Structural Studies of Ribonuclease. XX. Acrylonitrile. A Reagent for Blocking the Amino Groups of Lysine Residues in Ribonuclease*

Biochemistry ◽  
1966 ◽  
Vol 5 (1) ◽  
pp. 93-99 ◽  
Author(s):  
John P. Riehm ◽  
Harold A. Scheraga
Keyword(s):  
Structural Studies ◽  
Amino Groups ◽  
Lysine Residues

scholarly journals Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination

1981 ◽  
Vol 199 (2) ◽  
pp. 335-340 ◽  
Author(s):  
G C Dubois ◽  
E A Robinson ◽  
J K Inman ◽  
R N Perham ◽  
E Appella
Keyword(s):  
Amino Acid ◽  
Primary Structure ◽  
Half Life ◽  
Structural Studies ◽  
Amino Groups ◽  
Mouse Immunoglobulin ◽  
Lysine Residues ◽  
Rapid Removal ◽  
Proteins And Peptides

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

The Action of Thrombin on Modified Fibrinogen

1983 ◽  
Vol 49 (03) ◽  
pp. 208-213
Author(s):  
A J Osbahr
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Lysine Residue ◽  
Fibrinopeptide A ◽  
Amino Groups ◽  
Lysine Residues ◽  
Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway

2014 ◽  
Vol 458 (1) ◽  
pp. 119-130 ◽  
Author(s):  
Sai Chaitanya Chiliveri ◽  
Mandar V. Deshmukh
Keyword(s):  
Caenorhabditis Elegans ◽  
Rnai Pathway ◽  
Structural Studies ◽  
Lysine Residues

RDE-4 initiates the RNAi pathway in Caenorhabditis elegans. Structural studies on RDE-4 dsRBDs illustrate the indispensible role of dsRBD2 and linker in dsRNA recognition and Dicer activity. The structure-based mutagenesis identifies two tandem lysine residues that drive the dsRNA recognition.

scholarly journals Pitfalls in the use of carboxylic acid anhydrides for structural studies of nucleoprotein particles

1987 ◽  
Vol 241 (2) ◽  
pp. 621-623 ◽  
Author(s):  
M A Nieto ◽  
E Palacián
Keyword(s):  
Carboxylic Acid ◽  
Acetic Anhydride ◽  
Structural Studies ◽  
Amino Groups ◽  
Single Stranded Dna ◽  
Acid Anhydrides

During modification of protein amino groups with carboxylic acid anhydrides, these reagents cause a fall in pH, which can be prevented by addition of base. Although unmodified nucleosomal particles are not affected by the local transient changes in pH induced by the base (NaOH) added to prevent a fall in pH during modification, the nucleosomal particles modified by acetic anhydride are dissociated, with release of single-stranded DNA.

scholarly journals Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem II Membranes

2005 ◽  
Vol 37 (11) ◽  
pp. 737-742 ◽  
Author(s):  
Jin-Peng Gao ◽  
Zhen-Hua Yong ◽  
Feng Zhang ◽  
Kang-Cheng Ruan ◽  
Chun-He Xu ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Methyl Esters ◽  
Binding Activity ◽  
Water Soluble ◽  
Carboxyl Groups ◽  
Amino Groups ◽  
Charged Amino Acids ◽  
Lysine Residues ◽  
Glycine Methyl Ester ◽  
Positively Charged

Abstract To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with Nsuccinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4–10 and 10–14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3–4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

scholarly journals Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase

1977 ◽  
Vol 161 (1) ◽  
pp. 49-62 ◽  
Author(s):  
J M Lambert ◽  
R N Perham
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Ion Pair ◽  
Dimensional Structure ◽  
Ionic Interactions ◽  
Rabbit Muscle ◽  
Amino Groups ◽  
Muscle Enzyme ◽  
Lysine Residues

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these ‘surface’ amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.

scholarly journals Rearrangement of nucleosomal components by modification of histone amino groups. Structural role of lysine residues

Biochemistry ◽  
1984 ◽  
Vol 23 (19) ◽  
pp. 4280-4284 ◽  
Author(s):  
Juan Jordano ◽  
Francisco Montero ◽  
Enrique Palacian
Keyword(s):  
Amino Groups ◽  
Structural Role ◽  
Lysine Residues

REACTION OF FORMALDEHYDE AND THIOFORMALDEHYDE WITH MERCURIPAPAIN

1965 ◽  
Vol 43 (7) ◽  
pp. 1171-1177 ◽  
Author(s):  
W. A. Darlington ◽  
L. Keay
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Functional Groups ◽  
Tertiary Structure ◽  
Colorimetric Assay ◽  
Amino Groups ◽  
Full Activity ◽  
Amino Acid Side Chains ◽  
Lysine Residues ◽  
Secondary And Tertiary Structure

In a colorimetric assay with benzoyl-DL-arginine p-nitroanilide, acetylated and benzoylated papains retain full activity. Thus the ε-amino groups of the lysine residues are not required for enzyme activity. Intramolecular crosslinking of an enzyme could in theory stabilize secondary and tertiary structure and oppose denaturation. Thioformaldehyde is much more reactive with mercuripapain than formaldehyde, incorporating much more readily into the enzyme at equivalent concentrations. Incorporation is extensive, however, on reactive functional groups on the amino acid side chains, since acetylation decreased the incorporation markedly. In no case was there evidence of heat stabilization.

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