The application of chemical crosslinking for studies on cell membranes and the identification of surface reporters

Ji, Tae H.

doi:10.1016/0304-4157(79)90007-8

Cited by 192 publications

(65 citation statements)

References 171 publications

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“…Crosslinking was performed with cleavable crosslinkers as described (25)(26)(27). Purified proteasomes (500 g) were mixed with 100 M (final concentration) dithiobis(succinimidylpropionate) (DSP; Pierce) in a final volume of 1.25 ml 5 mM N-tris [hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS; Sigma).…”

Section: Methodsmentioning

confidence: 99%

Subunit arrangement in the human 20S proteasome

Köpp

Hendil

Dahlmann

et al. 1997

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

108

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In human 20S proteasomes two copies of each of seven different ␣-type and seven different ␤-type subunits are assembled to form a stack of four sevenmembered rings, giving the general structure ␣ 1-7 , ␤ 1-7 , ␤ 1-7 , ␣ 1-7 . By means of immunoelectron microscopy and chemical crosslinking of neighboring subunits, we have determined the positions of the individual subunits in the proteasome. The topography shows that for the trypsin-like, the chymotrypsin-like, and the postglutamyl cleaving activities, the pairs of ␤ type subunits, which are thought to form active sites, are nearest neighbors.

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Section: Methodsmentioning

confidence: 99%

Subunit arrangement in the human 20S proteasome

Köpp

Hendil

Dahlmann

et al. 1997

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

108

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“…Both receptors are functionally linked to adenylate cyclase [1,3-51 and have similar TSH binding characteristics [ 1,2] but no structural comparisons have yet been made. Consequently we have used affinity labelling [6] with 1251-labelled TSH to analyze both receptors, and our results suggest that they are structurally similar but not identical.…”

Section: Introductionmentioning

confidence: 94%

A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling

Buckland

Smith

1984

FEBS Letters

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TSH receptors from guinea pig thyroid and epididymal fat have been covalently crosslinked to 125I‐labelled TSH conjugated to N‐hydroxysuccinimidyl‐4‐azidobenzoate. Analysis by SDS—PAGE and autoradiography showed bands corresponding to TSH subunits (M r 14 000) and intact TSH (M r 28 000; subunits crosslinked) and two at higher M r separated by 14 000. The latter bands represented one or two subunits of TSH crosslinked to a subunit of the TSH receptor with M r 57 000 (fat) or 60 000 (thyroid). These M r values were reduced by trypsin treatment to 43 000 and 50 000, respectively. Analysis under non‐reducing conditions showed that both fat and thyroid receptors have a second disulphide linked subunit of M r 30 000.

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“…3). In some experiments, after the treatment with 2-mercaptoethanol, free -SH groups were irreversibly blocked by the addition of a molar excess of either iodoacetamide (not shown) or acrylonitrile, in order to prevent the disulphide exchange between the reduced bis-0-lactam XI* and proteins [30,31]. This procedure does not appear to be strictly necessary, since electrophoregrams of samples, either treated or untreated with these alkylating reagents, were similar (Fig.…”

Section: Labelling Of E Coli Membranes With Bis-p-lactam Antibioticsmentioning

confidence: 99%

Labelling and cross-linking of Escherichia coli penicillin-binding proteins with bis-beta-lactam antibiotics

1984

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We have synthesized a number of radioactively labelled bis-P-lactams, which are nearly symmetrical dimers of well-known p-lactam antibiotics and act as bifunctional specific cross-linking reagents for the penicillin-binding proteins of Escherichia coli envelopes. We have observed that some of our bis-/I-l.actam antibiotics cross-link two molecules of penicillin-binding protein 1 b or 1 c or 3. Furthermore our bis-/3-lactam antibiotics bind to E. coli proteins with higher affinities than the p-lactams from which they were derived. Therefore, a number of monomeric protein bands are consistently labelled with our bis-p-lactam antibiotics (190, 170, 145 and 124 kDa) as well as the previously described penicillin-binding proteins 1 a, 1 b, 1 c, 2, 3, 4, 5, 6, 7 and 8. We do not know yet the possible enzymic activities of the penicillin-binding proteins of 190 kDa, 170 kDa, 145 kDa and 125 kDa that were not described previously. We have also detected some protein bands moving very slowly, which appear to be dimers or cross-linking species of these new high-molecular-mass penicillin-binding proteins described above.It is widely accepted that a small number ofproteins located in the cell membrane catalyze the last stages of peptidoglycan biosynthesis in bacteria. Some of these proteins, which specifically interact with P-lactam antibiotics, known as penicillinbinding proteins (PBPs), can be selectively labelled and their activities blocked by p-lactam antibiotics (for a review, see [I]). Various PBPs can have either similar or different enzymic activities in peptidoglycan biosynthesis [2 -41. Mutations affecting a particular PBP, and the selective inhibition caused by P-lactams, produce well-defined alterations in the morphology of the cell [5,6]. Therefore, these findings stress the importance to cell morphology of either the localization of PBPs [7], or their enzymic activities [8], on the surface of the membrane. The localization of the PBPs is very relevant to the study of the critical processes of the cell cycle that only occur in separate places of its envelope, such as elongation of the sacculus, formation of the septum and cell lysis caused by /I-lactams [8-lo]. In order to study some aspects of the topography of the PBPs, we have synthesized a number of photoactivable P-lactams that labelled a 170-kDa PBP [l 11. Since then we have synthesized nearly symmetrical dimers of P-lactam antibiotics that act as bifunctional specific crosslinking reagents for the PBPs and serve as probes for the quaternary structure of these proteins. Bifunctional substrates have been used in protein purification [I2 -141. Also, a dimeric derivative of actinomycin D has been synthesized and used to study the binding nature of the antibiotic to DNA chains [15]. MATERIALS AND METHODS Microorganism and growth conditionsThe bacterial strain Escherichia coli W-7 (dupA, lysA) [16] was used in this work. Cells were grown in L medium [I71 supplemented with 10 pg/ml meso-diaminopimelic acid, at 37 "C under forced aeration.Abbreviation. PBP, pen...

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The application of chemical crosslinking for studies on cell membranes and the identification of surface reporters

Cited by 192 publications

References 171 publications

Subunit arrangement in the human 20S proteasome

Subunit arrangement in the human 20S proteasome

A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling

Labelling and cross-linking of Escherichia coli penicillin-binding proteins with bis-beta-lactam antibiotics

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