The bovine plasma retinol‐binding protein

Abstract

1. The primary structure of bovine plasma retinol-binding protein (RBP) has been determined and found to be more than 90% identica1 to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138 -148 and 2. The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (Kb = 0.34 0.02 pM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein-protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138-151 and 167-183) in the protein-protein recognition.3. Bovine RBP has been crystallized (space group P212121, with a = 4.61 nm, b = 4.91 nm, c = 7.61 nm) and the crystals are suitable for high-resolution X-ray diffraction studies. 169-183).Retinol-binding protein (RBP) is the retinol-specific transport protein present in blood that delivers the vitamin from the liver stores to the peripheral target tissues. RBP is a single polypeptide chain of molecular mass 21 kDa, containing one binding site for retinol, and in plasma interacts with transthyretin (previously called prealbumin), a tetrameric protein composed of four identical subunits [for a review, see [l, 21). The association of RBP with transthyretin, which has a molecular mass of 55 kDa, prevents its filtration through the kidney glomeruli. Human RBP and transthyretin have been crystallized and their three-dimensional structures determined [3,4]. However, the complex has not yet been crystallized and the mode of interaction of the two proteins is not known.Although human plasma RBP is the best characterized variant, there is also a substantial body of information on rat, rabbit and chicken RBPs [5-91. The three mammalian proteins have been sequenced and found to be highly similar [lo, 1 I]. It has also been found that mixed complexes between RBP and transthyretin from different species can form [9, 12, 131. Bovine RBP was first isolated by Heller [14], who found that its properties were very similar to those of human RBP with one notable exception: the formation of the complex between the holoprotein and transthyretin. The fact that, when using a procedure suitable for the isolation of the human RBP . transthyretin complex, uncomplexed RBP was purified from bovine serum, coupled to the observation that in this species there is no protein with a higher mobility than serum Facolta' di Scienze, Viale delle Scienze, 1-43100 Parma, Italy albumin, was taken as evidence for the lack of a protein with the binding properties of transthyretin [14]. This suggestion was later proved to be incorrect: bovine transthyretin has been purified and shown to interact with RBP [15, 161, although the c...

The piscine plasma retinol‐binding protein

“…The high degree of cross-interaction between RBP and transthyretin isolated from evolutionarily distant vertebrate species has been explained by the existence of a particularly conserved site in the RBP molecule which participates in the protein-protein recognition [7,91. Some regions appear especially conserved in mammalian RBPs [6, 71.…”

“…Whereas the primary structures of mammalian RBPs are approximately 90% identical [5 -71, the similarity between the primary structures of Xenopus laevis RBP, whose amino acid sequence has been deduced from its cDNA sequence [8], and human RBP is significantly reduced to 63%. Fluorescence polarization studies have shown that human RBP and transthyretin cross-interact with the bovine or chicken counterparts with high affinity [7,91; therefore, highly conserved regions on the two proteins participate in the protein -protein recognition. It has also been reported that the retinol-containing proteins in amphibian and reptilian plasma exhibit an apparent molecular mass of 60 -80 kDa (i.e.…”

“…Fluorescence polarization measurements were carried out with a Perkin Elmer MPF-3 spectrofluorometer, as described [7]. The anisotropy was corrected for blank readings.…”

“…The amino acid sequences were determined using a Beckman 890 M spinning-cup liquid-phase sequenator, as described [7]. Prior to Edman degradation, the N-terminalblocked peptides were treated with 25% trifluoroacetic acid according to [22].…”

“…Amino acid analysis was carried out by ion-exchange chromatography with post-column ninhydrin derivatization Zurich, Switzerland), as previously described [7]. …”