Antibodies to dehistonized chromatin of normal and fetal rat liver

Antisera to dehistonized adult or fetal rat liver chromatin were treated with electrophoretically separated chromosomal proteins of adult and fetal liver, Novikoff hepatoma and adult rat kidney. Both types of antisera reacted with numerous antigens in both tissue chromatins. However, immunoabsorption experiments have shown that while adult rat liver shared most of its nuclear antigens with other tissues, antisera to dehistonized fetal liver chromatins were more specific. In terms of antigenic specificity, the fetal rat liver and Novikoff hepatoma chromatins were similar; however, the former contained several antigens which could not be absorbed with Novikoff hepatoma chromatin.     

Comparison between different rabbit antisera against the glucocorticoid receptor

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.     

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.     

Studies with anti-growth hormone receptor antibodies.

Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.     

Specificities of heterologous antisera against human leukaemia cells. 2. Reactions against fetal liver cells and absorption studies with fetal tissue.

Rabbit or goat antisera directed to ALL and AML cells were investigated in cytotoxicity tests with fetal liver cells as targets. After absorption with erythrocytes and spleen cells from allogenic donors the antisera killed fetal liver cells. There was no reaction with remission leukocytes or blood leukocytes from normal donors. Treatment with fetal tissue removed the activity of the AML and ALL antisera against ALL cells but not of the AML antisera against AML cells. This indicates the existence of at least two antigens on the surface of AML cells, one antigen is common with ALL cells and of fetal origin and another one seems to be characteristic of AML cells and not of fetal origin. Because treatment with fetal tissue removed all activity of the ALL antisera it can be assumed that leukaemia-associated antigens on ALL cells are of fetal origin.     

Absorption of antisera for studies on specific enzyme turnover.

Antisera were raised to acetyl-CoA carboxylase and 6-phosphogluconate dehydrogenase from mammary glands of lactating rabbits, and cytochrome oxidase from rat liver. The enzymes were all highly purified but gave rise to multispecific antisera when tested against tissue extracts. Absorption procedures were devised to free the antisera of contaminating antibodies. Antisera to acetyl-CoA carboxylase and cytochrome oxidase were absorbed with fractions discarded during enzyme purification. The antiserum to 6-phospho-gluconate dehydrogenase was absorbed with a tissue extract from an early stage in mammary-gland differentiation. Monospecific antisera are essential for enzyme turnover studies and therefore antisera should be extensively tested and absorbed before use. A general procedure for the absorption of antisera to purified enzymes has been devised on the basis of accepted principles of antisera absorption.     

Orientation of rat liver cytochrome c oxidase subunits investigated with subunit-specific antisera

The orientation of rat liver cytochrome c oxidase subunits in the inner mitochondrial membrane was investigated with monospecific antisera against subunit II and nine nuclear-coded subunits. Mitoplasts were incubated with the antisera and the amount of bound antibodies was determined either directly with fluorescein-conjugated protein A or indirectly by back-titration of unbound antibodies with a nitrocellulose immunoassay. All subunits were found oriented to the cytosolic side, except subunits VIb and VIIc which did not react with their corresponding antisera. Antisera against subunits I, III and Vb were not available.     

Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.     

Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase

Rat liver cytochrome c oxidase was separated by SDS-gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIIa, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : II. Immunochemical Study of Larval and Adult Hemoglobins of Rana catesbeiana

Rabbit antibodies were prepared against the major hemoglobin components of the larval and adult stages of R. catesbeiana. The properties of the antisera were studied by double immunodiffusion, precipitation, and complement fixation. The antisera to tadpole and frog hemoglobins did not cross-react with either hemoglobin or apohemoglobin. The anti-serum against frog hemoglobin was used for the detection of frog hemoglobin in tadpoles undergoing either natural or thyroxine-induced metamorphosis. It was shown that frog hemoglobin is detectable first in the liver, indicating that the liver is the site of erythrocyte maturation during metamorphosis.     

Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver

Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver beta-glucosidase, beta-galactosidase, alpha-mannosidase, beta-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM(2)-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states.     

Glycogen synthase from human and bovine polymorphonuclear leukocyte Immunochemical characterization and comparison to glycogen synthase from rat and rabbit muscle and liver cells

Glycogen synthase from human and bovine polymorphonuclear leukocytes was purified to homogeneity. Rabbit antisera were raised against the two glycogen synthases and used for immunochemical analysis. Western blotting analysis showed that the subunit of glycogen synthase in crude homogenates of human and bovine leukocytes in both cases has an M_r of 85 000. The existence of a cross-reactivity between the two enzymes and the corresponding antisera demonstrates immunological similarities between bovine and human leukocyte glycogen synthase. In addition, both antisera recognize glycogen synthase in crude cellular extracts from rabbit and rat liver and from skeletal muscle. Leukocyte glycogen synthase, therefore, cannot be classified as either muscle (M-type) or liver (L-type) glycogen synthase and our results do not support the proposed immunochemical distinction between M- and L-type glycogen synthase.     

Antisera to individual histone fractions of the calf thymus. II. A study of the immunochemical specificity of the histones by an indirect immunofluorescence method]

Immune antisera to 5 fractions (H1, H2a, H2b, H3, H4) of calf thymus histone were assayed using indirect immunofluorescence (IIF). The analysis of such sera by this technique, as well as the data on complement fixation obtained previously, show that these antisera are highly active and specific for various test-objects: thymys, liver nuclei of rat, chicken, and calf, chicken erythrocytes, metaphase chromosomes of mouse fibroblasts. These antisera are of importance for the evaluation of species- and tissue-specificity of different histone fractions. Using the IIF reaction, a comparison was made between the nucleosome fraction H3, which is evolutionary stable, and fraction HI from calf thymus, rat and chicken liver, and chicken erythrocytes.     

L-type glycogen synthase. Tissue distribution and electrophoretic mobility

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.     

Selective changes in protein kinase C isoenzymes in rat liver nuclei during liver regeneration.

Using isoenzyme-specific antisera, protein kinase C (PKC) alpha and PKC delta were detected in total liver homogenate and in isolated nuclei. PKC beta I, beta II, epsilon, epsilon', and zeta were not detected. During liver regeneration, nuclear PKC alpha levels decreased while PKC delta levels increased. These studies demonstrate, for the first time, the presence of a calcium-independent PKC isoenzyme in liver nuclei and suggest that PKC alpha and PKC delta may have different roles in liver regeneration and cell proliferation.     

Erythrocyte Membrane-specific Antigens common to Several Species of Rodentia

THE rare existence of organ-specific antisera transcending species differences was pointed out by Coombs¹. Similar new organ, or “erythrocyte-specific”, antisera reacting selectively to the erythrocytes of several species of Myomorpha are described here. The antisera, “anti-A”, were prepared by the method of Adachi and Furusawa²: adult guinea-pigs were sensitized with the “antigen-A” (the protein portion of dd mouse erythrocyte membrane). The detailed procedure for preparing labelled antibody, the staining method and its specific reactivity to mouse erythrocytic cells have been reported before³. Circulating erythrocytes from various animals were stained with the fluorescein isothiocyanate-labelled “anti-A”. Smeared cell preparations were fixed with acetone at ? 20° C for 20 min and incubated with the labelled antisera, which had been thoroughly absorbed with the emulsion of mouse liver and kidney to remove the cross-reacting antibodies, at 37° C for 90 min. A light microscope with a darkfield condenser was used for the fluorescent observations using Orsen's interference filter system. Clear fluorescent reactions, though of varied intensities, were observed with those animals classified as Myomorpha, that is, mouse, rat, golden hamster and Chinese hamster (Table 1, Fig. 1). Mammalian erythrocytes other than those of the guinea-pig used as the sensitized animal showed faint fluorescences. Nucleated erythrocytes of the chick, quail, turtle, toad and goldfish were negative to the reaction. The erythrocyte-specific reactivity of the labelled antisera was examined with the liver, kidney and thymus cells of the animals whose erythrocytes displayed intense fluorescences; they showed, however, no fluorescence.     

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.     

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.     

The predicted proteinase furin is not the hepatic proalbumin convertase.

Rat and chicken liver microsomal membranes were used to investigate the relationship between proalbumin processing activity and the predicted proteinase furin. Two polyclonal antisera directed against the predicted catalytic domain of furin showed the highest level of immunoreactivity in a microsomal fraction that had minimal proalbumin converting activity. Extracts of the fraction containing most converting activity lacked detectable furin. In addition, the proalbumin convertase was not inhibited by the anti-furin antisera. These results strongly suggest that furin is not responsible for the in vivo cleavage of proalbumin.     

Specificity of antisera directed against rat liver cytochrome P-448

A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b₅, NADH-cytochrome b₅ reductase, NADPH-cytochrome c reductase or epoxide hydrase.