Sequence of amino acids in the major and minor β chains of the diffuse hemoglobin from BALB/c mice

The amino acid sequence of blastokinin, also called uteroglobin, has been determined by a combined study of both the intact native molecule and the peptide fragments resulting from tryptic and chymotryptic digestions. Sequence analyses performed by automated methods and by sequential digestion with leucine aminopeptidase and carboxypeptidase Y demonstrate that blastokinin is a dimer of identical 69-amino acid subunits held together in parallel orientation by two disulfide bridges at positions 3 and 68. The polypeptide chains are further characterized by the absence of tryptophan residues and by single residues of histidine and tyrosine at positions 8 and 21, respectively. Six of eight amino acids, positions 17-24, near the progesterone binding site of blastokinin contain a hydroxyl group. Knowledge of the chemistry of this receptor site should allow better perspectives of the chemistry of molecules in normal tissues that are dependent on progesterone for growth and development, as -well as compounds that could act as cancer antagonists for endocrine therapy of hormone-dependent tumors. Blastokinin (1), also termed uteroglobin (2), is a low molecular weight protein that was first isolated from the uterine fluid of pregnant rabbits. The synthesis and secretion of this protein have been shown to increase during the first 6 days of pregnancy, accounting for-more. than 50% of the total uterine protein by day 6 (day 0 = coitus) (3-5). The increase in blastokinin/uteroglobin (BK/UG)t levels at a time when endogenous levels of progesterone were rising implied that a correlation may exist between these two events. Several reports demonstrated that exogenous progesterone was capable of inducing the synthesis of this protein (2, 6, 7). The recent finding that BK/UG in turn binds progesterone with high affinity (7-9) implies that BK/UG may serve as a carrier molecule for the same steroid that induces its synthesis. These facts not only make BK/UG unique but they also make a further study of its structure important. In the paper we present the primary structure for BK/UG, its possible physiological role(s), and the significance of knowing its structure as a hormone receptor. amide gel electrophoresis and a single immunoprecipitin band in agar immunodiffusion plates. Prior to chemical analyses, an additional purification step was performed by molecular sieving through a Sephadex G-25 column (1.9 X 95 cm) equilibrated with 10 mM HC1. Elution with the same buffer (flow rate, 2 ml/min) allowed BK/UG to elute with the void volume while salts and other low molecular weight contaminants were still migrating. Peak fractions of BK/UG, identified by absorbancy at 280 nm, were pooled and lyophilized. Four samples, each containing 7-14 mg of BK/UG, were isolated. One sample was digested by trypsin without further treatment. The disulfide bonds of cystine in the other three samples were reduced and aminoethylated (11) prior to digestion by trypsin or chymotrypsin. MATERIALS AND METHODSAmino acid analyses were performed with a Be...

Section: Methodsmentioning

confidence: 99%

Amino acid sequence of a progesterone-binding protein.

Popp¹,

Foresman²,

Wise³

et al. 1978

“…Up to 1977, five genetic variants of mouse hemoglobin a chains were known from studies of hemoglobin solubility and polypeptide sequence analyses (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) …”

Section: Glymentioning

confidence: 99%

mentioning

confidence: 99%

“…We have shown previously (1) that isoelectric focusing-a method for the separation of proteins based upon differences in their isoelectric points-can resolve at least some proteins that differ from one another only by neutral amino acid substitutions. That demonstration relied upon the availability of a variety of mouse hemoglobins (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) of known amino acid sequence (15)(16)(17)(18)(19)(20) [and nowknown gene sequence (21,22)] that differ from one another only by one to four substitutions of neutral amino acids (valine, isoleucine, serine, threonine, asparagine, glycine, and alanine), Although several additional a-globin genotypes have been discovered since then (23)(24)(25)(26), all of the naturally occurring variant chains subsequently characterized by protein sequence analysis also have differed in neutral amino acids only (Table 1). That no charge variation among mouse a-globins has been found in nature is consistent with the deleterious effect that charged amino acid substitutions generally have in human hemoglobin a chains.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Detection of neutral amino acid substitutions in proteins.

Whitney¹,

Cobb²,

Popp³

et al. 1985

The field of biochemical genetics relies heavily upon the detection by electrophoresis of genetically determined variants of proteins. Most of these variants differ by substitutions that involve charged amino acids. Genetic variants of another large class, ones that involve substitutions among neutral amino acids, are not easily detected and are often ignored. Ampholyte isoelectric focusing in some cases can separate proteins indistinguishable by standard electrophoresis, including genetic variants of mouse hemoglobins that differ only by neutral amino acid substitutions. A revolutionary variation of isoelectric focusing, in which gradients covering a small pH range are fixed into place in a polyacrylamide gel, provides greater resolution of these nearly identical proteins. Mouse hemoglobin tetramers that differ only by the substitution of alanine for glycine in the a-globin chains are resolved by several millimeters with the new technique; by comparison, these tetramers are imperfectly resolved on a standard pH 7-9 isoelectric focusing gel. This improved technique of isoelectric focusing was used to identify a variety of previously unreported genetic variants of mouse hemoglobin ar chains. Immobilized gradients tailored to the requirements of the proteins being analyzed will extend greatly the ranges of protein variations that can be easily recognized for diverse applications, including genetic quality-control analyses and in studies of genetics, mutagenesis, and evolution.Standard electrophoretic techniques are often suitable for differentiating among proteins that differ from one another by the substitution of charged amino acids but generally are not capable of separating proteins that differ only by neutral amino acid substitutions. We have shown previously (1) that isoelectric focusing-a method for the separation of proteins based upon differences in their isoelectric points-can resolve at least some proteins that differ from one another only by neutral amino acid substitutions. That demonstration relied upon the availability of a variety of mouse hemoglobins (2-14) of known amino acid sequence (15)(16)(17)(18)(19)(20) [and nowknown gene sequence (21,22)] that differ from one another only by one to four substitutions of neutral amino acids (valine, isoleucine, serine, threonine, asparagine, glycine, and alanine), Although several additional a-globin genotypes have been discovered since then (23)(24)(25)(26)

“…Variation within these two classes of haplotype is also known. The Hbbs allele of SWR mice (3,4) has been shown by protein sequence analysis to produce a slightly different ,B sequence than that of C57BL mice. Au/SsJ mice carry the HbbP haplotype, which is distinct from Hbbd in that it specifies a slightly different minor chain (5).…”

mentioning

confidence: 99%

DNA fragments of the Mus musculus beta globin haplotypes Hbbs and Hbbd.

Weaver¹,

Haigwood²,

Hutchison³

et al. 1979

Two alternative haplotypes at the complex locus controlling hemoglobin a chain synthesis in Mus musculus were compared at the DNA level. As expected, Hbbd homozygotes-which as adults synthesize two species of 13 chain-have two genes for d3 globin. Adult mice homozygous for the Hbbs haplotype make only a single type of 1 polypeptide, yet they also have two 13 globin genes. Apparently the two Hbbs genes encode identical proteins, or one of the two genes is not detectably expressed. The Hbbs and Hbbd haplotypes are thus more similar at the DNA level than studies of their polypeptide products have indicated.The synthesis of the hemoglobin 1 chain in the laboratory mouse Mus musculus musculus is controlled by the Hbb locus, at which a number of allelic alternatives, or haplotypes, have been described (1). These are of two general types: Hbbs and Hbbd. The Hbbs haplotype determines the synthesis of a single 13 globin polypeptide, /ls, in the adult mouse. Two types of /3 globin are specified by the Hbbd complex: Mdmai and (3dmin; the relative amount of these two proteins is developmentally regulated (2). Variation within these two classes of haplotype is also known. The Hbbs allele of SWR mice (3, 4) has been shown by protein sequence analysis to produce a slightly different ,B sequence than that of C57BL mice. Au/SsJ mice carry the HbbP haplotype, which is distinct from Hbbd in that it specifies a slightly different minor chain (5). Finally, the Hbbd-like alleles of the interbreeding subspecies M. m. castaneus and M. m. molossinus produce minor chains that vary slightly from those of M. m. musculus (6). Closely linked to the Hbb locus are the genes for the ,B-like embryonic globin y (7,8).Digestion of a mammalian genome by the restriction endonuclease EcoRI generates several million fragments (9). Jeffreys and Flavell (10) were able to identify the EcoRI fragments of rabbit DNA that contained 3 globin sequences after spreading the fragments in a single dimension. They electrophoresed a denatured digest through an agarose gel (denaturation allows a larger amount of DNA to be loaded), transferred the DNA to a nitrocellulose membrane, and hybridized it with a cloned cDNA copy of ,B globin mRNA labeled in vitro with 32P by nick-translation. A similar analysis of human ,B globin genes has been reported by Mears et al. (11). The sensitivity and resolution afforded by such analyses may be considerably enhanced by spreading the digested DNA in two dimensions. The DNA is first subjected to reverse-phase chromatography on RPC-5 (12, 13), and the fractions so obtained are then run in an analytical agarose slab gel. The DNA in the pattern, or "spread," is denatured in situ, transferred to nitrocellulose (14) Hybridization probes were labeled with 32P by nick-translation to a specific activity of 50-200 cpm/pg (18). The probe for 1 globin was obtained by digesting the plasmid pCR1IBM9 (19), which contains a cloned cDNA copy of mouse 13 globin mRNA, with the endonuclease Hha I. The fragment bearing the inserted cDNA sequenc...