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Home / Equine Research Bank / Biochem J / The role of aromatic side chain residues in micelle binding ...
The Biochemical journal1987; 245(3); 821-829; doi: 10.1042/bj2450821

The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

Abstract: Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.
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Publication Date: 1987-08-01 PubMed ID: 3663193PubMed Central: PMC1148203DOI: 10.1042/bj2450821Google Scholar: Lookup
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  • Comparative Study
  • Journal Article
  • Research Support
  • U.S. Gov't
  • P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper investigates how aromatic side chain residues from pancreatic colipase influence micelle formation, focusing specifically on porcine and equine colipase. The researchers use fluorescence techniques to understand these interactions, discovering that Nitrotyrosine-55 of porcine colipase exhibits significant potential in this process.

Research Methodology

  • The researchers utilized fluorescence techniques to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles.
  • Nitrotyrosine-55 of porcine colipase was obtained by modifying the protein with tetranitromethane (in low excess, and in the presence of taurodeoxycholate) followed by gel filtration and ion-exchange chromatography.
  • Confirmation of the modified residue was done through h.p.l.c peptide purification and sequence analysis.

Key Findings

  • Upon reduction and reaction with dansyl chloride, a fluorescent derivative was produced that was twice as active with lipase as native colipase and exhibited a strong emission band at 550 nm.
  • Addition of micellar concentrations of taurodeoxycholate increased the emission maximum 4.3-fold and caused a 70 nm blue shift to 480 nm.
  • Creating a mixed micelle by including oleic acid reduced these spectral effects.
  • Data analysis via Scatchard analysis reveals a Kd of 6.8 X 10(-4) M, indicating a single colipase-binding site for taurodeoxycholate micelles.
  • Basing their observations on phospholipase systems, the researchers infer that dansyl-NH-tyrosine-55 would directly insert into the micelle.

Role of Equine Colipase

  • The presence of a single tryptophan residue (Trp-52) in equine colipase provided a natural probe for studying protein-micelle interaction.
  • The 345 nm emission maximum of horse colipase is indicative of a solvent-accessible tryptophan residue, which lessens upon micelle binding.
  • A blue shift of 8 nm, increase in amplitude (2-fold), and decrease in KSV for acrylamide quenching in the presence of micelles, hinted towards a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles.
  • This also suggested a loss of solvent accessibility of the residue due to micelle bonding.
  • Significant pH effects noticed during KI quenching are likely indicative of nearby negative charges to Trp-52.

Cite This Article

APA
McIntyre JC, Hundley P, Behnke WD. (1987). The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins. Biochem J, 245(3), 821-829. https://doi.org/10.1042/bj2450821

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 245
Issue: 3
Pages: 821-829

Researcher Affiliations

McIntyre, J C
  • Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, OH 45267.
Hundley, P
    Behnke, W D

      MeSH Terms

      • Acrylamide
      • Acrylamides / pharmacology
      • Animals
      • Binding Sites
      • Cesium / pharmacology
      • Chlorides
      • Colipases / metabolism
      • Deoxycholic Acid / analogs & derivatives
      • Horses
      • Kinetics
      • Micelles
      • Pancreas / metabolism
      • Potassium Iodide / pharmacology
      • Protein Binding / drug effects
      • Proteins / metabolism
      • Spectrometry, Fluorescence
      • Structure-Activity Relationship
      • Swine
      • Taurodeoxycholic Acid / metabolism
      • Tryptophan / analogs & derivatives
      • Tryptophan / analysis
      • Tryptophan / metabolism

      Grant Funding

      • HL-30431 / NHLBI NIH HHS

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      Citations

      This article has been cited 2 times.
      1. Ross LE, Xiao X, Lowe ME. Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.. Biochim Biophys Acta 2013 Jun;1831(6):1052-9.
        doi: 10.1016/j.bbalip.2013.02.009pubmed: 23470256google scholar: lookup
      2. Hermoso J, Pignol D, Penel S, Roth M, Chapus C, Fontecilla-Camps JC. Neutron crystallographic evidence of lipase-colipase complex activation by a micelle.. EMBO J 1997 Sep 15;16(18):5531-6.
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