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 Identification of ‘buried’ lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors

1981 ◽  
Vol 193 (2) ◽  
pp. 525-539 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Catalytic Activity ◽  
Quaternary Structure ◽  
Hydrophobic Interactions ◽  
Chloramphenicol Acetyltransferase ◽  
Ion Pair ◽  
Lysine Residue ◽  
Type I ◽  
Amino Groups ◽  
Type Iii ◽  
Lysine Residues

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or “buried” amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is “buried” in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.

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.

Fibrinogen Bern I: A Hereditary Fibrinogen Variant With Defective Conformational Stabilization By Calcium Ions

1981 ◽  
Author(s):  
C Rupp ◽  
C Kuyas ◽  
A Häberli ◽  
M Furlan ◽  
E A Beck
Keyword(s):  
Calcium Ions ◽  
Gel Electrophoresis ◽  
Calcium Binding ◽  
Negative Charge ◽  
Fibrinopeptide A ◽  
Isoelectric Focussing ◽  
High Calcium ◽  
Two Generations ◽  
Polypeptide Chains ◽  
Conformational Stabilization

Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.

scholarly journals Horse liver alcohol dehydrogenase. A study of the essential lysine residue

1975 ◽  
Vol 149 (3) ◽  
pp. 627-635 ◽  
Author(s):  
S S Chen ◽  
P C Engel
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Constant ◽  
Lysine Residue ◽  
Horse Liver Alcohol Dehydrogenase ◽  
First Order ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Lysine Residues ◽  
Modified Enzyme ◽  
Covalent Complex

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10°C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.

Diagonal Chromatography for the Selective Purification of Tyrosyl Peptides

1974 ◽  
Vol 52 (11) ◽  
pp. 1013-1017 ◽  
Author(s):  
William H. Cruickshank ◽  
Barry L. Malchy ◽  
Harvey Kaplan
Keyword(s):  
Acetic Acid ◽  
Stationary Phase ◽  
Enzymatic Digestion ◽  
Acid Water ◽  
Chromatographic Purification ◽  
Amino Groups ◽  
Chromatographic System ◽  
Original Direction ◽  
Acetic Acid Water ◽  
Citraconic Anhydride

Thiolysis of an O-dinitrophenyl-tyrosyl peptide results in an increased solubility in the stationary phase of a n-butanol – acetic acid – water – pyridine (15:3:12:10) (BAWP) paper chromatographic system. It is shown that this property can be used to form the basis of a diagonal paper chromatographic purification of tyrosyl peptides from enzymatic digests of proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is subjected to enzymatic digestion, applied to a strip of Whatman 3 MM paper, thiolyzed with 5% 2-mercaptoethanol in acetone, and subjected to chromatography using BAWP as solvent. A guide strip is removed, thiolyzed with 5% 2-mercaptoethanol in 25% pyridine, and resubjected to chromatography in BAWP at right angles to the original direction of chromatography. The tyrosyl peptides are displaced off the diagonal towards the origin. The off-diagonal peptides are isolated from the original chromatogram by thiolysis and chromatography using the diagonal chromatogram to locate the positions of the dinitrophenyl-tyrosyl peptides.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of β-galactosidase

2004 ◽  
Vol 82 (2) ◽  
pp. 275-284 ◽  
Author(s):  
Julia Xu ◽  
Mary A.A McRae ◽  
Scott Harron ◽  
Beatrice Rob ◽  
Reuben E Huber
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Sodium Potassium ◽  
The Difference ◽  
Potassium Binding ◽  
Covalent Intermediate ◽  
Catalytic Effects

The interactions between Na+ (and K+) and Asp-201 of β-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type β-galactosidase (0.36 ± 0.09 mM) was about 10× lower than for K+ (3.9 ± 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-β-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-β-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.Key words: β-galactosidase, sodium, potassium, binding, aspartic acid.

scholarly journals Modification of pig M4 lactate dehydrogenase by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue

1975 ◽  
Vol 149 (1) ◽  
pp. 107-113 ◽  
Author(s):  
S S Chen ◽  
P C Engel
Keyword(s):  
Lactate Dehydrogenase ◽  
Equilibrium Mixture ◽  
Lysine Residue ◽  
Original Activity ◽  
Complete Inactivation ◽  
Lysine Modification ◽  
Covalently Bonded ◽  
Lysine Residues ◽  
Enzyme Protection ◽  
Schiff Base Formation

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.

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