F-actin but that alpha-beta heterodimers formed by recombination of the chains did. We conclude that spectrin binding to F-actin in the presence of band 4.1 requires the participation of both of spectrin's polypeptide chains.     

Spectral, conformational and chemical properties of opossum methemoglobin

Opossum methemoglobin differs from methemoglobin A in spectral, spin state, conformational and chemical properties. The primary structural alterations in opossum hemoglobin, including the critical substitution at alpha 58 (E7) His leads to Gln result in the following properties. (a) Major contribution of the spectral transitions due to inositol hexakisphosphate binding arises from the alpha chains. (b) The aquomet to hydroxymet (high-spin to low-spin) transition as a function of pH is slightly retarded resulting in considerable high spin at alkaline pH. (c) The tertiary conformation (t) around the beta hemes, upon transition to a T quaternary state, differs from the known hemoglobin t tertiary structure. (d) Both alpha and beta hemes are susceptible to rapid reduction by ascorbic acid (the reduction rate being tenfold faster than that of methemoglobin A). These properties suggest that the heme environments in both the alpha and beta subunits of opossum hemoglobin are different from those of human hemoglobin A.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

Differential expression of laminin alpha chains during proliferative and differentiation stages in a model for skin morphogenesis.

The purpose of this study was to determine the mRNA and protein expression of laminin alpha chains at various stages of in vitro skin morphogenesis. Fibroblasts in mono-cultures express low levels of the mRNA of laminin alpha1,alpha2, alpha3 and alpha4 chains. When co-cultured with keratinocytes for 28 days, they expressed the mRNA for all these chains. Keratinocytes in monolayer expressed the laminin alpha3 chain mRNA and very low levels of the mRNA of the alpha1 and alpha2 chains, although, when recombined with fibroblasts they also expressed laminin alpha1and alpha2 mRNA, but not the laminin alpha4 mRNA. Immunocytochemistry of cells in co-culture showed that laminin alpha1, alpha3 and alpha5 chains were expressed in the epidermis, while the laminin alpha2, beta1, and gamma1 chains were noted in the dermis and at the epidermo-dermal interface. The laminin alpha1chain was first expressed during the proliferative stage (14-21 days) and the laminin alpha2 and alpha5 chains appeared later, during the differentiation stage (28-42 days). The above results suggest that epithelial-mesenchymal interactions are involved in the expression of laminin alpha chain mRNA during in vitro skin morphogenesis. In addition, there is distinct temporal and spatial expression of these chains during proliferative and differentiation stages, possibly reflecting different functions.     

Association of hemoglobin chains with the cell membrane as a cause of red cell distortion in thalassemia

Hemoglobin chains were separated and their interaction with membrane ghosts was studied using their ability to quench the fluorescence intensity of a membrane embedded probe. It was observed that alpha chains bind faster and with higher affinity to the membrane sites than do beta chains. The fast reversible interaction of both chains with the membrane was followed by a time-dependent partial loss of reversibility. Band 3 cytoplasmic fragments (B3F) were isolated and their reaction with separated Hb chains was studied using fluorescence quenching techniques as well. The data demonstrate that the relative affinity of the chains for B3F and loss of reversibility of the reaction followed patterns similar to the corresponding interaction of the chains with whole membranes. Band 3 cytoplasmic poles are therefore suggested as the high-affinity sites on the membrane for hemoglobin chains. When globin was reacted with B3F, it was observed that this protein binds strongly to the same membrane sites, but practically irreversibly. Exchange of the HbA content of normal cells by separated alpha or beta chains resulted in membrane distortions in both cases, but alpha chains caused greater morphological changes than did beta chains. The results of this study may provide one explanation for the differences in the thalassemia syndromes when excess of either alpha or beta chains is involved.     

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.     

Different levels of glycosylation contribute to the heterogeneity of alpha 1(II) collagen chains derived from a transplantable rat chondrosarcoma

Three collagen fractions, each of which contain molecules composed of alpha 1(II) chains, have been isolated from pepsin-solubilized rat chondrosarcoma collagen. One fraction could be selectively precipitated from the pepsin digest at 0.7 M NaCl. Two additional fractions were obtained on chromatography of the collagen precipitating at 1.2 M NaCl on carboxymethyl cellulose under nondenaturing conditions. When chromatographed on carboxymethyl cellulose under denaturing conditions, each fraction contained components eluting in the position expected for alpha 1(II) chains. One of the fractions precipitating at 1.2 M NaCl contained the recently described 1 alpha and 2 alpha chains in addition to material eluting as alpha 1(II) chains. Comparison of the chains eluting as alpha 1(II) chains in the various fractions with respect to amino acid composition, carbohydrate content, and cyanogen bromide-cleavage products showed that they differed only in the number of glycosylated hydroxylysyl residues. In this regard, alpha 1(II) chains obtained from collagens precipitated at 1.2 M NaCl exhibited significantly higher levels of glucosylgalactosylhydroxylysyl residues than alpha 1(II) chains precipitated at 0.7 M NaCl. These results indicate that molecules composed of alpha 1(II) chains are heterogeneous with respect to levels of hydroxylysine-linked carbohydrate moieties and that the more highly glycosylated molecules require higher salt concentrations for precipitation from acidic solutions. The data also indicate that a proportion of the more highly glycosylated alpha 1(II) chains are involved in the formation of one or more molecular species with 1 alpha and 2 alpha chains.     

Three novel collagen VI chains with high homology to the alpha3 chain

Here we describe three novel collagen VI chains, alpha4, alpha5, and alpha6. The corresponding genes are arranged in tandem on mouse chromosome 9. The new chains structurally resemble the collagen VI alpha3 chain. Each chain consists of seven von Willebrand factor A domains followed by a collagenous domain, two C-terminal von Willebrand factor A domains, and a unique domain. In addition, the collagen VI alpha4 chain carries a Kunitz domain at the C terminus, whereas the collagen VI alpha5 chain contains an additional von Willebrand factor A domain and a unique domain. The size of the collagenous domains and the position of the structurally important cysteine residues within these domains are identical between the collagen VI alpha3, alpha4, alpha5, and alpha6 chains. In mouse, the new chains are found in or close to basement membranes. Collagen VI alpha1 chain-deficient mice lack expression of the new collagen VI chains implicating that the new chains may substitute for the alpha3 chain, probably forming alpha1alpha2alpha4, alpha1alpha2alpha5, or alpha1alpha2alpha6 heterotrimers. Due to a large scale pericentric inversion, the human COL6A4 gene on chromosome 3 was broken into two pieces and became a non-processed pseudogene. Recently COL6A5 was linked to atopic dermatitis and designated COL29A1. The identification of novel collagen VI chains carries implications for the etiology of atopic dermatitis as well as Bethlem myopathy and Ullrich congenital muscular dystrophy.     

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.     

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Nonuniform distribution of myosin light chains within the thick filaments of lobster slow muscle: Immunocytochemical study.

The in situ distribution of the alpha and beta myosin light chains was investigated at the subsarcomeric and subfilament levels in individual fibers of the superficial flexor muscle (SFM) of the lobster, Homarus americanus. Polyclonal antibodies were produced against the two classes of myosin light chains and used for subsequent immunolocalization on thin sections of sarcomeres and on isolated filaments from both the medial and lateral fiber bundles of the SFM. The beta myosin light chains were uniformly distributed within the crossbridge region of sarcomeres of both medial and lateral bundles. The alpha myosin light chains were uniformly distributed within the crossbridge region of sarcomeres from the medial bundle, but were nonuniformly distributed over the crossbridge region of lateral bundle sarcomeres. In the latter, the number of alpha myosin light chains was highest toward the center of the thick filaments, diminishing towards the ends. Similar distributions of alpha light chains were found in isolated myosin filaments. These data demonstrate that heterogeneity in protein composition extends to the level of the myosin filament and suggest that the myosin filament substructure in lobster may be different than that found in vertebrate skeletal muscle.     

Secretion of non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains upon depletion of ascorbate by cultured human cells

Our previous report showed that human fetal lung fibroblasts secreted non-disulfide-bonded, non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains depending on culture conditions [Connective Tissue (1999) 31, 161-168]. The secretion of non-helical collagenous polypeptides is unexpected from the current consensus that such polypeptides are not secreted under physiological conditions. The absence of interchain disulfide bonds among alpha1(IV) and alpha2(IV) chains was always correlated with the absence of triple-helical structure of the type IV collagen. The finding corresponds with the fact that the interchain disulfide bonds are formed at or close to the completion of the type IV collagen triple-helix formation. The present report shows that ascorbate is the primary factor for the triple-helix formation of the type IV collagen. When human mesangial cells were cultured with ascorbate, only the triple-helical type IV collagen was secreted. However, when the cells were cultured without ascorbate, the non-helical alpha1(IV) and alpha2(IV) chains were secreted. Relative amounts of the secreted products were unchanged with or without ascorbate, suggesting that ascorbate is required for the step of the triple-helix formation. The ascorbate-dependency of the triple-helix formation of the type IV collagen was observed in all the human cells examined. The non-helical alpha1(IV) chain produced by the ascorbate-free culture contained about 80% less hydroxyproline than the alpha1(IV) chain from the triple-helical type IV collagen. The evidence for the non-association of the non-helical alpha1(IV) and alpha2(IV) chains in the conditioned medium was obtained by an anti-alpha1(IV) antibody-coupled affinity column chromatography for the conditioned medium. Although all the non-helical alpha1(IV) chains were found in the bound fraction, all the non-helical alpha2(IV) chains were recovered in the flow-through fraction. The present findings suggest that ascorbate plays a key role in the trimerization step of three alpha chains and/or in the subsequent triple-helix formation of the type IV collagen.     

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.     

Profile of Numbers of Sequence Differences Among V-Genes Coding for the Variable Regions of T Cell Receptor for Antigen Alpha and Beta Chains

When human T cell receptor for antigen (TCR) alpha chain V-genes were compared pair-wise, the numbers of nucleotide differences showed a characteristic distribution; most were in the range of 100 to 200 differences out of a total of about 300 bases. The same distribution was observed for mouse TCR alpha chains. Even more interesting was that comparing human alpha chains and mouse alpha chains gave essentially the same nucleotide difference pattern. It is inferred from the large number of differences and from the nonspecificity of trans-species (human and mouse) nucleotide sequence differences of TCR V-genes that TCR alpha chains probably diverged early during evolution. The same feature was also observed for human and mouse TCR beta chains, although the alpha and beta chain V-genes were distinct. This evolutionary preservation could be of vital importance to the fidelity of the complicated trimolecular interactions among TCR alpha and beta chains, the processed peptide, and the major histocompatibility complex (MHC) class I or II molecules. Received: 22 January 1996 / Accepted: 9 September 1996     

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.