Immunological cross-reactivity of gamma-glutamyltranspeptidases from human and rat kidney, liver, and bile

Antisera to purified gamma-glutamyltranspeptidase (gamma GTP) from human and rat kidney were prepared, and their reactivities toward purified gamma GTP from kidney, liver, and bile were tested. The following results were obtained: 1. On double immunodiffusion, Triton-solubilized gamma GTP, and papain-solubilized gamma GTP from rat kidney gave single precipitin lines which fused completely against antiserum to the purified enzyme from rat kidney. 2. An antigen-antibody complex of human kidney gamma GTP retained about 50% of the catalytic activity of the antigen. 3. Double immunodiffusion showed that the enzymes from human liver, kidney, and bile were immunologically identical. 4. Antiserum to rat kidney gamma GTP partially cross reacted with human gamma GTP, but antiserum to human gamma GTP reacted only very weakly with rat gamma GTP. It is concluded that gamma GTP of human liver, kidney, and bile are immunologically identical and that rat gamma GTP and human gamma GTP have certain antigenic determinants in common.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Localization of immunoreactive regions in the beef heart adenine nucleotide carrier using rabbit antisera against the carboxyatractyloside-liganded and the sodium dodecyl sulfate denaturated carrier forms

The existence of different antigenic determinants in the beef heart adenine nucleotide (AdN) carrier was demonstrated by exploring the reactivity of fragments of the carrier protein with rabbit antisera directed against either the beef heart AdN carrier denatured with sodium dodecyl sulfate (NaDodSO4 carrier) or the beef AdN carrier liganded by the specific inhibitor carboxyatractyloside (CATR-carrier). The antigenic determinants reacting with antiserum to the CATR-carrier appeared to be close to the N- and C-terminal ends of the carrier protein, whereas those reacting with antiserum to the NaDodSO4 carrier were located preferentially in the central region of the protein. The same antisera were used to study the immunogenic specificity of the beef liver AdN carrier, the rat heart AdN carrier, and the rat liver AdN carrier. The beef liver and rat heart AdN carriers were found to react with both antisera whereas the rat liver AdN carrier reacted essentially with the CATR-carrier antiserum.     

Detection of the principal protein target of a hepatic carcinogen

Antiserum was prepared against the principal liver protein conjugate of the hepatic carcinogen, 3′-methyl-4-dimethylaminoazobenzene. One precipitin band was obtained when the antiserum reacted with the purified conjugate in double immunodiffusion gel analysis. The same anti-serum detected two proteins in rat liver cytosol. Of these two proteins, one was immunoreactively identical to the purified antigen; in contrast, the other protein was only partly identical to it. Absorption of the antiserum with rat kidney cytosol yielded specific antiserum that reacted only with the protein that was immunologically identical to the purified conjugate. That protein, detected in normal rat liver cytosol, is apparently the principal protein target of the azocarcinogens in liver carcinogenesis.     

Heavy chain variable region allotypic sub-specificities of rabbit immunoglobulins. I. Identification of three subpopulations of a1 IgG molecules.

Four anti-al Ab subpopulations were isolated from an anti-al antiserum by sequential immunoadsorption chromatography. These four anti-al Ab subpopulations were differentially bound by two "limited heterogeneity" Abs having different components of the al allotypic specificity. Each of the four anti-al Ab subpopulations reacted with al IgG molecules obtained from a2 and a3 rabbits. A subpopulation designated anti-al Ab reacted with 100% of al IgG molecules. Thus, the anti-al-A Ab recognizes al determinants common to all al IgG molecules. Each of the other three subpopulations, designated anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab, reacted with only a fraction of the al IgG molecules but the sum of the percentages of al IgG molecules which reacted with each of these three anti-al Ab subpopulations approximated 100% of the al IgG molecules. Thus each of the anti-al-B Ab, anti-al-C Ab, and anti-al-D Ab recognizes non-common determinants distinct for each of three subpopulations of al IgG molecules. Although 65 to 90% of IgG molecules in al homozygous rabbits have the al allotypic specificity, these IgG molecules are heterogeneous with respect to their antigenic determinants comprising the al allotype; at least three kinds of al IgG molecules are identified. This heterogeneity probably reflects variation in the amino acid sequence of the Vh region of al IgG molecules and, therefore, poses a similar argument which had led to the hypothesis of two genes for one polypeptide chain and to the theory of episomal insertions for the genetic control of immunoglobulin synthesis.     

The distribution of surface antigens during fractionation of mouse liver plasma membranes

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified gamma-globulin antibody fraction was iodinated with (125)I. The reaction of the (125)I-labelled gamma-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The gammaglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ;Light' and ;heavy' mouse liver plasma-membrane subfractions bound similar amounts of gamma-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO(3)-Na(2)CO(3) buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.     

Radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) content of adult rat liver

1. Rabbit antiserum was raised against purified carbamoyl-phosphate synthase (ammonia) from rat liver. 2. The antiserum proved to be specific in double-diffusion test and reacted in an in situ immunohistochemical test on rat liver proteins fractionated on a sodium dodecyl sulphate polyacrylamide gel only in the region where carbamoyl-phosphate synthase (ammonia) migrated. 3. This antiserum was used for setting up a radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) in cetyltrimethylammonium bromide extracts of rat liver. To obtain reproducible results in this assay it was necessary to treat the unlabelled ligand with sodium dodecyl sulphate and dithiothreitol. This treatment led to a large increase in the percentage of labelled ligand displaceable by added unlabelled ligand. 4. Radioimmunochemical determination showed that adult rat liver (3-month old) contains 5.5 mg carbamoyl-phosphate synthase (ammonia) protein per gram wet weight.     

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49

Three antisera to myelin basic protein—a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP—were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69–84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69–71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69–71. S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.This work supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by NS-15322 from the National Institutes of Health of the U.S. Public Health Service; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Quantitation and Characterization of Peptides from the C-Terminal Flanking Region of Rat and Bovine Preprotachykinins

Sequence analysis of cDNAs has shown that the biosynthetic precursors of substance P (alpha-, beta-, and gamma-preprotachykinins) contain a common amino acid sequence in the C-terminal flanking region that has not been conserved between species. Antisera have been raised against the synthetic peptide Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, which represents rat beta-preprotachykinin-(117-126)-peptide, and used in radioimmunoassays. Antiserum R50 reacted strongly with C-flanking peptides in extracts of rat and bovine tissues whereas antiserum GP-4 reacted only with the rat peptides. The primary structure of the predominant molecular form of preprotachykinin C-flanking peptide in an extract of bovine corpus striatum was established as: Ala-Leu-Asn-Ser-Val5-Ala-Tyr-Glu-Arg-Ser10-Val-Met-Gln-Asp-Tyr1 5-Glu. This sequence represents beta-preprotachykinin-(111-126)-peptide which is equivalent to gamma-preprotachykinin-(96-111)-peptide. A C-flanking peptide with similar chromatographic properties was identified in extracts of rat brain and gut together with a second immunoreactive component that may represent a fragment or a posttranslationally modified variant. A peptide corresponding to the 37-amino-acid residue C-flanking peptide derived from alpha-preprotachykinin was not detected in the extracts as expected from the known low abundance of alpha-preprotachykinin mRNA in rat brain and gut.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

Cross-reactivity of metallothioneins from different origins with rabbit anti-rat hepatic metallothione in antibody.

Rat hepatic Cd-metallothionein was purified and isolated into its two components, metallothionein 1 and 2, by disc electrophoresis. Antibodies to metallothionein 2 were generated in rabbits. The antiserum reacted with the protein and formed a single precipitin band on a double diffusion plate. By ammonium sulfate precipitations, it was found that the antiserum cross-reacted with rat hepatic metallothionein 1. Cross-reactivity of the antiserum was also observed for components of rat renal Cd-metallothionein, rabbit hepatic Cd-metallothionein and human renal metallothionein.     

Type-specific and cross-reactive antigenicity of capsid proteins VP1 and VP2 of echovirus type 7

After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.     

Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.     

Subunit specific antisera to the Escherichia coli ATP synthase: effects on ATPase activity, energy transduction, and enzyme assembly

We obtained antisera to each of the five subunits (α, β, γ, δ, and ?) of the F₁ portion of the proton-translocating ATPase from Escherichia coli (ECF₁). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF₁ to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF₁ with trypsin removed intact γ from ECF₁, but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF₁ from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF₁. The e antiserum stimulated the ATPase activity of purified ECF₁ as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys.197, 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ? antiserum. Adsorbing the δ or ? antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF₁, and prevented it from restoring ATP-dependent proton translocation in ECF₁-depleted vesicles. Therefore, it appears that more of the δ and the ? subunit is exposed in the purified ECF₁ molecule than in the membrane-bound enzyme.     

The potential protective effect of immunization with outer-membrane protein I from Neisseria gonorrhoeae

Immunization of rabbits with outer membranes (OM) of Neisseria gonorrhoeae produced antibodies directed against outer-membrane proteins PI and PIII. The antibodies directed against PIII reacted equally well on Western blots with all strains tested, but antibodies directed against PI reacted only with the homologous strain. When purified PIB was used for immunization the immune response was quite different: the sera obtained reacted with both homologous and heterologous PIB types and also reacted with strains expressing PIA. Western blotting of peptides produced by sequential cleavage of PIB revealed that the antigenic determinants recognized by anti-OM sera were predominantly located in the central surface-exposed region of PIB, as is the epitope recognized by the protective anti-PIB monoclonal antibody SM24. In contrast antibodies produced by immunization with purified PI reacted with antigenic determinants in the N-terminal portion of PIB. Nevertheless these determinants are accessible to immune attack on the native protein since the anti-PI sera were opsonic and were strongly bactericidal for both PIA- and PIB-expressing strains.     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.