Microsequence analysis of the <i>N</i>‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting

Hirano, Hisashi; Komatsu, Setsuko; Kajiwara, Hideyuki; Takagi, Yoshiaki; Tsunasawa, Susumu

doi:10.1002/elps.11501401134

Cited by 51 publications

(39 citation statements)

References 40 publications

(13 reference statements)

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“…Their N-terminus is blocked by acetylation. The N-terminal amino acid is acetyl-Ser, as in the other inamrnalian [-chains, shown by Edman degradation after the intact chain was subjected to prolonged exposure to triflouroacetic acid which partialy removes the acetyl group (Hirano et al, 1993).…”

Section: Discussionmentioning

confidence: 89%

“…Both the zeta-chains (c and [), of Hbs 3 and 4 were N-terminally blocked, a feature common to the embryonic [ chains of mammals. To determine the N-terminal sequence, the Hb4 5 chain was first subjected to mild acid hydrolysis according to Hirano et al (1993) and subsequently sequenced by Edman degradation. To determine further partial primary structure analysis of one of Table 1.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii

Holland

Gooley

1997

European Journal of Biochemistry

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The embryonic hemoglobins of the marsupial Tammar wallaby (Macrop~s eugenii) are known to aggregate, which was shown by the finding that the Hill coefficient, h, was greater than 4.0 in the upper part of the oxygen equilibrium curve. Here, we have undertaken a detailed primary structure analysis of the Tammar wallaby pouch young hemoglobin complement, which we hoped might provide clues into the residues that cause aggregation and a high embryonic h.The Tammar wallaby embryonic hemoglobin complement is principally four major hemoglobins each with a different isoelectric point. Two early expressed hemoglobins contain the same embryonic p-like chain, epsilon ( E ) , but two separate n-like chains, termed zeta and zeta prime ([ and c) both of which are N-terminally blocked. The later two expressed hemoglobins contain the same adult u-chain, but different [Hike chains. The latest expressed hemoglobin contains the same b-like chain, e, as the two early expressed forms, but the third expressed hemoglobin contains a unique &like chain which we have termed omega (LO). A protein database similarity search using the first 54 N-terminal amino acids of the w-chain showed a range of sequence identities of 57-72% to all known mammalian /?-like chains, including the other marsupial c-chains. The closest identity, retlected by both the highest percentage identity and SmithWaterman score, was with the embryonic P-chains of the aves.While the primary structures of the hemoglobins reported here do not explain the low hemoglobinoxygen affinity in embryonic marsupial blood, the finding of the similarity with the bird globin-like sequence with one of the marsupial chains has implications on mammalian globin evolution. How many other marsupials and placental mammals are harboring a bird-like globin in their embryos'?

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Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii

Holland

Gooley

1997

European Journal of Biochemistry

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“…Ions were generated by irradiating the sample area with a nitrogen laser at a wavelength of 337 nm. Calibrations were carried out using a mixture of angiotensin I (1297.51 MH + ), adrenocorticotropic hormone ACTH (clip [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] …”

Section: Ms Analysismentioning

confidence: 99%

“…MALDI-TOF MS analysis of a tryptic digest of the protein indicated for the N-terminal peptide MDSQVR a value of m/z 777, which points to the presence of acetylmethionine. This residue is commonly found in the N-terminus of eukaryotic proteins together with N-acetyl Ala, Ser, Gly and Thr [17].…”

Section: Determination Of Amino-acid Sequencementioning

confidence: 99%

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M‐type homopolymer

Mignogna¹,

Chiaraluce²,

Consalvi³

et al. 2002

European Journal of Biochemistry

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Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is composed of a single subunit that contains both the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleation site, typical of mammalian L chains. Comparison of the amino-acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M-type homopolymer. Interestingly, the T. bernacchii ferritin chain shows 85.7% identity with a coldinducible ferritin chain of the rainbow trout Salmo gairdneri. The structural and functional properties indicate that cold acclimation and functional adaptation to low temperatures are achieved without significant modification of the protein stability. In fact, the stability of T. bernacchii ferritin to denaturation induced by acid or temperature closely resembles that of mesophilic mammalian ferritins. Moreover iron is taken up efficiently and the activation energy of the reaction is 74.9 kJAEmol )1 , a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJAEmol )1 ).

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“…Application of four different deblocking strategies [9] did not solve the problem. Thus the aim of the present investigation was to characterize this potential allergen further and, in particular, to obtain sequence data which may highlight its function in the plants.…”

Section: Introductionmentioning

confidence: 94%

Characterization of a New IgE‐Binding 35‐kDa Protein from Birch Pollen with Cross‐Reacting Homologues in Various Plant Foods

et al. 1998

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The present investigation was undertaken to obtain molecular data of a new immunoglobulin (Ig)E-binding birch pollen protein with a mass of 35 kDa. In a previous study, this protein showed IgE cross-reactivity with 34-and 35-kDa proteins in apples, pears, carrots, bananas and other exotic fruits. Since the protein was Nterminally blocked, it was purified by preparative SDS-PAGE, and multiple proteolytic fragments were subsequently generated by in-gel digestion with the endoproteinases Glu C, Lys C and Clostripain. After electrophoretic separation and blotting onto polyvinylidene difluoride (PVDF), the resulting polypeptides were subjected to N-terminal amino acid microsequencing. The internal sequences obtained showed a high degree of sequence identity to isoflavone reductases (IFR) and isoflavone reductase-like proteins (IRL) from several plants which also had a similar size. For a stretch of 25 consecutive residues this identity ranged from 56% for IFR from peas and chick peas and an IRL from maize, to 80% for a tobacco IRL. A 453 bp fragment was amplified from total birch pollen RNA by polymerase chain reaction (PCR) using primers derived from the nucleotide sequence of the tobacco IRL. The deduced 151 amino acid sequence represented Ϸ 50% of the protein and confirmed the sequence identities obtained by Edman degradation. Moreover, the 25 amino acid sequence was included in the cloned fragment. Deduced and determined amino acids showed only one mismatch, which was due to a single nucleotide exchange. At the antibody level, the immunological relationship of the birch pollen protein to IRL and IFR was demonstrated by immunoblotting with a rabbit antiserum against a pea IFR which recognized the same birch protein as patients' IgE. The rabbit antiserum also reproduced the cross-reactivity pattern previously observed with patients' IgE by recognizing related proteins in specific plant foods, including some exotic fruits. We therefore suggest that the 35-kDa birch pollen protein belongs to the IFR/IRL family and represents a minor allergen, possibly being responsible for less common pollen-related food allergies in patients allergic to birch pollen.

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Microsequence analysis of the N‐terminally blocked proteins immobilized on polyvinylidene difluoride membrane by Western blotting

Cited by 51 publications

References 40 publications

Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii

Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M‐type homopolymer

Characterization of a New IgE‐Binding 35‐kDa Protein from Birch Pollen with Cross‐Reacting Homologues in Various Plant Foods

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