Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 44.9 A, b = 57.0 A and c = 135.9 A, and diffract to beyond 2 A resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.     

Studies of rabbit serum transferrin

Rabbit serum transferrin is isolated by a procedure designed to preserve its conformation and disulfide linkages. A progress report is presented on the determination of its amino acid sequence, as part of studies on its primary, secondary and tertiary structure. The sequence of 378 residues, of the approximately 680 residues in the molecule, are determined. Observations are made on the site of carbohydrate attachment, iron binding sites and half-cystine residue location. The results are discussed in relation to the X-ray crystallographic studies of human lactoferrin (lactotransferrin) and of rabbit serum transferrin being made in other laboratories.     

Sequence studies of rabbit serum transferrin

Large fragments of rabbit serum transferrin have been prepared by enzymatic digestion with subtilisin and by chemical cleavage with BNPS-skatole. Sequence determinations on fragments from the N-terminal lobe lead to the assignment of 273 residues and those from the C-terminal lobe 267 residues. Together with previous determinations, a total of 614 of the ca 679 residues in rabbit transferrin have been assigned. A number of corrections are made to the preliminary sequence assignments of O.U. Beg, H.A. McKenzie and D.C. Shaw (1988) Biochemistry International 17, 1135-1142.     

The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin.

Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain.     

Isolation, characterization and N-terminal amino-acid sequence of rabbit transferrin

Rabbit serum transferrin has been isolated and purified by ion-exchange column and high-performance liquid chromatography. The N-terminal amino-acid sequence of 32 residues was determined by automatic Edman degradation in a liquid phase sequenator. Of the first twelve residues sequenced previously three identifications were corrected. Comparison with the known transferrin sequences shows 15 common amino-acid residues. Comparison to human serum transferrin revealed that 37% of amino-acid residues were exchanged. Cys9 and Cys19 which are supposed to be involved in disulphide bridges, are conserved.     

Preliminary X-ray study of crystals of human transferrin

Large crystals of human transferrin have been grown from polyethylene glycol solutions. The crystals are orthorhombic, space group P2₁2₁2₁, with $\text{a = 78}\text{A}\text{?}\text{, b = 94}\text{A}\text{?}\text{and}\text{c = 112}\text{A}\text{?}$. Density measurements indicate that there are four transferrin molecules in the unit cell.     

Removal of iron from diferric rabbit serum transferrin by rabbit reticulocytes.

When radioiron-labelled transferrin with 55Fe located predominantly in the N-terminal iron-binding site and 59Fe predominantly in the C-terminal iron-binding site was incubated with rabbit reticulocytes, both radioisotopes of iron were removed at similar rates. Electrophoresis of transferrin samples taken during the course of an incubation, in polyacrylamide gels containing 6 M-urea, showed that iron was removed in a pairwise fashion, giving rise to iron-free transferrin.     

Preliminary X-ray investigation of fragment 1 of bovine prothrombin

Crystals of fragment 1 of bovine prothrombin grown from phosphate at pH 7.5 are tetragonal, space group P4₁2₂2 or P4₃2₁2 with $\text{a = b = 79.5}\text{A}\text{?}\text{, c = 84.9}\text{A}\text{?}$, with probably one molecule of 22,000 molecular weight in the asymmetric unit. The presence of 17.5% carbohydrate in the fragment may account for the high liquid content (60%) of the crystals.     

Preliminary X-ray data for aspartate aminotransferase from Escherichia coli

Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221. Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees. There is one protein subunit of molecular weight 43,600 per asymmetric unit.     

Preliminary crystallographic data for adenylosuccinate synthetase from rabbit skeletal muscle

Single crystals of adenylosuccinate synthetase, an essential component of purine biosynthesis, extracted from rabbit skeletal muscle were prepared as suitable specimens for X-ray structure analysis. The crystal belongs to the space group P4₃2₁2 or P4₁2₁2 with unit cell dimensions $\text{a = b = 71.2}\text{A}\text{?}\text{, c = 194.8}\text{A}\text{?}$. The asymmetric unit contains one protein molecule of 54,000 molecular weight.     

Binding of an aptamer to the N-terminal fragment of VCAM-1

In vitro selection of 2'-fluoropyrimidine oligonucleotide aptamers was performed against the N-terminal two-domain fragment of mouse VCAM-1. The SELEX procedure enriched the starting pool in a family of homologous sequences. High binding affinity (10nM) of one member of this family, aptamer 12.11, was demonstrated in a filter binding assay.     

Metal-binding properties of a single-sited transferrin fragment

A single-sited iron-binding fragment of transferrin, prepared by proteolytic cleavage with thermolysin, has been characterized. The fragment, bearing no carbohydrate, must be derived from the N-terminal half of the protein's two homologous domains. The strength of iron-binding at pH 6.7 and 7.4, and the EPR spectroscopic features of its iron and copper complexes, establish it as carying the ‘b’ site of transferrin.     

Letter: X-ray data for four crystalline forms of serum albumin

Cell dimensions, space groups and crystal densities are reported for a monoclinic form of human serum albumin (a = 126.5, b = 39.2, c = 135.2Å, β = 93.3 °, C2, Z = 4), tetragonal human serum albumin (a = 84.0, c = 276Å, P4₁2₁2, Z = 8), a mercury dimer of human serum albumin (a = 155, b = 83, c = 122Å, P2₁2₁2₁, Z = 4) and a hexagonal form of equine serum albumin (a = 96.6, c = 144.5Å, P6₁, Z = 6).     

Preliminary X-ray data for the ribose binding protein from Salmonella typhimurium

Crystals of the periplasmic ribose binding protein of Salmonella typhimurium have been subjected to X-ray analysis. The crystals grow as rectangular parallelopipeds with the symmetry of space group P2₁. Unit cell dimensions are $\text{a = 64·4}\text{A}\text{?}\text{, b = 60·6}\text{A}\text{?}\text{, c = 62·8}\text{A}\text{?}$, and β = 91·25 °. There are two molecules of molecular weight 29,000 per asymmetric unit.     

Preliminary proteome analysis of rabbit serum with hepatic failure

The administration of dimethylnitrosamine (DMN) into rabbit induced liver fibrosis/cirrhosis and finally caused a lethal hepatic failure. Blood collected from the rabbit was centrifuged and the supernatant was analyzed by two-dimensional gel electrophoresis (2-DE) for the study of proteome in serum. Compared with 2-DE gel of serum from healthy rabbit, a significant reduction in the number of protein spots having molecular weights (MWs) below 21 kDa was observed in the gels of the serum from the rabbit treated with DMN, while the secretion of albumin was kept at a high level. Separated spots in the two-dimensional gel were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Serum amyloid A-3 protein precursor (SAA3) and other serum amyloid A (SAA) protein precursors were identified by matching the peptide masses with those in database. In the SAA family of acute-phase/inflammatory response proteins, SAA3 is mainly synthesized in the liver. The SAA3 secreted level in the serum decreased with time after DMN administration as the result of hepatic dysfunctions.     

Inhibition of insulin secretion by betagranin, an N-terminal chromogranin A fragment

Betagranin, an N-terminal fragment of chromogranin A, results from a proteolytic processing, and is co-secreted with insulin. While other chromogranin A-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic beta-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are down-regulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.     

Aluminum site structure in serum transferrin and lactoferrin revealed by synchrotron radiation X-ray spectroscopy

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site.     

Evidence for functional differences between the two sites of rabbit transferrin: effects of serum and carbon dioxide

A recently developed technique combining urea gel electrophoresis with Western immunoblotting has been modified for assessing the relative ability of each iron binding site of rabbit transferrin in delivering iron to rabbit reticulocytes. The two sites can be made to release iron at the same or differing rates, depending on the experimental conditions. In Hanks' balanced salts solution in an atmosphere of room air or 5% CO2, the acid-labile site in the N-terminal lobe of the protein was found to be 1.4- and 2.9-times more effective than its acid-stable counterpart in providing iron to reticulocytes after 90 min incubation. Both sequential and simultaneous release of iron from the two sites was observed, but sequential release was initiated only from the N-terminal site. The same site also proved to be a better iron donor by a factor of 2 when incubations were conducted in Hanks' medium enriched with 20% serum in 5% CO2. Only in 20% serum in air were the two sites found to be equivalent iron suppliers to reticulocytes. In the cases studied, an atmosphere of 5% CO2 increased 2-fold the effectiveness of iron donation by the acid-labile site to reticulocytes, while the presence of 20% serum enhanced the iron-donating ability of the acid-stable C-terminal site. Thus, the transferrin-reticulocyte interaction is sensitive to environmental variables, and such sensitivity may help account for apparent discrepancies in previous studies of the relative iron-donating abilities of the two sites of transferrin.