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...

No abstract

No abstract

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an α8β8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.