Antibody production to choline acetyltransferase purified from human brain

Choline acetyltransferase (CAT) was isolated from human caudate and putamen. The enzyme was highly purified by a series of steps involving fractionation by protamine sulfate and ammonium sulfate followed by chromatography on DEAE-Sephadex, hydroxyapatite and carboxymethyl cellulose columns. The isolated CAT gave a single protein band on polyacrylamide gel electrophoresis at pH 8.3 which corresponded with CAT activity. A single band was also obtained at pH 6.8. Rabbit antiserum was prepared to the purified homogeneous CAT from carboxymethyl cellulose columns. It exhibited a single sharp precipitin band on double diffusion tests on Ouchterlony I.D. plates when tested against the partially purified hydroxyapatite enzyme. On preincubation, the antiserum inhibited CAT activity to 50–60% of control independently of the concentration of enzymatic protein. Normal rabbit serum neither produced a precipitin band on double diffusion tests nor inhibited the CAT activity on incubation. The anti-CAT rabbit antibody thus appeared to be specific.     

ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

Characterization of a Novel Hemolytic Activity of Human IgG Fractions Arising from Diversity in Protein and Oligosaccharide Components

Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanin''s small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance.     

Ultrastructural localization of ‘anixiopsin’: a lectin of the fungus Anixiopsis stercoraria (Hansen) Hansen

In order to localize the anixiopsin, a lectin of the keratinolytic fungus Anixiopsis stercoraria, the authors used a monospecific antiserum prepared by immunization of rabbits with their own erythrocytes coated in vitro with anixiopsin. In light and scanning electron microscopies, lectinic sites were visualized by means of latex microspheres sensitized with anti-rabbit IgG antibodies. In transmission electron microscopy using the IgG fraction of the rabbit anti-anixiopsin immune sera and protein A-gold, anixiopsin seemed mainly present on the outermost cell wall layer of the ascospores, in a pseudomembraneous structure dense to electrons. Implications of these results on physical and biological properties of the lectin are discussed.     

Purification of carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius)

The enzyme carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short-chain acyl groups between coenzyme A and L-carnitine, and hence, plays an important role in the -oxidation of fatty acids. Purification and characterization of CAT from desert animal species may help in explaining the involvement of secondary pathways for energy production in these species. In this paper, we report the purification and partial characterization of CAT from the Arabian camel. CAT was purified from the skeletal muscle of the Arabian camel by ammonium sulfate and acetone fractionation, followed by chromatography on DEAF-Sepharose, agarose-Co A and Superose 12 gel filtration columns. CAT was purified by 2937-fold to a specific activity of 94 Units mg^–1. The purified CAT was a monomer of 59 kDa as judged by native and SDS-PAGE, and showed a pl of 5.2. The enzyme displayed maximum activity with propionyl-Co A. Apparent K_m for acetyl-, propionyl- and butyryl-Co A were 27.7, 17.3 and 29 M respectively, while palmitoyl-Co A was not a substrate.     

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

Summary Revertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

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.     

Membrane-bound choline acetyltransferase from human brain: Purification and properties

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into apotassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35–65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChAT, indicating that both forms of this enzyme are related. Furthermore, as previously reported for soluble ChAT, Fab-Sepharose chromatography could be used for the purification of mChAT and this preparation also resolved into two bands of 10% SDS gel.Special Issue dedicated to Prof. Eduardo De Robertis.     

Uptake of methotrexate linked to an anti-EL4-lymphoma antibody by EL4 cells

Summary To study the mechanism of tumor inhibition, the uptake of methotrexate (MTX) covalently linked to a rabbit IgG antibody against a tumor-associated antigen on the surface of mouse EL4 lymphoma cells (AELG) has been compared with the uptake of free MTX and of MTX covalently linked to normal rabbit IgG (NRG). When EL4 cells were incubated at 37°C with 10 M free MTX uptake leveled off after 30 min, at 30 pmol/mg protein. In contrast, uptake of both conjugates under these conditions continued throughout an observation period of 6 h. At 6 h the net uptake of MTX bound to AELG was 40 pmol/mg protein and that of MTX bound to NRG was 24 pmol/mg protein. These results show that both MTX-AELG and MTX-NRG conjugates are taken up by EL4 cells. The rate at which EL4 cells took up bound MTX was much slower than that of free MTX but, at 6 h, the net uptake of MTX-AELG exceeded that of the free drug. Abbreviations used in this paper: AELG, antiEL4 IgG; NRG, normal rabbit IgG; MTX, methotrexate; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride     

Marek's Disease Herpesviruses II. Purification and Further Characterization of Marek's Disease Herpesvirus A Antigen

Marek's disease herpesvirus A antigen was purified greater than 200-fold with a 24% recovery by ion exchange column chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The antigen had an isoelectric point of 6.68 ± 0.03 in the presence of 1 M urea and 0.05% Brij 35, a nonionic detergent, and approximately 6.5 in the absence of dissociating agents. When analyzed by electrophoresis on analytical polyacrylamide gels, the purified antigen migrated as a single broad band which stained for both protein and carbohydrate, suggesting that it was a highly purified heterogeneous glycoprotein. However, the antigen was not purified to homogeneity as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and by immunodiffusion analysis. Antibody to Marek's disease herpesvirus A antigen was prepared in a rabbit, and antibody to two contaminating antigens was removed by adsorption to yield monospecific antisera.     

Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.     

Comparative Study of Induction of iNOS mRNA Expression in Vascular Cells of Different Species

To determine the difference in induction of inducible nitric oxide synthase (iNOS) mRNA expression in cultured vascular cells of different species, the expression of iNOS genes and their regulatory mechanisms in rat, human, bovine, and rabbit vascular endothelial cells and smooth muscle cells (SMC) were studied by Northern blotting, chloramphenicol acetyltransferase (CAT) assay, and electrophoretic mobility shift assay (EMSA). Qualitative estimation of iNOS mRNA by Northern-blot analysis demonstrated that the combination of interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and lipopolysaccharides (LPS) drastically induces iNOS expression in rat and human SMC, and a more moderate effect was observed for endothelial cells; the effect of IL-1 alone was much weaker than that of the three factors. IL-1 alone or a mixture of IL-1, TNF-, and LPS both showed negligible effect on iNOS expression in bovine and rabbit vascular endothelial cells and SMC. Results of CAT assay corresponded well with Northern analysis indicating 7-fold increase in CAT activity by the mixture of IL-1, TNF-, and LPS in SMC and more moderate, 2-fold increase, in endothelial cells. IL-1 alone produced an intermediate effect (less than 2-fold) on vascular SMC of rats and humans. The results of EMSA showed that two shifted bands appeared when the nuclear protein from rat and human vascular endothelial cells bound to the region from –1037 to –787 of the rat iNOS gene, while vascular SMC nuclear protein only produced a single shifted band under the same conditions. These results suggest that cell- and species-specific mechanisms exist in the induction of iNOS expression.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Restriction of a sperm surface antigen's mobility during capacitation.

Fluorescent Fab¹ [immunoglobulin G (IgG) fragment with antigen binding capacity from papain digestion of IgG] antibody fragments and globulin from antisera prepared against a single rabbit sperm surface membrane glycoprotein antigen (MGP) were used to study surface antigen mobility. Epididymal and ejaculated spermatozoa exhibited a redistribution of MGP surface antigen over the acrosomal region when labeled at 4°C and warmed to 37°C. Following in vivo capacitation, spermatozoa did not exhibit MGP surface antigen redistribution over the acrosome. The restricted mobility of this surface antigen implies a physiological change in plasma membrane fluidity, which may be a necessary preliminary to the acrosome reaction. Furthermore, it is suggested that the presence or absence of specific peripheral membrane proteins may control the positional relationship between mobile and nonmobile integral membrane components.     

Isolation of human nerve growth factor from placental tissue

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the supernatant was sequentially chromatographed, at neutral pH, on Sephadex G-100, DEAE-11, and Sephadex G-150. A high-molecular-weight protein fraction (150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1–3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse -NGF.To whom reprint requests should be addressed.     

A simple purification method for enterotoxin F produced by Staphylococcus aureus and some properties of the toxin

Staphyloccoccus aureus enterotoxin F (SEF), which is associated with S. aureus strains isolated from toxic-shock-syndrome patients, was purified by successive chromatography on CM sephadex C-25 and gelfiltration on sephadex G-75. When tested by disc-polyacrylamide gel-electrophoresis the toxin migrated as a homogeneous protein. In SDS-polyacrylamide gel-electrophoresis three protein bands were observed. The main component had a mol wt of 23000 and the two minor components had a mol wt<13 000. By iso-electric focussing a main protein band with an iso-electric point of 7.2 was obtained. The LD₅₀ for rabbits (3–3.5 kg) by subcutaneous and intravenous application of SEF was 6 g and 180 g, respectively. Antibodies to SEF prepared in a sheep did not react with other staphylococcal enterotoxins (A to E).     

Cell surface labelling of mononuclear cells with antisera associated to turnip yellow mosaic virus or alphalpha mosaic virus particles. A freeze-etch study

Synopsis Turnip yellow mosaic virus (TYMV) and alphalpha mosaic virus (AMV) were used as immuno-electron microscopical markers to detect cell surface receptors on mononuclear cells in freeze-etch replicas. TYMV particles were conjugated with vacuum-distilled glutaraldehyde to rabbit IgG anti-mouse immunoglobulins (TYMV-RAMIg conjugate) or to rabbit IgG anti-mouse antigen (TYMV-RAMTh conjugate). B-lymphocytes incubated with TYMV-RAMIg conjugate showed either randomly distributed particles or patches of virus particles on the etched surface of the cell membrane. Mouse thymocytes incubated with TYMV-RAMTh conjugate, however, showed only a random distribution of the virus particles. Human mononuclear cells incubated with rabbit IgG anti-AMV and AMV for the demonstration of the receptors for the Fc fragment of IgG showed the oblong shape of the AMV particles on the etched cell membrane. Fc receptors were either randomly distributed or aggregrated into patches. It is concluded that both types of virus particles are useful markers for the demonstration of membrane receptors in freeze-etch replicas of labelled cells.     

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O₂ have been studied with ten freshwater and marine strains. Generally, O₂ was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O₂ concentrations of below 15 M (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O₂ as electron acceptor (up to one doubling of protein). However, O₂ was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O₂ tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O₂ concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 M. The specific rates of O₂ uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O₂ concentrations in the gas phase.