Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A.     

Purification of a Chlamydia trachomatis-specific antigen by immunoadsorption with monospecific antibody.

This study describes the isolation and partial characterization of a Chlamydia trachomatis specific antigen. A species-specific antigen of C. trachomatis (antigen-0.65) was identified by two-dimensional immunoelectrophoresis. Antiserum specific for antigen-0.65 was prepared in rabbits by immunizing with agarose-gel precipitates excised from two-dimensional immunoelectrophorograms. Purified gamma-globulins from antigen-0.65 specific serum were coupled to the N-hydroxysuccinimide ester derivative of agarose which was then used for the immunoadsorbent purification of antigen-0.65 from Triton X-100 solubilized lymphogranuloma venereum (L2/434/Bu) organisms. The isolated antigen was immunochemically pure when tested against rabbit antiserum prepared to LGV-434 organisms by using rocket and two-dimensional immunoelectrophoresis. Antigenicity was destroyed by protease treatment and heating at 56 degrees C for 30 min, but the antigen was stable to ribonuclease, deoxyribonuclease, periodate oxidation and pH extremes of 2.2 and 10.6. Polyacrylamide gel electrophoresis of purified antigen showed a major protein band with an apparent m.w. of 155,000.     

Isolation of the terminal complement complex from target sheep erythrocyte membranes.

(1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.     

Acid-Precipitated M Protein Compared with a Column-Eluted M Protein Preparation from Type 12, Group A Streptococci

An M protein preparation of group A streptococci, precipitated with 0.03 m sodium acetate buffer (pH 4.0) was compared with a column-eluted M protein preparation. Absorption spectra and methyl pentose content were similar in both preparations. Acrylamide gel electrophoresis patterns were different. Gel diffusion demonstrated two lines of fusion in the preparations. More antigens could be demonstrated in both preparations by using immunoelectrophoresis. Neither the pH 4 precipitate nor the column-eluted preparation appeared to be a pure M protein preparation.     

Calcium-binding proteins from human platelets. A study using crossed immunoelectrophoresis and 45Ca2+

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45Ca2+ and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45Ca2+. These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4. Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45Ca2+ prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was weakly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelet-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIb-IIIa precipitate also became apparent. No increased incorporation of calcium occurred in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45Ca2+.     

Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations

The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions.     

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides.

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll a . protein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

Experimental allergic aspermatogenic orchitis. 1. Isolation of a spermatozoal protein (AP1) which induces allergic aspermatogenic orchitis.

A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions.     

Isolation and partial characterization of rabbit plasma alpha1-antitrypsin.

Alpha1-Antitrypsin was isolated from rabbit plasma by salting out with (NH4)2SO4 followed by ion-exchange chromatography either on DEAE-Sephadex or DEAE-cellulose (each at pH8.8 and 6.5), and affinity chromatography on Sepharose-Cibacron Blue and Sepharose-concanavalin A. The protein thus obtained was homogeneous during crossed immunoelectrophoresis by using an antiserum to whole rabbit plasma, but it migrated as two broad bands when electrophoresed in alkaline polyacrylamide gels. Under optimal loading conditions, two or three subcomponents could be distinguished in each band. The two major forms of rabbit alpha1-antitrypsin, designated components F and S, were separated by preparative polyacrylamide-gel electrophoresis, and some of their physico-chemical properties were established. Both forms reacted with trypsin at a molar ratio of 1:1. Their elution volumes from a Sephadex G-200 column were identical, corresponding to a mol.wt. of 58000; however, some heterogeneity was observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing in polyacrylamide gel in a pH 4-6 gradient revealed a multiple-band pattern for each form in the range of pH4.4-4.9. The two forms of rabbit alpha1-antitrypsin possessed the same N-terminal amino acid (glutamic acid) and had very similar amino acid and carbohydrate compositions.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen.

Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.     

Role of a streptococcal antigen in the pathogenesis of acute poststreptococcal glomerulonephritis. Characterization of the antigen and a proposed mechanism for the disease.

We studied the significance of a streptococcal protein (preabsorbing Ag) (PA-Ag) in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN). This protein was isolated from nephritogenic streptococci. Purification of PA-Ag was achieved by chromatography, followed by Sephadex IEF. A single protein band at pH 4.7 was identified as PA-Ag. The m.w. was 43,000. Rabbit antisera against PA-Ag and sera of patients with APSGN showed identical precipitation lines by immunodiffusion. Antibodies to PA-Ag were found to be present in 30 of 31 patients with APSGN, in 1 of 36 patients with uncomplicated group A streptococcal upper respiratory tract infections, and in 1 of 36 normal adults. By using immunoelectrophoresis, it was found that PA-Ag activates the alternate pathway of C. Other water-soluble streptococcal fractions, used as controls, did not activate the C system. The demonstration that PA-Ag is present in the glomeruli in the early phase of APSGN and its ability to activate C3 and factor B suggest that PA-Ag may be involved in the pathogenesis of APSGN, via in situ C activation.     

Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidyl-AH-sepharose.

L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique.     

Isoelectric focus analysis of rat anti-phosphocholine antibodies.

Anti-phosphocholine (PC) antibodies in sera from four strains of rats were examined before and afterimmunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodSO4-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin protein (pI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S. pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Purification and characterization of Aleutian disease virus

Virus was isolated from infected mink organs by a combination of tissue homogenization, fluorocarbon extraction and ultracentrifugation. The final preparation was analysed by crossed immunoelectrophoresis and electronmicroscopy. Virions had a capsid diameter of 22 nm. Preparative agarose electrophoresis separated virions from contaminating ferritin. Crossed immunoelectrophoresis of virus gave a single precipitate with sera from infected mink. Crossed immunoelectrophoretic analysis with intermediate gels showed that a part of the virus preparation was complexed with antibody. Serum from a certain mink was found to contain precipitating antibody to (poly)nucleotid. Virus and virus-antibody complexes were found to focus at pH 4.0-4.4 in isoelectric focusing. In SDS-polyacrylamide gel-electrophoresis the main virus protein was found to have a molecular weight of 69000. This study gives further support to the classification of aleutian disease virus as a parvovirus.