Rat plasma selenoprotein P properties and purification

A selenoprotein in rat plasma, selenoprotein P, was fractionated and characterized. Plasma collected from rats 3 h post injection of 75SeO3(2-) contained one 75Se-labeled protein, selenoprotein P. Selenoprotein P was fractionated using salt precipitation, Affi-Gel Blue, and DEAE chromatography. The 75Se-containing subunit of selenoprotein P was purified to 90% homogeneity using SDS-polyacrylamide gel electrophoresis followed by electroelution. This isolation resulted in an 850-fold purification of the 75Se-containing subunit of selenoprotein P with a 15% yield of 75Se radioactivity. The molecular weight of selenoprotein P in plasma was 98,000. The 75Se-containing subunit of selenoprotein P had a molecular mass of 57 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing under nondenaturing conditions resulted in a band of 75Se radioactivity at pH 5.4. A comparison of Coomassie Blue- and silver-staining properties of selenoprotein P in SDS-polyacrylamide gels was made. Reverse-phase HPLC and Sephadex G-50 chromatography of tryptic peptides of the 57 kDa subunit of selenoprotein P yielded several peaks of 75Se radioactivity. These results indicate that 75Se is present in several locations within the 57 kDa subunit of selenoprotein P.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii.

In order to determine whether microtubular proteins of flagellar axonemes were phosphorylated, cells of Chlamydomonas reinhardtii were grown in medium containing [32P]orthophosphate for several generations. Only one (alpha subunit) of the two tubulin polypeptides separated by Na dodecyl-SO4-polyacrylamide gel electrophoresis appeared labeled, as detected by autoradiography of the dried gel. 3H- and 32P-labeled alpha tubulin subunit purified by preparative Na dodecyl-SO4-polyacrylamide gel electrophoresis and Na dodecyl-SO4-hydroxyapatite chromatography contained about 0.2 mol of phosphate per mol of polypeptide. Upon partial acid hydrolysis, radioactivity could be accounted for as serine and threonine phosphate. By altering the conditions of the Na dodecyl-SO4-polyacrylamide gel electrophoresis is was possible to resolve the purified alpha-tubulin subunit into five or more components: a major band comprising approximately 65% of the total mass, not phosphorylated, and four or more minor bands comprising together 35% of the mass. Among the minor components at least two were phosphorylated.     

Purification and characterization of a novel abundant protein in rat bile that binds azo dye metabolites and copper

Following intravenous administration to rats of the azo dye hepatocarcinogen 3'-methyl-N,N-dimethyl-4-aminoazo-[14C]benzene, 60-70% of the injected dose was recovered in bile in 2 h. Approximately 10% of bile radioactivity was trichloroacetic acid-precipitable, not extracted by n-butanol and non-dialyzable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bile followed by fluorography revealed two major and several minor proteins to which radiolabelled azo dye metabolites were bound; one of these major proteins (50 kDa) was purified from bile and shown to be homogeneous by SDS-polyacrylamide gel electrophoresis, isoelectric focusing (pI 7) under denaturing conditions and N-terminal analysis. The protein (MBP) comprises 15% of the total bile protein. Amino acid analysis revealed a preponderance of acidic and hydrophobic amino acids. The absorption spectrum of the native protein had a major peak at 280 nm and minor peaks at 345, 400, 600 and 650 nm. The fluorescence spectrum showed a major excitation maxima at 285 and 350 nm and corresponding emission maxima at 345 and 440 nm. Atomic absorption spectroscopy revealed 5 atoms of Cu per mol protein. Approximately 90% of the Cu was EPR silent. MBP did not react with antisera directed against rat serum IgA, albumin or ceruloplasmin; nor did these proteins react against antisera to MBP. Seven distinct peptide bands ranging from 5 to 18 kDa were obtained when MBP was subjected to CNBr cleavage and the digests were analyzed by SDS-polyacrylamide gel electrophoresis. The [14C]-Me-DAB derived radioactivity was present in only two of the peptides, indicating specific binding sites for azodye metabolites.     

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.     

Ontogeny of glucagon-like immunoreactive peptides in rat intestine

The ontogeny of the intestinal glucagon-like peptides was investigated in rats between 16 days of gestation and 4 postnatal days. The intestinal content of glucagon-like immunoreactive (GLI) peptides increased from 0.09 +/- 0.02 pmol/nmol protein at 16-17 days to plateau at 2.8 +/- 0.4 pmol/nmol protein by 20 days of gestation (P less than 0.001). The apparent immunoreactive glucagon (IRGa) content of the gut ranged from 0.03 +/- 0.01 to 0.08 +/- 0.01 pmol/nmol protein. No developmental trends in IRGa peptide content were observed. Following gel filtration of intestines extracted from rats of 18 days of gestation or greater, two main GLI peptides were detected with apparent mol. wts. of 11-12 and 5-6 kDa. Significant peaks of GLI peptides were not detected following gel filtration of intestines extracted from 16- or 17-day fetuses, nor were peaks of IRGa found at any age. In conclusion, the fetal rat intestine undergoes maturational development between 17 and 19 days of gestation to produce the GLI peptides.     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

A study of ferritin iron formation in the rabbit alveolar macrophage

In order to investigate the intracellular pathway of iron to ferritin, rabbit alveolar macrophages were incubated with ⁵⁹FCl₃, homogenized by sonification, and a soluble cell fraction separated from the stroma by centrifugation at 23 000 g. The soluble fraction was examined by gel filtration using Sephadex. Two peaks were identified in the eluate at 254 nm; peak I contained a group of proteins, including ferritin, and most of the eluted radioactivity. The ⁵⁹Fe in this peak was confined to ferritin; no other ⁵⁹Fe-binding protein was identified. Peak II contained a small amount of ⁵⁹Fe. Chase experiments with ‘cold’ iron showed that peak I ⁵⁹Fe was derived from ⁵⁹Fe associated with the cell stroma. A protein carrier for ⁵⁹Fe between the stroma and ferritin was not identified in the eluate of the soluble fraction. Rather it appeared that iron moved from the stroma through the cytoplasm to ferritin in a low molecular weight form.     

Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi.

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Studies on the fatty acid binding proteins in cytosol of rat liver.

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.     

Identification of the major sites of enzymic glycosylation of myelin basic protein

In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2-1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guinea pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studied. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent Km values of the three peptides were within a narrow range of 0.52-0.63 mM, whereas the Vmax values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences: (formula; see text)     

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose C1 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca²⁺/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase V1.     

拟南芥细胞异三聚体G蛋白的分离纯化

以拟南芥哥伦比亚Columbia(Col-0)野生型悬浮培养细胞为材料,采用超声波破碎、匀浆、离心、40%~60%饱和度硫酸铵分步沉淀、Sephadex G-25脱盐、DEAE-Sepharose Fast Flow离子交换、Sephadex G-200凝胶过滤,最后经过Sepharose CL-6B得到纯化的目的蛋白,蛋白收率为0.097%。纯化的蛋白质经非变性聚丙烯酰胺凝胶电泳鉴定显示为一条带,经Western blotting证实为G蛋白。把经Native-PAGE鉴定的蛋白质的条带回收,进行SDS-PAGE显示有3条带。一条是Gα亚基,其分子量为60kDa左右;另外2条带分子量为45kDa和35kDa,可能是β、γ亚基,初步证实拟南芥中存在异三聚体G蛋白。G蛋白提取方法的建立为在基因突变型拟南芥中G蛋白功能的研究奠定基础。     

Purification of selenoprotein P from human plasma

Selenoprotein P was partially purified (> 1000-fold) from human plasma in four chromatographic steps using ⁷⁵Se-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 μmol Se/l at a total selenium concentration of 1.1 μmol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid hydroperoxide glutathione peroxidase is the 18-kDa selenoprotein expressed in human tumor cell lines

Human tumor cell lines cultured in 75Se-containing media demonstrate four major 75Se-labeled cellular proteins (57, 22, 18, and 12 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Among these selenoproteins, an enzymatic activity is known only for the 22-kDa protein, since this protein has been identified as the monomer of glutathione peroxidase. However, all tested cell lines also contained a peroxidase activity with phospholipid hydroperoxides that is completely accounted for by the other selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX) (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of 75Se-labeled proteins separated by gel permeation chromatography supported the identification of PHGPX as the monomeric protein matching the 18 kDa band. This paper is the first report on the identification of PHGPX in human cells.     

Collagen synthesis by cloned pulmonary artery endothelial cells

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.     

Cellulosome-like entities inBacteroides cellulosolvens

Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (M_r 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium.     

Characterization of the detergent solubilized receptor for gastrin-releasing peptide

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.