Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

Production of an antibody against rat liver nuclear T3 receptor

Rabbits were immunized with rat liver nuclear L-triiodothyronine (T3) receptor purified by preparative sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis using bromoacetyl[125I]T3 as an affinity label. SDS-PAGE confirmed the presence of two receptor forms of the apparent molecular weights 57,000 and 45,000. We describe here a specific antibody, raised against the 57,000 receptor type, which reacts with both receptor forms as assessed by electroimmunoblotting and immunoprecipitation in liquid medium.     

Protection of sodium dodecyl sulfate-induced aggregation of concanavalin a by saccharide ligands

Concanavalin Å is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin Å requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl β-d-galactopyranoside did not inhibit the aggregation of concanavalin Å by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin Å may have an additional, ligand-binding site which is netal-dependent and which can be modified by the addition of a saccharide ligand.     

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.     

Integration of alkaline phosphatase in the intestinal brush border membrane

Alkaline phosphatase has been solubilized from porcine intestinal mucosa by two different methods: treatment of the mucosa by Emulphogen BC 720 and papain hydrolysis of enterocyte brush border membrane vesicles. Two different enzyme forms have been obtained by these methods.The two enzyme forms (‘detergent form’ and ‘papain form’) have been purified to homogeneity by similar techniques and exhibit closely related molecular characteristics. However, the detergent form displays a hydrophobic behaviour and aggregates in media free of detergent. The two forms can be differentiated by their electrophoretic mobility on polyacrylamide gel in the absence of sodium dodecyl sulphate.By electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate, it has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively. The difference between these molecular weights has been attributed to the existence of a hydrophobic region in the detergent form which is present on each subunit.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

Neither amino nor carboxyl termini are required for function of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain.

Antibodies were raised against synthetic peptides corresponding to several regions of the rat brain gamma-aminobutyric acid (GABA) transporter. According to our model, this glycoprotein has 12 transmembrane alpha-helices with both amino and carboxyl termini located in the cytoplasm. The antibodies recognized the intact transporter on Western blots. Upon papain treatment, a reconstitutively active transporter can be isolated upon lectin chromatography (Kanner, B. I., Keynan, S., and Radian, R. (1989) Biochemistry 28, 3722-3728). The papainized transporter runs on sodium dodecyl sulfate-polyacrylamide gels as a broad band with an apparent molecular mass between about 58 and 68 kDa as compared to 80 kDa for the untreated transporter. The transporter fragment was recognized by all the antibodies, except for that raised against the amino terminus. Pronase cleaves the transporter to a relatively sharp 60-kDa band, which reacts with the antibodies against the internal loops but not with either the amino- or the carboxyl-terminal. This pronase-treated transporter, upon isolation by lectin chromatography, was reconstituted. It exhibits full GABA transport activity. This activity exhibits the same features as the intact system including an absolute dependence on sodium and chloride as well as electrogenicity. We conclude that the amino- and carboxyl-terminal parts of the transporter, possibly including transmembrane alpha-helices 1, 2, and 12, are not required for the transport function.     

T antigen of BK papovavirus in infected and transformed cells.

BK virus T antigen from BKV-transformed rat and hamster cells and from productively infected monkey cells has been examined by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forms of the antigen that migrate as proteins of 86,000 and 92,000 daltons have been identified. Both forms can be labeled by 32P.     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Species- and Brain Region-Specific Dopamine Transporters: Immunological and Glycosylation Characteristics

Abstract: Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for cross-reactivity to four epitope-specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of ∼80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N-linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms <80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti-rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.     

Charge separation of proteins complexed with sodium dodecyl sulfate by acid gel electrophoresis in the presence of cetyltrimethylammonium bromide.

Globular proteins, casein, and membrane proteins which were reacted with sodium dodecyl sulfate were studied by acid urea gel electrophoresis. The sodium dodecyl sulfate bound tightly to the proteins, producing a more acidic charge which prevented migration into the gel. When cetyltrimethylammonium bromide was added to the sodium dodecyl sulfate-protein complexes, the sodium dodecyl sulfate apparently reacted with cetyltrimethylammonium bromide and dissociated so that the proteins migrated in acid gel in a normal manner as compared to the proteins without any added detergent. The sodium dodecyl sulfate-cetyltrimethylammonium bromide complex could be removed from the proteins by centrifugation. Thus, cetyltrimethylammonium bromide used in conjunction with acid gel electrophoresis allows direct comparison by charge of proteins fractionated in the presence of sodium dodecyl sulfate with the starting mixture of proteins not exposed to detergent. The reaction of cetyltrimethylammonium bromide with sodium dodecyl sulfate in acidic urea also provides a simple convenient method of removal of sodium dodecyl sulfate from proteins.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

Identification of the post-translational modifications of the core-specific lectin. The core-specific lectin contains hydroxyproline, hydroxylysine, and glucosylgalactosylhydroxylysine residues

The core-specific lectin (CSL) synthesized and secreted by rat hepatocytes and the rat hepatoma H-4-II-E shows affinity for mannose and N-acetylglucosamine residues in the "core" region of asparagine-linked oligosaccharides. The CSL undergoes two stages of post-translational modification which result in an increase in its Mr from 24,000 to 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have determined that the lectin undergoes hydroxylation of proline and lysine and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GlcGalHyLys). CSL metabolically labeled with [3H]lysine and [3H]proline contains hydroxylated forms of proline and lysine. The mature form of the lectin can also be metabolically labeled with [3H]galactose. alpha,alpha'-Dipyridyl, an inhibitor of collagen prolyl and lysyl hydroxylases, prevents the metabolic incorporation of [3H]galactose and the post-translational increases in the Mr of the CSL, indicating that both events are dependent upon hydroxylation of proline and lysine. Virtually all of the hydroxylysine present in the CSL is recovered as glucosylgalactosylhydroxylysine after alkaline hydrolysis. The post-translational modifications of the CSL place it in a select family of secreted proteins which contain collagen-like sequences, including the pulmonary surfactant proteins, complement component C1q, and the 18 S asymmetric form of acetylcholinesterase.     

Structural similarity between the macrophage lectin specific for galactose/N-acetylgalactosamine and the hepatic asialoglycoprotein binding protein

The rat peritoneal macrophage lectin specific for galactose/N-acetylgalactosamine was shown to be a homologue of the hepatic asialoglycoprotein binding protein (rat hepatic lectin, RHL). The macrophage lectin was immunochemically crossreactive with the major form of RHL (RHL-1) but not with the minor forms (RHL-2 and -3). The overall homology between the macrophage lectin and RHL-1 was confirmed by peptide maps of their lysyl endopeptidase digests on reverse-phase HPLC. Despite these similarities, however, the macrophage lectin was distinct from HRL-1 as revealed by the differences in the NH2-terminal 20 amino acid sequences of these two lectins.     

Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.     

A two-cycle immunoprecipitation procedure for reducing nonspecific protein contamination.

A two-cycle immunoprecipitation procedure is described that markedly reduces nonspecific protein contamination occurring during the precipitation of hepatic lipase from rat H4 hepatoma cells. In this method, the precipitation of immune complexes during both cycles is achieved by utilizing a sodium dodecyl sulfate (SDS)-washed preparation of lyophilized Staphylococcus aureus cells (Staph A); this washed preparation effectively removes Staph A contaminants without compromising the ability to bind immune complexes. Following initial immunoprecipitation of the antigen, the Staph A/IgG/antigen complex containing coprecipitated nonspecific proteins was dissociated with SDS. Triton X-100 was added to the dissociated immunoprecipitate at a concentration (by weight) of at least 5 parts Triton X-100 to 1 part SDS. A second cycle of immunoprecipitation was then initiated by addition of fresh antibody, followed by Staph A precipitation of immune complexes and analysis by SDS-polyacrylamide gel electrophoresis. The two-cycle procedure is shown to be reproducible and suitable for the quantitative determination of relative amounts of hepatic lipase. The procedure described here is generally applicable to the immunoprecipitation of other antigens.     

Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen.

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.     

Human alpha-L-iduronidase. I. Purification and properties of the high uptake (higher molecular weight) and the low uptake (processed) forms

Two major forms of human alpha-L-iduronidase have been individually purified over 175,000-fold to apparent homogeneity by sequential anion exchange, lectin affinity, and gel filtration chromatography. The two forms, initially designated as soluble and membrane-associated, were extracted from human lung in approximately equal amounts. Optimal solubilization of the membrane-associated form was facilitated by use of a non-ionic detergent or mannose 6-phosphate and saponin. Following detergent homogenization, the two forms were separated by anion exchange chromatography and then individually purified. The more electronegative form was membrane-associated, had a pI of approximately 5.9, and was selectively taken up (high uptake) by cultured Hurler syndrome fibroblasts; the more electropositive soluble form had a pI of about 6.6 and was incorporated into Hurler fibroblasts at a markedly lower rate (low uptake). After treatment with alkaline phosphatase, the pI values of both enzymes were about 7.8. Using 4-methylumbelliferyl-alpha-L-iduronide as substrate, the low and high uptake forms were each purified in milligram quantities to specific activities of 284,000 and 202,000 units/mg, respectively, with a combined yield greater than 35%. Each purified enzyme form migrated as a single protein band which also stained for enzymatic activity when electrophoresed in 7% native polyacrylamide disc gels at pH 4.3. By gel filtration, the high uptake form had an Mr = 85,000 whereas the Mr for the low uptake form was 68,000. Molecular weight estimates by analytical polyacrylamide gel electrophoresis were 82,000 and 70,000 for the high and low uptake forms, respectively. Rabbit anti-human low uptake alpha-L-iduronidase antibodies cross-reacted with the high uptake form as demonstrated by both immunotitration and Ouchterlony double immunodiffusion. Amino acid analysis revealed that the high uptake (higher molecular weight) form contained more arginine, glycine, alanine, glutamate or glutamine, leucine, isoleucine, histidine, and proline residues per molecule than the low uptake (lower molecular weight) form. Automated Edman degradation determined that the NH2-terminal residues of both forms were blocked. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography demonstrated that each purified form was composed of several components; each post-high performance liquid chromatographic component retained catalytic activity and was immunologically cross-reactive with antibodies against the low uptake form.(ABSTRACT TRUNCATED AT 400 WORDS)