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Title: Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein

Abstract

Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. ({sup 14}C)Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H{sub 2}O{sub 2} is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.

Authors:
;  [1]; ; ;  [2];  [3]
  1. (Washington Univ., St. Louis, MO (United States))
  2. (Univ. of Illinois at Chicago (United States))
  3. (Univ. of Missouri, Kansas City (United States))
Publication Date:
OSTI Identifier:
5933429
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States); Journal Volume: 88:14
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROTEINS; CROSS-LINKING; CARBON 14 COMPOUNDS; FERRITIN; HYDROGEN PEROXIDE; PEPTIDE HYDROLASES; RECEPTORS; STAPHYLOCOCCUS; STOICHIOMETRY; TRACER TECHNIQUES; BACTERIA; CARBON COMPOUNDS; CHEMICAL REACTIONS; COMPLEXES; ENZYMES; HYDROGEN COMPOUNDS; HYDROLASES; IRON COMPLEXES; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MEMBRANE PROTEINS; METALLOPROTEINS; MICROORGANISMS; ORGANIC COMPOUNDS; OXYGEN COMPOUNDS; PEROXIDES; POLYMERIZATION; TRANSITION ELEMENT COMPLEXES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Lin, Jihjing, Thach, R.E., Patino, M.M., Gaffield, L., Walden. W.E., and Smith, A.. Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein. United States: N. p., 1991. Web. doi:10.1073/pnas.88.14.6068.
Lin, Jihjing, Thach, R.E., Patino, M.M., Gaffield, L., Walden. W.E., & Smith, A.. Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein. United States. doi:10.1073/pnas.88.14.6068.
Lin, Jihjing, Thach, R.E., Patino, M.M., Gaffield, L., Walden. W.E., and Smith, A.. 1991. "Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein". United States. doi:10.1073/pnas.88.14.6068.
@article{osti_5933429,
title = {Crosslinking of hemin to a specific site on the 90-kDa ferritin repressor protein},
author = {Lin, Jihjing and Thach, R.E. and Patino, M.M. and Gaffield, L. and Walden. W.E. and Smith, A.},
abstractNote = {Incubation of a 90-kDa ferritin repressor protein (FRP) with small amounts of radiolabeled hemin resulted in the formation of a strong interaction between the two that was stable to SDS/PAGE. Of seven other proteins tested individually, only apohemopexin and bovine serum albumin showed similar crosslinking ability, albeit to a much lower extent. ({sup 14}C)Hemin specifically crosslinked to FRP in the presence of a 50-fold excess of total wheat germ proteins. Inclusion of catalase did not prevent the reaction of hemin with FRP, suggesting that H{sub 2}O{sub 2} is not involved. The subsequent addition of a stoichiometric amount of apohemopexin did not reverse the reaction. Exhaustive digestion of the complex with Staphylococcus aureus V8 protease produced a major labeled peptide of 17 kDa. These results show the existence of a highly specific, uniquely reactive hemin binding site on FRP.},
doi = {10.1073/pnas.88.14.6068},
journal = {Proceedings of the National Academy of Sciences of the United States of America; (United States)},
number = ,
volume = 88:14,
place = {United States},
year = 1991,
month = 7
}
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  • The development of reagents capable of selective cleavage of large double-stranded DNA would greatly facilitate the manipulation and mapping of genomic DNA. We report here the design and synthesis of a {lambda} repressor-staphylococcal nuclease hybrid protein capable of efficient hydrolysis of both linear and supercoiled duplex DNA. The repressor domain of the hybrid nuclease binds specifically to its recognition sequence, {lambda} operator O{sub R}1 or O{sub L}1, and delivers the nuclease activity to DNA sequences adjacent to the repressor binding site.
  • Specific antisera were obtained by injecting rabbits or goats with dehistonized HeLa cell chromatin. Gamma irradiation (10 to 100 krad) of the isolated chromatin increased its immunological reactivity with these specific antisera. Conversely, irradiation of the isolated chromosomal nonhistone proteins or DNA followed by reconstitution resulted in a nearly complete loss of immunological reactivity of the reconstituted complexes. Selective protein dissociation experiments indicated that radiation crosslinked the active nonhistone proteins to the DNA. This interpretation was further substantiated by polyacrylamide gel electrophoresis of the crosslinked proteins.
  • Highlights: • Hemin prevents Aβ42, α-synuclein and RCM-κ-casein forming amyloid fibrils. • Hemin inhibits the β-sheet structure formation of Aβ42. • Hemin reduces the cell toxicity caused by fibrillar Aβ42. • Hemin dissociates partially formed Aβ42 fibrils. • Hemin prevents amorphous aggregation by ADH, catalase and γs-crystallin. - Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin preventsmore » amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.« less