The heterogeneity of human immunoglobulins

Milstein, C.

doi:10.1042/bj1100026p

Cited by 7 publications

(8 citation statements)

References 13 publications

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“…All the ovalbumins contained two different phosphoserine sequences similar to those reported by Milstein [3]. It is apparent from the complete sequence of hen ovalbumin that the site 1 phosphoserine is residue 68, and the site 2 phosphoserine residue 344 [4].…”

Section: Pur If Cu T Ion Of Phosp Hoser Ine Pep T Idesmentioning

confidence: 51%

“…The diagonal method of Milstein [3] was used. Fig.1 shows a general scheme for the identification and purification of the phosphoserine peptides, and of their subpeptides.…”

Section: Irlmtification and Purification Of' Phosphorylated Peptidesmentioning

confidence: 99%

“…Milstein [3] isolated two phosphoserine peptides from hen ovalbumin by a diagonal electrophoresis method, and determined the amino acid sequences of these peptides. It is now known from the complete amino acid sequence of hen ovalbumin, which consists of 385 residues [4,5], that the site 1 phosphoserine is residue 68, and site 2 phosphoserine is residue 344.…”

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confidence: 99%

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Sequences of Sixteen Phosphoserine Peptides from Ovalbumins of Eight Species

Henderson

Moir²,

Fothergill

et al. 1981

European Journal of Biochemistry

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Phosphoserine peptides have been isolated by a diagonal electrophoresis method from proteolytic digests of ovalbumins from hen, grouse, turkey, golden pheasant, magpie goose, Chinese goose, Aylesbury duck and fulvous whistling duck. The amino acid sequences of these peptides have been determined. There are two sites of phosphorylation in each ovalbumin, which are located in two different regions of the ovalbumin molecule. Amino acid replacements are more frequent in the site 1 sequences than in the site 2 sequences. Both site 1 and site 2 sequences contain invariant residues. Sequence variations occur near the serine residues that are phosphorylated, but the amino acid two residues C-terminal to the phosphoserine is always glutamic acid, suggesting that this may be a recognition signal for the phosphorylating enzyme. Variations in amino acid sequence among the species are consistent with differences in the ovalbumins determined by peptide mapping and quantitative immunoprecipitation assays. A phylogenetic tree has been constructed from a comparison of the sequences of 248 residues from the eight ovalbumins.Hen ovalbumin is known to contain two phosphoryl groups [l] that are attached to serine residues [2]. Milstein [3] isolated two phosphoserine peptides from hen ovalbumin by a diagonal electrophoresis method, and determined the amino acid sequences of these peptides. It is now known from the complete amino acid sequence of hen ovalbumin, which consists of 385 residues [4,5], that the site 1 phosphoserine is residue 68, and site 2 phosphoserine is residue 344. The site 2 region contains the sequence that corresponds to the heptapeptide that is released by the action of subtilisin on ovalbumin to form plakalbumin [6].The present paper describes the isolation of phosphoserine peptides from ovalbumins from eight related species, and the results of amino acid sequence studies on these peptides. These results have implications concerning the specificity of phosphorylation, and concerning the phylogeny of the species studied. Moreover, the postsynthetic modification of ovalbumin by phosphorylation is of interest, since it is one of the final steps in the production of mature ovalbumin by a system which has proved lo be particularly suitable for studying eukaryotic gene organisation [7] and the control of transcription and translation [8]. MATERIALS AND METHODS OvalhuminsOvalbumins from each species were purified from single egg-whites by chromatography on CM-cellulose followed Abbreviations. Dansyl, 5-dimethylaminonaphthalene-1-sulphonyl; Cm-Cys, S-carboxymethylcysteine; Cys(O3H), cysteic acid; Met(Oz), methionine sulphone; m6.5, mz, electrophoretic mobilities at pH 6.5 and pH 2 respectively. EnzymesAlkaline phosphatase, chymotrypsin, pepsin, trypsin and carboxypeptidase A were obtained from Worthington Biochemical Corporation. Subtilisin (protease VII) was from Sigma Chemical Company. Thermolysin was a gift from Dr R. P. Ambler. Staphylococcal serine proteinase was from Miles Laboratories Ltd. Digestions of Ovalbum...

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Section: Pur If Cu T Ion Of Phosp Hoser Ine Pep T Idesmentioning

confidence: 51%

“…The diagonal method of Milstein [3] was used. Fig.1 shows a general scheme for the identification and purification of the phosphoserine peptides, and of their subpeptides.…”

Section: Irlmtification and Purification Of' Phosphorylated Peptidesmentioning

confidence: 99%

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Sequences of Sixteen Phosphoserine Peptides from Ovalbumins of Eight Species

Henderson

Moir²,

Fothergill

et al. 1981

European Journal of Biochemistry

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“…The ovalbumin or its cyanogen bromide fragments were digested at a concentration of approximately 2 mg/ml with enzyme (1 : 50 by weight) at 37 'C for [12][13][14][15][16] [28]. Carboxypeptidase Y digests were done according to Kuhn et al [29].…”

Section: Overall Strategy and Methods Qf' Cleavugementioning

confidence: 99%

“…Dephosphorylation of peptides using alkaline phosphatase was done on paper strips according to Milstein [16].…”

Section: Rcm-ovalbuminmentioning

confidence: 99%

The Complete Amino‐Acid Sequence of Hen Ovalbumin

Nisbet

Saundry

Moir

et al. 1981

European Journal of Biochemistry

451

245

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The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699.Ovalbumin has four sites of postsynthetic modification ; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn-292, and the two phosphorylated serines are at residues 68 and 344. The 'signal sequence' of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346 -352. The hen ovalbumin polymorphism characterised by an Asn-tAsp replacement results from a mutation at residue 31 1.The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723 -728 (1978)].Ovalbumin is the most abundant protein in egg white, and is readily purified and crystallised in gram quantities [l].

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