Bullous pemphigoid antigen. II. Isolation from the urine of a patient.

This report concerns the isolation of bullous pemphigoid antigen from the nondialyzable urinary components of a patient with the disease. The isolation was accomplished by ion exchange chromatography and gel filtration. Pemphigoid antigen was found to be a basic glycoprotein that on SDS gel electrophoresis showed two major bands, one in the 18,000 m. w. region and the second with a m. w. of 74,000. Between these two bands, two additional bands appeared; one of 35,000 daltons and the other of 68,000 daltons. The 18,000 m. w. band was eluted from the gel and rerun on SDS gels. These gels showed the 18,000 m.w. band and also the appearance of the 35,000 and 74,000 m.w. bands. This finding indicates that urinary pemphigoid antigen may exist both as a single monomeric form and in polymeric aggregates.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

A histamine-releasing factor from activated human mononuclear cells

Human mononuclear cells activated by streptokinase-streptodornase have been shown to elaborate a factor capable of releasing histamine from human basophils. We have developed reproducible methods for its production in large quantities by using cells obtained from leukapheresis packs, by detection utilizing donor basophils known to release well with anti IgE, and by quantitation of histamine by the radioenzyme method. Human histamine-releasing factor (HRF) gave a single peak upon gel filtration with an estimated m.w. of 32,000; SDS gel electrophoresis revealed a single major band as seen at m.w. 30,000. HRF can be resolved into at least two forms separable by ion-exchange chromatography on QAE Sephadex, and two peaks of activity were obtained by chromatofocusing or isoelectric focusing in gels at pH 6.9 and between 7.4 and 8.3. This factor represents an important potential link between cellular immunity and immediate hypersensitivity.     

Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels

Outer membrane protein of Escherichia coli prepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Isolation and characterization of a bovine liver tyrosine-O-sulfate-binding protein--a putative receptor molecular for tyrosine-sulfated proteins?

Golgi-enriched microsomal membrane fraction was prepared from bovine liver. Sodium choleate extract of this membrane preparation was subjected to fractionation using Sepharose gel covalently bonded with tyrosine-O-sulfate. SDS gel electrophoresis of the fractionated sample revealed the presence of a major protein with an apparent molecular weight of 175,000. The protein appears to be specific for tyrosine-O-sulfate as it binds neither the unmodified tyrosine nor the structurally similar tyrosine-O-phosphate. pH-dependence study showed the binding of the protein to tyrosine-O-sulfate-Sepharose gel to be strong from pH 8.0 down through 6.0. At pH 5.5, the binding affinity became dramatically reduced. A similar tyrosine-O-sulfate-binding protein was also detected in the choleate extracts of the Golgi-enriched microsomal membrane fractions prepared from bovine pancreas and from both liver and pancreas of dog.     

Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.     

Isoelectric analysis of 3H-pargyline-labelled monoamine oxidase in rat and carp

1. After selective binding of [3H]pargyline to either monoamine oxidase (MAO) A or MAO B in the rat liver, MAO B alone in the rat brain and MAO in carp brain and liver, molecular weight and isoelectric points (pI) of these MAO were determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing and results obtained were compared. 2. For all tissues tested, SDS-polyacrylamide gel electrophoresis of [3H]pargyline-bound samples revealed a labelled protein band of an apparent mol. wt of 60,000 da. 3. Estimation of radioactivity of [3H]pargyline bound after isoelectric focusing revealed a single protein band with acidic pI values of about 5.5 for rat brain and liver MAO B. 4. Moreover, the pI values of about 7.5 were obtained for carp brain and liver MAO. This basic value was also found for MAO A in the rat liver MAO A.     

Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme

Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 mumol acetylcholine (ACh) formed min-1 mg-1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000-dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000- and 56,000-dalton bands after SDS gel separation of crude extract.     

Analysis of proteins synthesized in respiratory syncytial virus-infected cells.

The spectrum of respiratory syncytial virus-encoded proteins was examined in infected cell extracts by standard polyacrylamide gel electrophoresis and by two-dimensional gel analysis. Polyacrylamide gel electrophoresis analysis of a variety of respiratory syncytial virus-infected, actinomycin D-treated cell lines revealed the presence of as many as nine virus-encoded proteins. Seven of these nine proteins were immunoprecipitated by anti-respiratory syncytial serum. Only one major band of [3H]glucosamine was detected in infected cell extracts (Vp86), whereas the reported major virion glycoprotein (Vp48-53) was difficult to detect in infected cells when carbohydrate labels were employed. Two-dimensional gel analysis easily identified seven viral proteins, and one other was tentatively identified. The reported major virion glycoprotein again was not consistently detected. The results of this study confirm the existence of a virus-coded glycoprotein (Vp86) in infected cell extracts. The existence of this glycoprotein in the purified virion has been in dispute, but the apparent low methionine content of this protein may be the reason for this controversy.     

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.     

Characterization of tubular basement membrane antigens in human kidney

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.     

Biochemical characterization of six trisomics of grain sorghum,Sorghum bicolor (L.) Moench

To determine protein differences of grain sorghum disomics and trisomics, we analyzed leaf extracts from six trisomics and a disomic control by disc gel, gel isoelectric focusing, and SDS gel electrophoresis. Based on the number and position of protein bands revealed by Commassie blue staining, the disomic control could be differentiated from the trisomics, and trisomics could be shown to differ among themselves in most cases. SDS gels revealed the most protein bands, followed by isoelectric focusing and disc gel. However, disc gel electrophoresis was the simplest technique of the three and was just as effective in identifying trisomics and differentiating trisomics from the disomic control.Contribution 1596-j, Department of Agronomy, and 182-j, Department of Biochemistry, Kansas State University, Manhattan, Kansas.     

Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Characterization of a protein from Trypanosoma cruzi trypomastigotes that cleaves non-immune IgG bound through its Fab fragment

A protein from Trypanosoma cruzi bloodstream trypomastigotes that binds IgG from man and several other animal species was isolated and characterized. The 52-kDa protein obtained from different T. cruzi cell extracts showed saturable binding with a K of 3.72 nM. Immunoblot analysis revealed that Fab, but not Fc, fragments of the Ig were recognized. When the protein was added to an unrelated C-fixation reaction, lysis was abolished in a dose-dependent fashion. When freshly prepared T. cruzi extracts were run in a 10% acrylamide SDS gel into which normal rabbit IgG was incorporated before polymerization, proteolytic activity, as seen by a transparent band after Coomassie blue staining, migrated in the same m.w. range of the 52-kDa protein. These data provide further clues to the mechanisms through which this pathogen escapes the host's immune response, thus maintaining a long-standing infection.     

Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.     

Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.     

Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.