The piscine plasma retinol-binding protein. Purification, partial amino acid sequence and interaction with mammalian transthyretin of rainbow trout (Oncorhynchus mykiss) retinol-binding protein.

1. Retinol-binding protein (RBP) has been isolated from the pooled plasma or rainbow trouts (Oncorhinchus mykiss) by gel filtration, hydrophobic interaction chromatography and ion-exchange chromatography. By this procedure two forms of the protein, both with a molecular mass (approximately 20 kDa) similar to that of mammalian RBP, were purified to homogeneity. Five amino acid substitutions have been found in the partial (about 60%) sequences of the two forms of trout RBP, which are presumably acetylated at their N terminus. The apparent participation of six conserved cysteines in the formation of disulphide bridges, as in human RBP, and the similarity (about 60%) of the amino acid sequence of trout and mammalian RBPs, indicate the existence of a similar overall structure organization in evolutionary distant RBPs. 2. Although the two forms of trout RBP are not physiologically involved in the formation of any protein--protein complex in plasma, they are capable of interacting with mammalian transthyretin, albeit with a binding affinity (K'd = 15-40 microM) considerably lower than that of mammalian RBP. Our data indicate that the two forms of trout RBP also possess the region that in mammalian RBP has the functional role of binding transthyretin. It is suggested that transthyretin (or a homologous protein) was modified, during phylogenetic development of the non mammalian vertebrates, to acquire a binding site for such a region of the RBP molecule.     

Crystallization and preliminary X-ray data of human plasma retinol-binding protein

Crystals of human plasma retinol-binding protein have been obtained from 4.5 m-NaCl buffered at pH 6.8 with 20 mm-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system. $\text{a = b = 104.2}\text{A}\text{?}\text{and}\text{c = 74.5}\text{A}\text{?}$. The crystals diffract to a resolution of 2.0 Å.     

Crystallization of and preliminary X-ray data for the plasma retinol-binding protein

Crystals of the human and rabbit plasma retinol-binding proteins have been grown from solutions of polyethylene glycol 6000 and CdCl2. Two crystal forms have been observed for the human protein, while the rabbit protein has only crystallized in one form which is isomorphous with one of the human serum retinol-binding protein crystals. The crystals differ in their morphologies, but are both in space group P212121 and have similar unit cell sizes (a = 45.9, b = 53.3, c = 72.0 A and a = 45.7, b = 48.7, and c = 76.5 A). The crystals diffract to approximately 2.0 A resolution. In both cases there is 1 molecule/asymmetric unit.     

Rubredoxin from Clostridium thermosaccharolyticum. Amino acid sequence, mass-spectrometric and preliminary crystallographic data.

Rubredoxin isolated from the thermophilic bacterium Clostridium thermosaccharolyticum has been sequenced and crystallized. The 52-residue sequence is similar to those of rubredoxins occurring in other anaerobic bacteria, but displays some unique features, including a tryptophan residue in position 4, two consecutive proline residues in positions 25 and 26, and an aspartic acid residue in position 41. The molecular mass (5988 Da) of the native rubredoxin has been measured by electrospray-ionization m.s., thus establishing the applicability of the technique to this type of iron-sulphur protein. C. thermosaccharolyticum rubredoxin crystallizes as dark-red elongated prisms with a flat diamond cross-section. The X-ray diffraction shows symmetry consistent with space group P2(1)2(1)2(1). Cell parameters are: a = 2.73 nm, b = 2.98 nm, c = 6.49 nm.     

Fatty-acid-binding protein from bovine brain. Amino acid sequence and some properties

A fatty-acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary-derived growth inhibitor (MDGI).     

Purification, crystallization, and preliminary X-ray data for porcine fumarase

Single crystals of fumarase purified from pig heart have been prepared from solutions containing polyethylene glycol. The crystals give diffraction data corresponding to Bragg spacings of 2.0 A and contain a single subunit of the enzyme in the asymmetric unit of the C222 unit cell. Therefore, the subunits of this tetrameric molecule are arranged with the point symmetry group 222. The present purification scheme and studies of the NH2-terminal amino acid sequences suggest that only a single form of the enzyme is present, and it is thought to be the mitochondrial enzyme.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Isolation, crystallization and preliminary X-ray diffraction data of human progastricsin

Human progastricsin, a zymogen of one of the gastric aspartic proteinases, was isolated and crystallized. The crystals belong to the tetragonal space group P4(2)2(1)2, and have unit cell dimensions a = b = 105.5 +/- 0.1 A, c = 70.6 A. The native crystals of progastricsin diffract X-rays at least to 2.5 A and are suitable for a high-resolution X-ray analysis.     

In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences.

The synthetic retinoid fenretinide (4-HPR; N-[4-hydroxyphenyl] all-trans-retinamide) interacts with plasma apo-retinol-binding protein (RBP) to form a tight complex (K'd approximately 0.2 microM) which does not exhibit binding affinity to transthyretin (TTR). Therefore, a substantial modification of the retinol hydroxyl group does not appear to affect the interaction with RBP but does drastically interfere with the protein-protein recognition. The remarkable early reduction in plasma retinol level induced by fenretinide administration may be associated with the high binding affinity of this retinoid to RBP and to its interference with the RBP-TTR complex formation.     

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.     

The interaction of retinol-binding protein with its plasma-membrane receptor.

125I-labelled retinol-binding protein (RBP) bound to specific receptors in human placental brush-border membranes. Binding at 22 degrees C reached equilibrium within 15 min, but prolonged incubation caused a subsequent decline. Scatchard analysis of the equilibrium binding data at 22 degrees C and 15 min showed high-(3.0 +/- 2.7 x 10(-9) M) and low-(9.5 +/- 3.5 x 10(-8) M) affinity binding components. 125I-RBP, bound to membranes at 22 degrees C for 15 min and subsequently dissociated with excess unlabelled RBP, exhibited biphasic dissociation kinetics consisting of fast and slow components of release. In contrast, Scatchard analysis and dissociation kinetics of the binding that had taken place at 37 degrees C for 1 h showed the fast-dissociating/low-affinity binding component, but little of the slow-dissociating/higher-affinity binding component. When 125I-RBP, after incubation with membranes at 37 degrees C for 1 h, was re-isolated and subjected to dissociation kinetic analysis using a fresh batch of membranes, the fast-dissociating phase was unchanged, but the slow phase was almost absent. The complex kinetics were interpreted in terms of a heterogeneity in RBP consisting of high- and low-affinity binding forms. The higher-affinity-binding form is thought to be converted into the lower-affinity state on binding to the receptor. Transthyretin inhibited 125I-RBP binding to the membrane, suggesting that free, rather than transthyretin-associated, RBP bound to the receptor. The RBP receptor was trypsin-, heat- and thiol-group-specific-reagent sensitive and was highly specific for RBP.     

Guanylate kinase from Saccharomyces cerevisiae. Isolation and characterization, crystallization and preliminary X-ray analysis, amino acid sequence and comparison with adenylate kinases

This paper describes a large-scale purification of guanylate kinase (ATP + GMP in equilibrium ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X-ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences. 1. Guanylate kinase was purified in five steps to homogeneity: crude extract, ion-exchange chromatography, affinity chromatography and gel filtration twice. 2. The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 x 200 x 150 microns 3. Preliminary X-ray results are given. 3. The final sequence shows 186 amino acids (Mr = 20,548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N-terminal blocking group was identified by 1H-NMR spectroscopy. 4. Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases.     

Purification of a bovine counterpart to human prealbumin and its interaction with a bovine retinol-binding protein

A bovine counterpart to human prealbumin was purified from bovine serum by thiol-disulfide exchange chromatography on thiol-Sepharose 4B and affinity chromatography on human retinol-binding protein linked to Sepharose 4B. The bovine prealbumin had alpha1-mobility on agarose gel electrophoresis at pH 8.6. It has the same molecular weight as human prealbumin on gel filtration and consisted of subunits with a molecular weight of 12 500. This is compatible with a tetrameric structure for the bovine protein. Antiserum against human prealbumin cross-reacted with bovine prealbumin and vice versa. The bovine prealbumin formed at high ionic strength complexes with another bovine serum protein which were dissociated at low ionic strength. This property was used to isolate a protein from bovine serum, by chromatography on bovine prealbumin linked to Sepharose which cross-reacted with antiserum against human retinol-binding protein; had a molecular weight of 21 000 and alpha 2-mobility on agarose gel electrophoresis. It was concluded that the latter protein was a bovine retinol-binding protein.     

Amino acid sequence of human protein Z, a vitamin K-dependent plasma glycoprotein

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

Purification, crystallization and preliminary X-ray data of the bacteriophage MS2

Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4). These are the first bacteriovirus crystals diffracting to high resolution. The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees. The asymmetric unit contains one half of the virion. The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A. The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.     

Amino acid sequence of bovine neurophysin-II: a reinvestigation.

The entire amino acid sequence of bovine neurophysin-II has been redetermined by manual Edman degradation of tryptic peptides obtained from performic acid-oxidized neurophysin. Electrophoretically homogeneous bovine neurophysin-II was found to be a mixture of two species of protein molecules both containing 95 amino acid residues. The only difference between the two species of the neurophysin molecules is a single amino acid substitution at residue 89. Of the bovine neurophysin-II used in this work 70% of the protein material contained valine and 30% contained isoleucine at residue 89 in their sequences. The redetermined sequences of bovine neurophysin-II shown in Fig. 2 differ substantially from the reported sequence of bovine neurophysin-II but resemble closely that of porcine neurophysin-I and ovine neurophysin-III (Fig. 3).     

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.