Immunochemical characterization of casein from rabbit mammary gland.

1. Excellent precipitating antibodies to rabbit recombined casein polypeptides were obtained in a sheep after 8 weeks of immunization with rabbit recombined polypeptides coupled to Sepharose-albumin. 2. The antiserum was assessed for specificity by several immunochemical techniques and was monospecific when tested against acid-precipitated casein, recombined casein and extracts of lactating rabbit mammary tissue. 3. A specific anti-casein immunoglobulin fraction was prepared by immunoadsorption of the antiserum by using Sepharose-recombined casein as immunoadsorbent. 4. The specific anti-casein immunoglobulin was used to prepare a Sepharose-anti-casein immunoadsorbent for the isolation of casein from extracts of rabbit mammary tissue.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

Functional characterization of beta-adrenoceptor subtypes in rabbit right atria

The advent of radioligand binding studies has allowed the classification of receptor subtypes in various tissues. However, the presence of a receptor subtype in a heterogenous tissue does not insure that the receptor has a significant physiological role. beta 1- and beta 2-Adrenoceptors have been reported to coexist in the rabbit right atria. The purpose of the present investigation was to determine the physiological role of beta-adrenoceptor subtypes in catecholamine-induced chronotropic responses in the rabbit right atria through comparison of data from functional and radioligand binding studies. Rank order of potency was determined using isoproterenol, epinephrine and norepinephrine for both chronotropic and inotropic responses in the rabbit right atria and right ventricular papillary muscles, respectively. These studies indicated that the beta 1-adrenoceptor was primarily responsible for catecholamine-induced responses. Next, the beta 1-selective antagonist, atenolol, was found to inhibit the chronotropic responses of the nonselective beta-agonist, isoproterenol, and the beta 2-selective agonist, terbutaline, to the same extent. These data indicate that terbutaline produces its chronotropic effects in the rabbit right atria through stimulation of beta 1-, not beta 2-adrenoceptors. Finally, competition studies for [125I]iodocyanopindolol and the relatively selective beta 1- and beta 2-adrenoceptor antagonists (ICI 89406 and ICI 118551, respectively) indicated that the ratio of beta 1- to beta 2-adrenoceptor subtypes is 6:1. It is concluded that while both receptors may be present in the rabbit right atria, the beta 1-adrenoceptor is the predominant subtype both in density and physiological significance, while the beta 2-adrenoceptor plays little, if any role, in the chronotropic responses induced by catecholamines.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium

CGP 42 112 A and DuP 753 block [125I]-angiotensin II binding in rabbit ventricular myocardial membranes in a clearly biphasic manner, indicating the existence of high- and low-affinity sites for these highly selective agents. Assays using concentrations of either agent large enough to prevent high-affinity binding show that their respective high-affinity sites are distinct, and each corresponds to the low-affinity site of the other. The two receptor subsets, present in nearly equal proportions, are also distinguishable by their different sensitivities to dithiothreitol. These findings afford strong evidence for the existence of two distinct angiotensin II receptors in rabbit myocardium, corresponding to the A and B subtypes recently described in adrenals.     

Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide 'maps' prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.     

Immunochemical purification and characterization of ovine alpha-fetoprotein.

Ovine alpha-fetoprotein was successfully isolated from fetal sheep serum by using rabbit anti-ovine alpha-fetoprotein linked to an agarose immunoadsorbent column. Antibody used in this affinity chromatography column was produced by immunizing a rabbit with highly purified alpha-fetoprotein-antibody complex to yield a monospecific antiserum to ovine alpha-fetoprotein. Following affinity chromatography, alpha-fetoprotein was further purified by preparative polyacrylamide disc gel electrophoresis ultimately yielding a 105-fold purification. The purified alpha-fetoprotein was homogeneous on analytical polyacrylamide disc gel electrophoresis. Ovine alpha-fetoprotein was found to be immunochemically related to human alpha-fetoprotein and to exhibit a molecular weight and amino acid composition similar to other mammalian alpha-fetoproteins.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Immunochemical analysis of cartilage proteoglycans. Radioimmunoassay of the molecules and the substructures.

Antibodies specifically reacting with the link proteins, the hyaluronic acid-binding region and chondroitin sulphate-peptides were used to design specific radioimmunoassay procedures. The sensitivity of the method used for the link protein was about 20 ng/ml, and the other two components could be determined at concentrations of about 2 ng/ml. The radioimmunoassay procedures were tested by using proteoglycan subfractions or fragments thereof. The procedures used to quantify link protein and hyaluronic acid-binding region showed no cross-interference. Fragments of trypsin-digested proteoglycan monomers still reacted in the radioimmunoassay for hyaluronic acid-binding region. Subfractions of proteoglycan monomers separated according to size had a gradually higher relative content of the hyaluronic acid-binding region compared with both chondroitin sulphate-peptides and uronic acid, when the molecules were smaller. The proteoglycans therefore may contain a variably large chondroitin sulphate-rich region, which has a constant substitution with polysaccharide side chains.     

Immunochemical characterization of rat testicular asparagine synthetase.

We studied immunochemical properties of rat testicular asparagine synthetase. Western blot analysis of testis extract with polyclonal antibody raised against purified asparagine synthetase revealed an immunoreactive band at 62 kDa. The pancreas, brain, thymus, and spleen also showed 62-kDa bands. The intensities of these bands were roughly proportional to the specific activities of the enzyme in these tissues. The antibody showed some degree of cross-reactivity to asparagine synthetases from human, beef, pig, mouse, guinea pig, chicken, and frog, but not carp. But the enzyme from human HL-60 cells and lower vertebrates reacted with the antibody less strongly than enzyme from rats. The N-terminal amino acid sequence of the enzyme, determined by the Edman degradation method, in 10 recovered residues was identical to that of human asparagine synthetase deduced from corresponding cDNA (I.L. Andrulis et al., 1987, Mol. Cell. Biol. 7, 2435-2443). Immunohistochemical staining of the testis showed the presence of asparagine synthetase mainly in Sertoli cells in the seminiferous tubules.     

Immunochemical characterization and cellular localization of pepsinogens in cat and dog.

The antigenic relationships and cellular localization of cat and dog pepsinogens were investigated by electrophoretic analysis, immunodiffusion, immunoelectrophoresis, immunoabsorption, and by immunofluorescence, respectively. Rabbit antiserum to human and hog group I (Pg I) and group II pepsinogens (Pg II) had been previously prepared. Electrophoretic analysis revealed at least eight distinct proteases in extracts of gastric and proximal duodenal mucosa, resistant to alkalinization but destroyed by sequential accidification and neutralization. Rabbit antiserum to Pg I (anti-Pg I) and Pg II (anti-Pg II) produced a single precipitin arc against each extract forming a line of nonidentity. Immunoelectrophoresis of extracts produced a single precipitin arc against anti-Pg I or anti-Pg II. The specificity of the antibodies for the group I or group II pepsinogens was confirmed by immunoabsorption. By immunofluorescnece, both Pg I and Pg II were present in mucous neck and chief cells in fundic mucosa, in the pyloric gland cells in antral mucosa, and Brunner's glands in the proximal duodenum. The results indicate that canine and feline pepsinogens are electrophoretically heterogenous, that canine and feline Pg I share antigenic determinants with each other but not with Pg II, that a similar positive relationship exists for Pg II, and that both Pg I and Pg II are localized to the peptic cell mass, consisting of four types of cells.     

Immunochemical and biochemical distinction of subtypes of atrial natriuretic peptide receptor

The presence of subtypes of atrial natriuretic peptide (ANP) receptor has been demonstrated by examining the immunological features and ligand specificities of the receptors in various bovine tissues. The antibody probe used was the antiserum raised against the bovine lung ANP receptor, and the tissues examined for the possible presence of different types of ANP receptor were the lung, kidney, adrenal cortex, ovary, choroid plexus, and vascular tissues. When incubated with Triton extracts of these tissues, the antiserum strongly cross-reacted with the ovary, kidney, and choroid plexus receptors as well as the homologous lung receptor (type I). The adrenal and vascular receptors were recognized only weakly, however, suggesting the presence of distinct ANP receptors (type II). In support of this immunochemical subtyping, type I and type II receptors showed a marked difference in their ability to bind the ANP analog atriopeptin I (ANP5-25): type I receptors in the lung exhibited a moderate affinity for atriopeptin I with a KD of 10(-9) M; however, type II receptors in adrenal and artery showed only a weak affinity for the analog with a KD of 10(-6) M. Structural analysis of affinity-labeled ANP receptors by SDS-PAGE indicated that type I and type II receptors have similar disulfide-linked dimeric structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.     

Venom exonuclease. III. Immunochemical characterization and modification by specific antibody

The immunoelectrophoretic purity of the exonuclease preparation, isolated from Crotalus adamanteus venom according to a procedure previously published (Dolapchiev, L.B., Sulkowski, E. and Laskowski, M., Sr. (1974) Biochem. Biophys. Res. Commun. 61, 271-281), is reported. The enzyme showed one precipitin line against antibody prepared against partially purified venom. The exonuclease is unstable in dilute (1.25 microgram/ml and below) solutions. Bovine serum albumin stabilized the enzyme nonspecifically whereas the homologous antibody demonstrated a specific stabilizing effect under the same conditions. The binding of the anti-enzyme with the enzyme caused inhibition of both its activities--phosphodiesterase and pyrophosphatase. The inhibition of the exonuclease when attacking high molecular weight substrates is similar to the above and is of the same noncompetitive type. The thermal stability of venom exonuclease is reported.