Limited proteolysis and active-site labeling studies of soybean lipoxygenase 1

Biochemistry ◽  
1992 ◽  
Vol 31 (33) ◽  
pp. 7700-7706 ◽  
Author(s):  
Sampath Ramachandran ◽  
Richard T. Carroll ◽  
William R. Dunham ◽  
Max O. Funk
Keyword(s):  
Active Site ◽  
Limited Proteolysis ◽  
Soybean Lipoxygenase

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 105-111 ◽  
Author(s):  
R. D. Moreno ◽  
M. Hoshi ◽  
C. Barros
Keyword(s):  
Zona Pellucida ◽  
Active Site ◽  
Acrosome Reaction ◽  
Penetration Rate ◽  
Limited Proteolysis ◽  
Sulphated Polysaccharides ◽  
Functional Interactions ◽  
Sperm Binding ◽  
Sperm Penetration ◽  
Sperm Acrosome Reaction

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1†

Biochemistry ◽  
2004 ◽  
Vol 43 (41) ◽  
pp. 13063-13071 ◽  
Author(s):  
Viola C. Ruddat ◽  
Rakesh Mogul ◽  
Ilya Chorny ◽  
Cameron Chen ◽  
Noah Perrin ◽  
...  
Keyword(s):  
Active Site ◽  
Soybean Lipoxygenase ◽  
Site Binding

scholarly journals Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis

1998 ◽  
Vol 329 (3) ◽  
pp. 545-550 ◽  
Author(s):  
Sreenivas KANUGULA ◽  
Karina GOODTZOVA ◽  
E. Anthony PEGG
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bound States ◽  
Limited Proteolysis ◽  
Cysteine Residue ◽  
Acceptor Site ◽  
Cleavage Sites ◽  
Wild Type ◽  
Alkylation Damage ◽  
Increased Susceptibility

Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with the low-molecular-mass pseudosubstrate O6-benzylguanine, increased susceptibility to proteases was observed. A mutant C145A hAGT protein, which cannot repair O6-alkylguanine because the Cys-145 acceptor site in the active site of the protein is changed to Ala, showed identical trypsin cleavage to the wild type, but its digestion was not affected by O6-benzylguanine. These results suggest that alkylation of hAGT leads to an altered conformation. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation demonstrates that hAGT undergoes considerable conformational changes in its structure upon binding to DNA and after repair of alkylation damage.

scholarly journals Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen)

1988 ◽  
Vol 256 (3) ◽  
pp. 1013-1019 ◽  
Author(s):  
C S Greenberg ◽  
J J Enghild ◽  
A Mary ◽  
J V Dobson ◽  
K E Achyuthan
Keyword(s):  
Active Site ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Coagulation Factor ◽  
Cysteine Residue ◽  
Factor Xiiia ◽  
Platelet Factor ◽  
Transglutaminase Activity ◽  
A Chain

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.

Lipoxygenase Inhibitory Activity of 6-Pentadecanylsalicylic Acid without Prooxidant Effect

2010 ◽  
Vol 5 (1) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Isao Kubo ◽  
Tae Joung Ha ◽  
Kuniyoshi Shimizu
Keyword(s):  
Active Site ◽  
Competitive Inhibitor ◽  
Anacardic Acid ◽  
Soybean Lipoxygenase ◽  
Inhibition Kinetics ◽  
Iron Ion ◽  
Prooxidant Effect ◽  
Specific Structural Feature ◽  
Hydrophobic Tail

6-Pentadecanylsalicylic acid, referred to as anacardic acid (C15:0), was found to inhibit the linoleic acid peroxidation competitively catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC50 of 14.3 μM (4.88 μg/mL). This inhibition is a reversible reaction without pro-oxidant effects. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:0) is a competitive inhibitor and the inhibition constant, KI, was established as 6.4 μM. The hydrophilic head (salicylic acid) portion first chelates the iron in the active site and then the hydrophobic tail portion begins reversibly interacting with the C-terminal domain where the iron is located. The inhibition of anacardic acid (C15:0) can be explained by a combination of iron ion-chelation and hydrophobic interaction abilities because of its specific structural feature.

scholarly journals Evidence for common structural changes in thrombin induced by active-site or exosite binding

1993 ◽  
Vol 290 (3) ◽  
pp. 665-670 ◽  
Author(s):  
M A A Parry ◽  
S R Stone ◽  
J Hofsteenge ◽  
M P Jackman
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Intrinsic Fluorescence ◽  
Thrombin Inhibitors ◽  
Site Specific ◽  
Fluorescence Measurements ◽  
Fold Reduction ◽  
Flexible Surface ◽  
Fractional Increase

The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.

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