An analyzer and monitor for rapid microscale peptide separations

Nika, Heinz; Hultin, T.

doi:10.1016/0003-2697(79)90723-1

Cited by 10 publications

(11 citation statements)

References 18 publications

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“…Under the experimental conditions described, approximately 20-40 % of the ribosomes were induced to disulfide interaction between proteins L6 and L29 [8,9]. When analyzed by two-dimensional gradient gel electrophoresis ( Fig.…”

Section: Cyanogen Bromide Susceptibility Of Electrophoretic L6-l29 Prmentioning

confidence: 96%

“…The disulfide-linked L6-L29 complex was isolated from the ribosomal extracts by preparative elec-trophoresis in acid/urea gels at pH4.3 as described [9] but without buffer circulation between the electrode compartments. Methionine oxidation was prevented by pre-electrophoresis of the gels [21] using the following extensively modified scheme.…”

Section: Non-oxidizing Preparative Gel Electrophoresismentioning

confidence: 99%

“…Proteins L6 and L29 prepared by electrophoresis in pretreated or non-pretreated [9] gels were incubated for 24h (20 "C) with 25 "/, (v/v) thioglycolic acid (cf. [23,30]), or for 15 min (20 "C) with an excess of indolyl-3-propionic acid (100equivalents) in acetic acid/12M HCI (2:l) [31] for reduction of oxidized methionine.…”

Section: Reversal Of Methioniiie Oxidationmentioning

confidence: 99%

“…Previous experiments using limited chymotryptic cleavage of the isolated L6-L29 complex in the presence of SDS [I41 showed that a structurally shielded segment of L6, comprising about 80 amino acids, is involved in the disulfide cross-linking [9,10]. The reversibly bound L29 was not attacked under these conditions.…”

mentioning

confidence: 96%

“…The L6-L29 interaction is enhanced by incubating the ribosomes at increased ionic strength or in the presence of intercalating heteroaromatics [8]. The treatment has been shown to produce a reversible unmasking of structurally shielded parts of protein L6 [12,13].Previous experiments using limited chymotryptic cleavage of the isolated L6-L29 complex in the presence of SDS [I41 showed that a structurally shielded segment of L6, comprising about 80 amino acids, is involved in the disulfide cross-linking [9,10]. The reversibly bound L29 was not attacked under these conditions.…”

mentioning

confidence: 99%

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Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29

Nika¹,

Hultin²

1984

European Journal of Biochemistry

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Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high-efficiency gel pre-electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross-linked CNBr fragments of L6 and L29 could be isolated in high yield ( M , approx. 13 000 and 7000, respectively). After ['4C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two-dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic.The spatial arrangement of the various components in ribosomal subparticles and their interfaces is of basic interest for an understanding of ribosomal functions. Protein L6 in the large subparticle of mammalian ribosomes occupies a central position in the ribosomal structure. It is closely adjacent to 28-S rRNA [1,2], but also to the 18-S rRNA across the ribosomal interface [I]. In addition, it is one of the proteins implicated in the ribosomal domains which provide specific binding sites for 5-S rRNA, 5.8-S rRNA, initiator and elongator rRNAs [3 -71. An involvement of L6 in the binding of aminoacyl-tRNA to the ribosomal A and P sites has been suggested [6].Under gentle conditions L6 is reversibly cross-linked by disulfide interaction to the neighboring protein L29 [8 -101. This protein is probably located close to the peptidyltransferase center at the ribosomal A-site [Ill. The L6-L29 interaction is enhanced by incubating the ribosomes at increased ionic strength or in the presence of intercalating heteroaromatics [8]. The treatment has been shown to produce a reversible unmasking of structurally shielded parts of protein L6 [12,13].Previous experiments using limited chymotryptic cleavage of the isolated L6-L29 complex in the presence of SDS [I41 showed that a structurally shielded segment of L6, comprising about 80 amino acids, is involved in the disulfide cross-linking [9,10]. The reversibly bound L29 was not attacked under these conditions.The efficiency of the interaction in situ between proteins L6 and L29 makes this system uniquely favorable in attempts to identify and characterize the contact regions between individual ribosomal proteins. The aim of the present study was to isolate a disulfide-linked peptide pair of relatively small size representing the contact regions of the two proteins. Attempts to use cyanogen bromide for this purpose were only moderately successful, until an extensive scheme was developed for the effective prevention of methionine oxidati...

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Section: Cyanogen Bromide Susceptibility Of Electrophoretic L6-l29 Prmentioning

confidence: 96%

Section: Non-oxidizing Preparative Gel Electrophoresismentioning

confidence: 99%