Isolation and Identification of Heterogeneous Nuclear Ribonucleoproteins (hnRNP) From Purified Plasma Membranes of Human Tumour Cell Lines as Albumin-Binding Proteins

Biochem Pharmacol. 2004 Feb 15;67(4):655-65. doi: 10.1016/j.bcp.2003.09.027.

Abstract

Since albumin is being developed as a drug carrier to target tumours the search for albumin-binding proteins (ABPs), which play a role in cell surface binding and endocytosis of native and conjugated albumins becomes more and more interesting. We isolated five different proteins from purified plasma membranes from three different human tumour cell lines (CCRF-CEM, MV3 and MCF7) by albumin affinity chromatography and identified them as four members of the heterogeneous nuclear ribonucleoproteins (hnRNP) family and calreticulin by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Contamination of the plasma membrane preparation by nuclear membranes was excluded with anti-nucleopore antibodies. Western blot analyses of plasma membranes showed ABPs with the same molecular weights as the albumin-affinity isolates. Tryptic digestion of intact cells was used to determine the sidedness of the albumin-binding property, which is oriented to the exterior of the cell. Localisation to the plasma membrane and albumin binding is a novel property of hnRNP.

MeSH terms

  • Albumins / metabolism*
  • Amino Acid Sequence
  • Blotting, Western
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Chromatography, Affinity
  • Heterogeneous-Nuclear Ribonucleoproteins / isolation & purification*
  • Heterogeneous-Nuclear Ribonucleoproteins / metabolism
  • Humans
  • Iodine Radioisotopes
  • Molecular Sequence Data
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / metabolism

Substances

  • Albumins
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Iodine Radioisotopes
  • Trypsin