[50] Protein labeling by reductive alkylation

Jentoft, Neil; Dearborn, Dorr Gellatly

doi:10.1016/s0076-6879(83)91052-2

Cited by 217 publications

(143 citation statements)

References 37 publications

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“…If this interpretation holds, reductive methylation with formaldehyde and sodium cyanoborohydride should greatly reduce the complexity of the polyamine population. This reaction methylates primary as well as secondary amines to yield exclusively tertiary amino groups (14). By conversion of all of the partially N-methylated polyamine species to the completely N-methylated derivatives (Fig.…”

Section: Resultsmentioning

confidence: 99%

Species-specific polyamines from diatoms control silica morphology

Kröger

Deutzmann²,

Bergsdorf³

et al. 2000

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

636

629

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Biomineralizing organisms use organic molecules to generate species-specific mineral patterns. Here, we describe the chemical structure of long-chain polyamines (up to 20 repeated units), which represent the main organic constituent of diatom biosilica. These substances are the longest polyamine chains found in nature and induce rapid silica precipitation from a silicic acid solution. Each diatom is equipped with a species-specific set of polyamines and silica-precipitating proteins, which are termed silaffins. Different morphologies of precipitating silica can be generated by polyamines of different chain lengths as well as by a synergistic action of long-chain polyamines and silaffins.T he biological formation of inorganic structures, termed biominerals, is a widespread phenomenon in nature (1). Among the most famous examples are unicellular algaediatoms-that possess a cell wall composed of silica and organic molecules or macromolecules (2). Most interestingly, diatom biosilica displays a dazzling variety of species-specific silica patterns that are structured on a nanometer-to-micrometer scale (3-6). Elucidating the mechanism that controls production of nanostructured biosilica is a fascinating biochemical problem and, in addition, is of great interest in materials chemistry. Biomimetic approaches are believed to allow the production of advanced materials at ambient temperature and with high precision, which are expected to exhibit superior properties in a wide range of applications (7,8). Reaching this goal, however, requires a detailed knowledge of the organic molecules that govern silica biomineralization.Recently, cationic polypeptides (called silaffins) isolated from purified cell walls of the diatom Cylindrotheca fusiformis were shown to generate networks of silica nanospheres within seconds when added to a solution of silicic acid (9). The silaffins are tightly associated with the biosilica so that they can only be solubilized after dissolution of the cell wall in hydrogen fluoride (HF). Silaffin-1 contains a previously undescribed type of protein modification, a polyamine consisting of 6-11 repeats of the N-methyl-propylamine unit (a mass increment of 71 Da per repeat) covalently attached to specific lysine residues (9). It is probably this structural element that accelerates silicic acid polymerization and promotes production of nanosphere networks. Previously, ultrastructural work on cell wall biogenesis in a number of diatoms led to the conclusion that silica appears to be deposited in different forms during valve morphogenesis. Especially evident was the participation of silica spheres ranging up to 100 nm in diameter (4, 10). If silaffins are indeed involved in the process of pattern formation that creates the amazingly rich variety of diatom shell silica structures, then different diatom species are expected to contain different types of silaffins or silaffin-like molecules. We therefore analyzed the HFextractable organic cell wall components from a wide range of diatom species. Surprisingly, th...

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Section: Resultsmentioning

confidence: 99%

Species-specific polyamines from diatoms control silica morphology

Kröger

Deutzmann²,

Bergsdorf³

et al. 2000

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

636

629

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“…The reaction with formaldehyde and NaCNBH 3 is specific for primary amino groups; for peptides or proteins only, the N terminus and lysine residues are converted to dimethyl amino groups (7). In peptides, this conversion results in a mass increase of 28 Da.…”

Section: Discussionmentioning

confidence: 99%

“…The vaccine quality depends to a considerable extent upon the chemical modifications caused by the formaldehyde treatment (1,5,6). Formaldehyde is also used for isotope-labeling of proteins (7)(8)(9), for studying protein-protein interactions, e.g. histone organization in nucleosomes (10 -12), and for fixation of cells and tissues (13).…”

mentioning

confidence: 99%

Identification of Formaldehyde-induced Modifications in Proteins

Metz

Kersten²,

Hoogerhout³

et al. 2004

Journal of Biological Chemistry

525

307

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Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatographymass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.Aldehydes, such as formaldehyde and glutaraldehyde are widely employed reagents in the biochemical, biomedical, and pharmaceutical fields. Formaldehyde, for example, is applied to inactivate toxins and viruses for the production of vaccines, such as diphtheria, tetanus toxoid, hepatitis A, anthrax, and inactivated polio vaccine, and to stabilize recombinant pertussis toxin (1-4). The vaccine quality depends to a considerable extent upon the chemical modifications caused by the formaldehyde treatment (1, 5, 6). Formaldehyde is also used for isotope-labeling of proteins (7-9), for studying protein-protein interactions, e.g. histone organization in nucleosomes (10 -12), and for fixation of cells and tissues (13). Glutaraldehyde is utilized for the preparation of bioprostheses such as heart valves and vascular grafts (14 -16) and for conjugation of enzymes to carrier systems (17). These examples demonstrate the wide range of roles of aldehydes in the biomedical field. Besides the use of aldehydes in diverse applications, they can also destroy important sites of pro...

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“…Hsc70 and peptides were trace-labeled by reductive alkylation using NaCNBH 3 and [ 14 C]formaldehyde as described by Jentoft and Dearborn (23).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Auxilin with the Molecular Chaperone, Hsc70

Jiang

Greener

Barouch

et al. 1997

Journal of Biological Chemistry

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We have studied the direct interaction of the constitutive isoform of Hsp70 (Hsc70) with the DnaJ homolog, auxilin, a cofactor that binds to clathrin-coated vesicles and is required for their uncoating by Hsc70. Auxilin caused a 5-fold increase in Hsc70 ATPase activity and a corresponding increase in steady-state levels of bound ADP; the dissociation constant for this effect was 0.6 M. Auxilin also induced polymerization of Hsc70 and bound to the resulting polymer at a 1:1 molar ratio; here too the dissociation constant was 0.6 M. Both this binding and polymerization required ATP; the Hsc70 depolymerized with a 4-min half-life when ATP was completely hydrolyzed to ADP. Although auxilin induces polymerization stoichiometrically and other DnaJ homologs induce polymerization catalytically, these data show that auxilin is similar to other DnaJ homologs in its ability to activate the Hsc70 ATPase activity, to polymerize Hsc70, and in the nucleotide dependence of this polymerization. Furthermore, the 70-amino acid J-domain of auxilin polymerized Hsc70 with the same nucleotide dependence as intact auxilin. Therefore, although only auxilin and not other DnaJ homologs support uncoating, our data suggest that various DnaJ homologs share a common mechanism of interaction with Hsc70, perhaps because their J-domains interact similarly with Hsc70.

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[50] Protein labeling by reductive alkylation

Cited by 217 publications

References 37 publications

Species-specific polyamines from diatoms control silica morphology

Species-specific polyamines from diatoms control silica morphology

Identification of Formaldehyde-induced Modifications in Proteins

Interaction of Auxilin with the Molecular Chaperone, Hsc70

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