Methyl 4-mercaptobutyrimidate as a cleavable crosslinking reagent and its application to the Escherichia coli 30S ribosome

Rr, Traut; Bollen, Alex; Sun, Tao; Jw, Hershey; Sundberg, John P.; Lr, Pierce

doi:10.1021/bi00741a019

Cited by 272 publications

(153 citation statements)

References 12 publications

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“…The F(ab 0 ) 2 /GAH was thiolated with iminothiolane (Traut et al, 1973). Immunoliposomal doxorubicin was prepared as depicted in Figure 1, essentially in the manner as described by Suzuki et al (1995) with some modifications.…”

Section: Preparation Of Ild and Liposomal Dxr (Ld)mentioning

confidence: 99%

Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner

Hosokawa

Tagawa

Niki³

et al. 2003

Br J Cancer

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We have recently established a cancer-reactive human monoclonal antibody, GAH, with a positive ratio of over 90% against stomach cancer. GAH was formulated as polyethyleneglycol (PEG)-modified immunoliposomal doxorubicin (DXR) (ILD) and its efficacy was examined against gastrointestinal human cancers. In in vitro studies, a comparison of ILD with PEG-modified liposomal DXR (LD) demonstrated that ILD had dose-dependent cytotoxicity for GAH-reactive B37 cancer cells, but not LD. In concordance with this result, microscopic observations showed that ILD was bound to and GAH-dependently internalised by B37 cells. In in vivo studies, ILD exhibited significantly greater antitumour activity on cancer xenograft models than LD or free DXR. The relation between efficacy and antigen density was examined on 10 xenograft models bearing cancer cells with varying GAH reactivity. Immunoliposomal doxorubicin therapeutic activity correlated with the antigen density, with a minimum number being required. Also, ILD revealed strong antitumour activity on cancers with low sensitivity to DXR or LD, suggesting that ILD overcame the DXR resistance of antigen-positive cancer cells. Thus, these results show that GAH endows liposomes with targeting activity, resulting in strong efficacy against gastrointestinal cancers.

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Section: Preparation Of Ild and Liposomal Dxr (Ld)mentioning

confidence: 99%

Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner

Hosokawa

Tagawa

Niki³

et al. 2003

Br J Cancer

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“…Under identical conditions, dimethyl 3,3'-dithiobispropionimidate consistently produced a greater degree of crosslinking than the irreversible reagent of corresponding length, dimethyl suberimidate, for which we have no direct explanation (see also [31]). Reversible crosslinking with two other reagents which form disulfide-containing crosslinks of similar length, namely dithiobis(succinimidy1 propionate) and 'methyl 4-mercaptobutyrimidate' (= 2-iminothiolane [32]) according to [33], leads to nearly identical results. Concentrations in excess of 5 mM of any of these three reversible reagents lead to the formation of aggregates of very high M,, which do not penetrate the dodecylsulphate gels.…”

Section: Which Subunits Are Crosslinked?mentioning

confidence: 99%

The Quaternary Structure of Bovine α‐Crystallin

Siezen

Bindels

Hoenders

1980

European Journal of Biochemistry

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Reaction of calf a-crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0-8.5 and room temperature leads to inter-subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from Cg to CI2). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical a-crystallin molecule are in quasiequivalent positions.Molecular weight analysis by dodecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the a-crystallin molecule.Not all of the subunits can form inter-chain crosslinks, as a limiting value of about 600/, of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.In recent years we have investigated various aspects of the quaternary structure of bovine a-crystallin, a major structural lens protein, with the purpose of gaining insight into its function and the developments which take place on a molecular level upon aging of this multisubunit protein [ l -61.The a-crystallin molecule has a quasi-spherical shape with a diameter of 16nm [1,2], and it is composed of about 30 A subunits (19830 M, [7]) and about 10 B subunits (20070 Mr [S]), which show nearly 60 0/, sequence homology [8]. Limited information is available concerning the quaternary structure of the protein. Since a-crystallin molecules are very large and microheterogeneous [l] it is impossible to obtain such information by X-ray crystallography. Attempts to obtain the fine structure of the protein by electron microscopy have also been unsuccessful [I, 61. Therefore, one has to rely upon indirect methods, such as small-angle X-ray scattering [2], chemical modification of specific side-chain groups [5], partial digestion with proteolytic enzymes [3,4], and partial dissociation [9]. Different orientations of A subunits are distinguished according to accessibility to thiol reagents [ 5 ] and proteolytic enzymes [3,4], whereas Previous papers in this series appeared earlier in thih journalsimilar positions for all B subunits are inferred from their susceptibility to proteolysis in vitro at the carboxyl-terminal end [4]. We have suggested a model for the quaternary structure, which is based on a core of A subunits surrounded by two different layers of A and B subunits [9].Crosslinking with bifunctional reagents can provide information about the number and arrangement of subunits in oligomeric proteins, as well as the distribution of certain amino acid side-chains near the intersubunit contact areas and the symmetry of the olig...

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“…Many analytical techniques require the regeneration of the individual macromolecules. Cleavable crosslinking reagents that allow such regeneration include disulfides (11), glycols (12), and bisimidoesters (13,14).…”

mentioning

confidence: 99%

High yield photoreagents for protein crosslinking and affinity labeling

Jelenc

Cantor

Simon

1978

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

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4-Nitrophenyl ethers are proposed as new hig-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with &mines at pH 8 upon irradiation with 368nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free aicobol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the pnitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy4nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at 'y-cysteine F9 stoichiometrically. Subseuent ultraviolet irradiation yielded a y-y crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high afrinity conformation and that all cooperativity is lost.Crosslinking and affinity labeling of biological macromolecules have been used with increasing frequency for structural and functional studies. The first reagents employed in these techniques were derived from common acylating and alkylating reagents (1, 2). The fate of such reagents in solutions of bio-

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Methyl 4-mercaptobutyrimidate as a cleavable crosslinking reagent and its application to the Escherichia coli 30S ribosome

Cited by 272 publications

References 12 publications

Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner

Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner

The Quaternary Structure of Bovine α‐Crystallin

High yield photoreagents for protein crosslinking and affinity labeling

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