scholarly journals Studies on the biotin-binding site of avidin. Lysine residues involved in the active site

1987 ◽  
Vol 242 (3) ◽  
pp. 923-926 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Active Site ◽  
Amino Acid Analysis ◽  
Egg White ◽  
Reversed Phase ◽  
Complete Loss ◽  
Tryptic Peptides ◽  
Lysine Residues ◽  
Biotin Binding

Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

scholarly journals Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site

1988 ◽  
Vol 256 (1) ◽  
pp. 279-282 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Reversed Phase ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Biotin Binding ◽  
Peptide Fractions ◽  
Specific Reagent

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

scholarly journals Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site

1988 ◽  
Vol 250 (1) ◽  
pp. 291-294 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Egg White ◽  
Binding Activity ◽  
Complete Loss ◽  
Tryptophan Residue ◽  
Tryptophan Residues ◽  
Biotin Binding ◽  
Specific Reagent

Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

MODIFICATION OF GLUTAMIC AND ASPARTIC ACID RESIDUES OF PLASMINOGEN INHIBITS ITS ABILITY TO FORM AN ACTIVE PLASMINOGEN-STREPTOKINASE COMPLEX

1987 ◽  
Author(s):  
D S Holloway ◽  
L Summaria ◽  
R C Wohl ◽  
J A Caprini
Keyword(s):  
Amino Acid ◽  
Reaction Time ◽  
Glutamic Acid ◽  
Binding Site ◽  
Plasminogen Activator ◽  
Aspartic Acid ◽  
Amino Acid Analysis ◽  
Molar Excess ◽  
Maximum Inhibition ◽  
Time Courses

Plasminogen binds to streptokinase in a 1:1 molar complex that has activity as a plasminogen activator. This function of plasminogen, as a cofactor for streptokinase conversion of plasminogen to plasmin, was studied after treatment of Glu-, Lys-, and Mini-plasminogens with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). Amino acid analysis showed that both aspartic and glutamic acid residues were modified by EDC. Activity of the complex formed between streptokinase and the modified plasminogen was measured using the cfhromogenic substrate H-D-Val-Leu-Lys-pNA. Plasminogen, 2.8 uM, was incubated with 40 mM EDC in 50 mM MES buffer, pH 6.0, at 25°C. At various times while reacting plasminogen with the EDC, aliquots were removed for assay. Plasminogen function was assayed by mixing with a slight molar excess of streptokinase for 1 min at 37 C, followed by reaction with 0.1 M substrate, and absorbancy monitored at 405 nm. Modifications of 20% of the glutamic and aspartic acid residues occurred after treatment of plasminogen with EDC. This resulted in 80 to 90% inhibition of activation in all three types of plasminogen. Glu- and Lys-plasminogens reacted more quickly with the EDC than did Mini-plasminogen, with 50% inhibition occurring after 16 ± 5, 16 ± 4, and 67 ± 13 min reaction time with EDC for Glu-, Lys-, and Mini-plasminogens, respectively. Maximum inhibition of activation occurred within 1 hr reaction with EDC for Glu- and Lys-plasminogens but required 2.5 hr for Mini-plasminogen. The time courses for activation inhibition and the modification of the glutamic and aspartic acids of treated Mini-plasminogen were compared. A significant decrease in activation occurred (52%) concomitant with modification of only one or two glutamic acids, followed on further reaction with EDC by more loss of activatability as more glutamic and aspartic acids were modified. The inability of plasminogen to form an active plasminogen-streptokinase complex after modification with EDC indicates that glutamic and aspartic acid residues are involved in the binding site of plasminogen for streptokinase.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Amino acid analysis by reversed-phase high-performance liquid chromatography

1987 ◽  
Vol 407 ◽  
pp. 273-279 ◽  
Author(s):  
Kazuo Iwaki ◽  
Noriyuki Nimura ◽  
Yayoi Hiraga ◽  
Toshio Kinoshita ◽  
Kazuyoshi Takeda ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
High Performance ◽  
Reversed Phase
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