A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel

Lambin, P.; Rochu, Daniel; Fine, J.M.

doi:10.1016/0003-2697(76)90239-6

Cited by 157 publications

(58 citation statements)

References 13 publications

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“…components containing glycan or a high content of some amino acids such as proline (35,36). In contrast, ESI-MS analysis of Riftia Hbs provides a complete and self-consistent description of their constituent subunits and polypeptide chains, together with mass measurements with an unrivaled accuracy (0.01%).…”

Section: Polypeptide Chains Composition Determined By Esi-ms-mentioning

confidence: 99%

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

Zal

Lallier

Green³

et al. 1996

Journal of Biological Chemistry

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The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (ϳ3500 kDa) and V2 (ϳ400 kDa), and one in the coelomic cavity, C1 (ϳ400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at ϳ16, 23-27, and 30 kDa, corresponding to ( Several models have been proposed for the quaternary structure of annelid hexagonal bilayer hemoglobins (Hbs) 1 (1-5) and vestimentifera Hbs (6) but no definitive agreement exists. However, it is well established that HBL Hbs have a hexagonal symmetry and consist of two types of chains, globin-chains (ϳ17,000 Da) accounting for approximately 70% of total mass and heme-deficient linker chains (ϳ24,000 -32,000 Da) necessary for assemblage into the HBL structure (7). A detailed understanding of this molecular structure requires the knowledge of: (i) the molecular mass of the native molecule; (ii) the number and proportions of all constitutive polypeptide chains; (iii) the number and relation between subunits; and (iv) the determination of linker proportions. In a companion study (8) we have confirmed that Riftia pachyptila (Jones), a vestimentiferan living around deep-sea hydrothermal vents (9 -11), possesses three hemoglobins, two of them dissolved in the vascular blood (V1 and V2), and one in the coelomic fluid (C1). Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi-angle laser light scattering (MALLS). Both methods yielded approximately the same molecular weights with mass significantly higher than the literature data for V1. V1, V2, and C1 had M r of 3396 Ϯ 540 ϫ 10 3

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Section: Polypeptide Chains Composition Determined By Esi-ms-mentioning

confidence: 99%

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

Zal

Lallier

Green³

et al. 1996

Journal of Biological Chemistry

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“…SDS-PAGE was done at pH 8.8 on linear gradient gels (7.5-15%, w/v) by the method of Lambin (1976) in the buffer system of Laemmli (1970). Staining of gels for haemoproteins was performed by the method of Thomas et al (1976), and protein was stained with Coomassie Brilliant Blue R-250 as described by Weber & Osborn (1975).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer

Long

Anthony

1991

Microbiology

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This paper describes periplasmic c-type cytochromes from two strains of Paracoccus denifr@cans NCIB 8944 grown in heterotrophic or methylotrophic conditions. It is shown that the functions of two monomeric, monohaem cytochromes induced during growth on methanol have been wrongly designated in previous work. The CO-reactive cytochrome c553 (30 kDa) is not the electron acceptor for methanol dehydrogenase; this is shown to be the role of the cytochrome C552 (22 kDa). The monomeric 45 kDa cytochrome induced in conditions of oxygen insufficiency is a dihaem c-type cytochrome and does not contain haem b as previously assumed. In addition to these cytochromes, the Oxford strain of NCIB 8944 contains two cytochrome c complexes. One of these (150 kDa), produced in relatively small amounts, consists of a non-haem protein plus four haemoproteins (28, 33, 41 and 47 kDa). The second complex is a novel dimeric multi-haem cytochrome c (46 kDa) which constitutes about 25% of the periplasmic c-type cytochrome. It reacts with CO and has no methionine ligands. One subunit (16 kDa) has two low-spin haems; the larger subunit (30 kDa) has three haems which have low-spin characteristics in the oxidized state and are high-spin in the reduced state. The subunits were readily separated at pH 12 and could be subsequently reconstituted into a complex indistinguishable from the original. The 30 kDa subunit was denatured on prolonged exposure to high pH, which also converted it to a low-spin cytochrome. No function could be designated for these novel c-type cytochrome complexes.

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“…Electrophoresis of the photolabeled samples was performed according to the method of Laemmli (12) modified as described (7). The molecular weights of the receptor polypeptides were determined according to the method of Lambin et al (13). The effectiveness of a p-adrenergic receptor photoaffinity ligand is dependent in part on high affinity for the receptor and in part on the absence of nonspecific effects on the inhibition of adenylate cyclase activity.…”

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confidence: 99%

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

Rashidbaigi¹,

Ruoho²,

Green³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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The /3-adrenergic antagonist ['"I]iodoazidobenzylpindolol (['"I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc-cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc-cells with 1.0 jLM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18-to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a >90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc-cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the ['5I]IABP-labeled receptors after either short-or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the ,B-adrenergic receptor in lymphoma cells. The wild-type S49 lymphoma cell line (WT) and its coupling protein-deficient variant line (cyc-) have proven to be an invaluable model system for the study of the mechanism of hormonal regulation (1) and desensitization of adenylate cyclase (2-4). We have demonstrated that short-term (0-60 min) pretreatment of both WT and cyc-cells with epinephrine leads to similar patterns of desensitization of adenylate cyclase in both cell lines (2, 4). Long-term pretreatment of the lymphoma cells with (3-adrenergic agonists produced a greater than 90% depletion of epinephrine-saimulated adenylate cyclase and /3-adrenergic receptor antagonist binding in WT membranes, whereas decreases in these parameters in cyc-membranes were no greater than after a 60-min pretreatment (2). Thus it appeared that while the lack of the subunit of the adenylate cyclase G/ F coupling protein that is ADP-ribosylated by cholera toxin had no effect on short-term desensitization in cyc-, G/F may have been required for down-regulation (2). The absence of a change in, or need for, the functional coupling protein complex during the early stage(s) of desensitization, combined with the near total loss of receptor stimulation of adenylate cyclase and ,B-antagonist binding with prolonged agonist pretreatment, strongly suggest that the receptor is either altered or occluded during agonist pretreatment.Direct analysis of the /3-adrenergic receptor structure either with or without desensitization has proven more difficult than MATERIALS AND METHODS Materials.[125I]IABP was synthesized at the same specific activity as carrier-free Na'25I (New England Nuclear) by the method described for the chemical synthesis (6). The source of other chemicals has been described (2, 7).Cell Culture and Preparation of Membranes. WT and cyccells were grown in spinner culture as previously described (2). Cell inc...

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A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel

Cited by 157 publications

References 13 publications

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

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