Analysis of polypeptide molecular weights by electrophoresis in urea

Poole, Toy S.; Leach, Bonnie Strayer; Fish, Wayne W.

doi:10.1016/0003-2697(74)90272-3

Cited by 42 publications

(16 citation statements)

References 20 publications

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“…To examine the possible subunit structure of the two AEF components, 125I-labeled fractions 17, 19, and 17-19 were subjected to electrophoresis under dissociating conditions on a 1-D acid-urea 10% polyacrylamide slab gel in the absence of SDS (20). Only the 68,000-and 10,000-to 15,000-mol wt components appeared on this gel (T. Deiovitch.…”

Section: Fraction Numbermentioning

confidence: 99%

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

Delovitch¹,

Watson²,

Battistella³

et al. 1981

The Journal of Experimental Medicine

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An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated A/WySn (H-2a) stimulator spleen cells helps an in vitro primary anti-erythrocyte plaque-forming cell PFC response of BALB/c nude spleen cels and also A/WySn but not A.SW T cell-depleted spleen cells. AEF activity is adsorbed by anti-Ik and anti-I-Ak but not by anti-I-Jk, anti-I-ECk, and anti-Is. Gel filtration of ACA 54 resolves AEF into two main components that which appear in the 50,000- to 70,000-mol wt (component I) and 30,000- to 35,000-mol wt (component II) regions, respectively. Component I has a mol wt of 68,000, elutes from DEAE-Sephacel at 0.05-0.1 M NaCl, and has an isoelectric point (pI) of 5.8. It helps A/WySn but not A.SW B cells and, therefore, is H-2 restricted. Component II is not H-2 restricted, because it helps both A.SW and A/WySn B cells. It also stimulates (a) the growth of a long-term cytotoxic cell line in vitro, (b) Con A-induced thymocyte mitogenesis, and (c) the generation of cytotoxic T cells. The latter three properties of component II are not shared by component I. In addition, component II elutes from DEAE-Sephacel at 0.15-0.2 M NaCl and has a pI of 4.3 and 4.9. Ia determinants and Ig VH, CH, L-chain, and idiotypic determinants are not present on either component I or component II. The properties of component II are identical to that of a T cell growth factor produced by Con A-stimulated spleen cells. It is suggested that the H-2-restricted component I of AEF might be an MLR-activated responder T cell-derived Ia alloantigen receptor.

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Section: Fraction Numbermentioning

confidence: 99%

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

Delovitch¹,

Watson²,

Battistella³

et al. 1981

The Journal of Experimental Medicine

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“…of globin denatured in 6 M-guanidine hydrochloride and reduced with 5 mM-dithiothreitol was determined on a column (1.4 cm x 83cm) of Sephacryl S-200 equilibrated with 6 M-guanidine hydrochloride, 0.03 M-Tris/HCI, pH 7.0, and 0.5 mM-dithiothreitol as described by Fish et al (1969). The (Teale, 1959) was performed at pH 2.2 in the presence of 6.25 M-urea (Panyim & Chalkey, 1969;Poole et al, 1974). Globin was incubated overnight at room temperature in a solution of lOM-urea (deionized with Amberlite MB-1 resin), 5% (v/v) in acetic acid and 1% (v/v) in 2-mercaptoethanol.…”

Section: Molecular-weight Analysesmentioning

confidence: 99%

The quarternary structure of an unusual high-molecular-weight intracellular haemoglobin from the bivalve mollusc Barbatia reeveana

Grinich

Terwilliger

1980

Biochemical Journal

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The arcid clam Barbatia reeveana contains an intracellular haemoglobin with an unusual structure. First, compared with other intracellular haemoglobins, it is extremely large, with a mol.wt. of 430000 and an s20,w of 13.6S. A minor component (mol.wt. = 220000; s2O,w = 9.7S) is also present as a probable dissociation product of the major component. Secondly, this haemoglobin has an unusual subunit structure. It contains 1 mol of haem per 16000 g of protein, in common with most other haemoglobins. However, the smallest polypeptide that could be obtained after treatment with sodium dodecyl sulphate or 6 M-guanidine with reducing agent has a mol.wt. of 32000-37000. Digestion of the haemoglobin with the proteinase subtilisin produces both 57000-and 30000-mol.wt. aggregates that contain 1 mol of haem per 16000 g of protein and that can be dissociated into 16 500-mol.wt. polypeptides by treatment with sodium dodecyl sulphate. The intact polymer shows slight co-operativity (h = 1.7), lacks a Bohr effect between pH 7 and 8, and has a low oxygen affinity [P50 = 4.8 kPa (36mmHg) at 200C] relative to other haemoglobins. The 30000-mol.wt. aggregate obtained by digestion of the polymer binds oxygen reversibly with an affinity greater than that of the polymer, but with some co-operativity (h = 1.7). These results are consistent with the hypothesis that the subunits of this unusually large intracellular haemoglobin are 32000-mol.wt. polypeptides that in turn are composed of two covalently linked haem-containing oxygen-binding domains. This is the first report of an intracellular haemoglobin with such a structure.Most intracellular haemoglobins of both vertebrates and invertebrates consist of haem-containing polypeptides with mol

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“…5). This finding implies that each standard protein and polypeptide had been converted into a random-coil configuration by the urea, as is necessary for the use ofgels for molecular-weight estimations with urea (Tanford, 1968;Poole et al, 1974). A least-squares linear regression of molecular weight on KR based on the standard proteins with 95 % confidence limits is: Molecular weight = [-(3.28 ±0.16) x 104] + [(1.06±0.04) x 106] X KR, from which the molecular weights were estimated to be: peak C monomer band, 52000; peak D monomer band (polypeptide la), 57000; peak E monomer band (polypeptide Ib), 59000.…”

Section: U1508mentioning

confidence: 99%

“…Proteins of known molecular weight were converted into their thiol forms (Steinert, 1975) or S-carboxymethyl derivatives (Steinert & Rogers, 1973a) and were used to establish the standard line only if electrophoretically homogeneous on both gel systems. The use of globular proteins as standards for molecular-weight estimates of the fibrous keratin polypeptides is valid only 1975 if all disulphide bonds are broken and all species are converted into rigid structures by the sodium dodecyl sulphate (Reynolds & Tanford, 1970a) or are fully denatured to random coils by urea (Tanford, 1968;Poole et al, 1974). The same gel system with sodium dodecyl sulphate was used for preparative polyacrylamide-gel electrophoresis by using a Uniphor 7900 apparatus (LKB Instruments Inc., Rockville, Md., U.S.A.).…”

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confidence: 99%

The polypeptide composition of bovine epidermal α-keratin

Steinert

Idler

1975

Biochemical Journal

155

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1. The polypedtide chains that comprise the subunits of the tonofilaments, or th alpha-keratin component, of bovine epidermis were fractionated by combination of chromatography on DEAE-cellulose and preparative polyacrylamide-gel electrophoresis. 2. The seve polypeptide chains investigated had generalyy similar properties; all contained two residues per molecule of tryptophan and N-acetylserine was the common N-terminal amino acid residue. 3. On the basis of close similarities in alpha-helix content and amino acid composition, the polypeptide chains were classified into three distinct groups. Each group contained approximately one-third of the total polypeptides on a molar basis. The groups and designated polypeptides chain numbers were: group one, polypeptides 1a and 1b, which had moleculae weights of 58,000, contained about 25% alpha-helix, 86 glutamic acid and 8 cysteine residues per molecule, but which differed in net charge, extinction coefficients and tyrosine contents; group two, polypeptides 2, 3, and 4, which hadmolecular weights within thewithin the range of 52,00-56,000, contained about 48% alpha-helix, 54 glutamic acid and 6 cysteine residues per molecule, but which differed in extinction coefficients and tryosine contents; and group, polypeptides 5 and 6, which had molecular weights of 47000-48000, contained about 56% alpha-helix, 64 glutamic acid and 4 cysteine residues per molecule, but which differed in extinction coefficients and tyrosine contents...

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Analysis of polypeptide molecular weights by electrophoresis in urea

Cited by 42 publications

References 20 publications

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

The quarternary structure of an unusual high-molecular-weight intracellular haemoglobin from the bivalve mollusc Barbatia reeveana

The polypeptide composition of bovine epidermal α-keratin

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