Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

Dottavio-Martin, Diane; Ravel, Joanne M.

doi:10.1016/0003-2697(78)90706-6

Cited by 315 publications

(104 citation statements)

References 7 publications

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“…Purification of Peptides Generated by Proteasomal Degradation of Casein-Peptides were first generated by digestion of ␤-[ 14 C]casein by proteasomes as described by Kisselev et al (3), and then they were purified from undigested substrate and proteasomes by successive size exclusion HPLC (S.E.-HPLC) and RP-HPLC, as described below. The ␤-[ 14 C]casein was prepared by reductive methylation as described by Dottavio-Martin and Ravel (31) and had a specific activity of 3.9 ϫ 10 7 cpm/mg. This procedure radiolabeled the casein exclusively on lysine residues; the 95% of other amino acids (as calculated from the frequency of lysine residues in bovine ␤-casein) remained unmodified.…”

Section: Methodsmentioning

confidence: 99%

Pathway for Degradation of Peptides Generated by Proteasomes

Šarić¹,

Graef²,

Goldberg

2004

Journal of Biological Chemistry

174

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The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from ␤-[ 14 C]casein and studied in HeLa cell extracts the degradation of the 9 -17 residue fraction and also of synthetic deca-and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14 C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30 -50%. Pure TOP failed to degrade proteasome products 18 -24 residues long but degraded the 9 -17 residue fraction to peptides of 6 -9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9 -17 residue proteasome products were also converted to peptides of 6 -9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9 -17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9 -17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6 -9 residue fragments are further digested to amino acids by aminopeptidases.

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Section: Methodsmentioning

confidence: 99%

Pathway for Degradation of Peptides Generated by Proteasomes

Šarić¹,

Graef²,

Goldberg

2004

Journal of Biological Chemistry

174

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“…To protect free amino groups, TT or DT was treated with a 2.4 weight excess of succinic anhydride, while manually maintaining a pH of 7 (15). Reduction of TT (1 mg/ml) with sodium cyanoborohydride (CBH; 1 mg/ml) was performed under gentle stirring at pH 7 (0.04 M sodium phosphate) and 37°C for 1 h as described (16) Assays. The exposed free thiols of the vaccines were quantitated by Ellman's reagent, 5,5'-dithio-bis(2-nitrobenzoic acid), under nondenaturing conditions (17).…”

Section: Methodsmentioning

confidence: 99%

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Schwendeman

Costantino

Gupta

et al. 1995

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

103

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The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37°C and elevated humidity-i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

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“…Apocytochrome c was radiolabelled by reductive alkylation with [14C]formaldehyde (New England Nuclear, specific activity 10 Ci/mol) and sodium cyanoborohydride [16][17][18]. By this procedure the net charge of the protein is not altered.…”

Section: Methodsmentioning

confidence: 99%

Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study

Pilon¹,

Jordi²,

Kruijff³

et al. 1987

Biochimica et Biophysica Acta (BBA) - Biomembranes

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(1) The interaction of apocytochrome c with different molecular species of phosphatidyiserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 raN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidyiserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidyiserine is indicated by the low K~ of 0.017/zM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using ~4C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interracial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 .AZ/molecule in the absence of lipid to 700-900 ,A2/molecule after penetration of dioleoyiphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidyiserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.

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Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

Cited by 315 publications

References 7 publications

Pathway for Degradation of Peptides Generated by Proteasomes

Pathway for Degradation of Peptides Generated by Proteasomes

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study

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