On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--I. Recognition of a small-molecular weight (mol. wt 11,000) protein

1. An extract from the livers of both normal chickens (N) and chickens infected with avian erythroblastosis virus (Eb) contains a small molecular weight protein (SMWP, mol. wt 11,000). 2. Double immunodiffusion studies with rabbit antiserum against fowl serum proteins shows a precipitin arc for SMWP in N and Eb extracts, which is continuous with one from one of the normal chicken serum proteins. 3. When treated with 60% saturated ammonium sulphate the SMWP in the liver extracts divides between the precipitate and the supernatant although the specific serological activity of Eb extracts (gag--or COFAL--determined antigenic activity) is restricted to the precipitated SMWP fraction. 4. The COFAL activity of Eb liver extracts could be associated with SMWP by its attachment to this protein, or this phenomenon of "association" could represent the result of changes in synthesis of SMWP or post-synthetic changes.     

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis-IV. Homology of small molecular weight (mol.wt 11,000) protein with serum albumin based on the content of amino acid residues

A small molecular weight (mol.wt 11,000) protein (SMWP) was obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. SMWP, which had been shown to have no biochemical homology with beta 2-microglobulin, is homologous with chicken serum albumin. SMWP shows a similar order of homology with another small molecular weight protein isolated by others from chicken plasma. Like chicken albumin, SMWP is more closely homologous with bovine albumin than with human albumin.     

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--V. Studies on an immunological relationship of a small molecular weight liver protein (SMWP) to serum albumin

An extract of the livers of normal chickens (N) and chickens (Eb) infected with avian erythroblastosis virus (EbV) contained a small molecular weight protein (SMWP, mol. wt 11,000). When the extract was not dehaeminized, SMWP in agarose electrophoresis was shown to have peroxidase activity probably due to the presence of haem. When this non-dehaeminized extract was chromatographed on Con-A Sepharose neither SMWP nor an antigen (EbAg) present in Eb livers were retained. The association of EbAg with SMWP is still unexplained. Immunoelectrophoresis shows a reaction of identity between chicken SMWP and serum albumin. Chicken SMWP is thus not a beta 2-microglobulin. The finding of an immunological relationship between SMWP and albumin confirms the biochemical homology of SMWP with serum albumin in terms of amino acid residues. It has been suggested that SMWP may be a precursor or fragment of albumin but the possibility of its being a distinct entity, a microalbumin, should not be discounted.     

On the possible presence of beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis--II. Amino acid analysis of the protein

1. Amino acid analyses are presented for a small molecular weight (mol. wt 11,000) protein (SMWP) obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. It is resistant to acid denaturation and was isolated following acidification of liver homogenates and removal of haem and lipids. 2. Close homology exists between SMWP of fowl and human liver, and a protein of the same size obtained from chicken serum, (similar in size to human beta 2-microglobulin). 3. Human beta 2-microglobulin, virus core proteins and chicken prealbumin show a lower order relationship. 4. The amino acid residues of SMWP from healthy and erythroblastosis infected chicken livers are identical despite immunologic differences.     

Rat testicular testibumin is a protein responsive to follicle stimulating hormone and testosterone that shares immunodeterminants with albumin

During a search for hormonally responsive products in media from Sertoli cell enriched cultures, a major follicle stimulating hormone responsive and testosterone-responsive protein was identified and designated CMB-1. The results of the present study indicate that this protein is related immunologically to rat albumin and rat alpha-fetoprotein (AFP) and is concentrated in the testis of the adult rat. CMB-1 was therefore termed testibumin. Testibumin was purified from Sertoli cell enriched cultures to apparent homogeneity by sequential high-performance liquid chromatography on anion-exchange, chromatofocusing, gel permeation, and hydroxylapatite columns. The purified protein consists of two concanavalin A (Con A) reactive forms: one which does not interact with Con A and the other which binds to this lectin and is eluted with methyl alpha-mannoside. Testibumin is a monomer with an apparent molecular weight of 69,000 and a pI ranging between 4.5 and 4.85. The heterogeneity of this protein was further demonstrated by crossed-immunoelectrophoresis and two-dimensional gel electrophoresis. A monospecific antiserum and highly purified testibumin were used to develop a specific radioimmunoassay which permitted studies of the hormonal responsiveness of Sertoli cell enriched culture and of the content of testibumin in the reproductive tract fluids in vivo. Even though testibumin was found in serum of both sexes, it was highly concentrated in the testicular and epididymal compartments in adult rats. This protein was compared to rat serum albumin and rat AFP immunologically. With the use of immunoblots, antiserum developed against testibumin showed partial cross-reactivity with albumin and AFP when these latter proteins were denatured and were present in amounts several orders of magnitude greater than testibumin. The extent of this cross-reactivity was then examined by comparing the ability of native and S-carboxymethylated albumin to compete with 125I-testibumin for binding to a monospecific testibumin antiserum. It was shown that the unfolded derivative of albumin showed partial cross-reactivity with testibumin. We conclude that testibumin is immunologically related to albumin and AFP as these latter proteins are related to one another and that testibumin is possibly the homologue of albumin in the seminiferous tubular compartment.     

Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.     

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.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.     

Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein.

Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related.     

Two major serum components antigenically related to complement factor H are different glycosylation forms of a single protein with no factor H-like complement regulatory functions.

Factor H is a 150-kDa serum glycoprotein with key regulatory functions in the alternative pathway of complement activation. Two glycoproteins with a molecular mass of approximately 42 and 37 kDa that react with an antiserum against factor H were purified from human plasma. The two glycoproteins have identical N-terminal amino acid sequences but differ in glycosylation. Sequence comparisons indicated that they both correspond to a 1.4-kb mRNA recently cloned from human liver cDNA. The serum concentration of the two glycoproteins together was estimated to be approximately 40 mg/liter. They were found not to exert factor H-like regulatory functions in the alternative pathway. Thus, the 42-kDa glycoprotein described here appears to be distinct from the previously characterized factor H-related protein of similar size, suggesting that human serum contains two factor-H related molecules which both have a molecular mass of 41 to 43 kDa but which differ largely in structure.     

Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Senile cardiac amyloid: demonstration of a unique fibril protein in tissue sections.

Antisera were raised against degrading amyloid fibrils isolated from the heart of a patient with senile cardiac amyloidosis (SCA), and from a medullary carcinoma of the thyroid (MCT). The antisera were absorbed and used in indirect immunofluorescence to identify an amyloid fibril protein (ASCA) in heart tissue from patients with senile cardiac amyloidosis and to identify the amyloid fibril protein (AMCT) found in association with medullary carcinomas of the thyroid. Absorbed anti-ASCA antiserum did not react with normal tissue such as heart, liver, spleen, and striated muscle, or with amyloid tissue known to contain amyloid fibril proteins AA, AlambdaI, AlambdaIV, AlambdaV, AMCT or with pancreatic tissue containing islet amyloid deposits. The reactions with senile amyloid he,rt tissue could be blocked completely by degraded amyloid fibrils extracted from senile amyloid heart tissue or by amyloid fibril protein ASCA isolated from such fibrils. The anti-AMCT antiserum showed a similar specific reaction restricted to amyloid associated with MCT. In addition, antisera specific for amyloid fibril proteins AA, AlambdaI, AlambdaIV, and AlambdaV failed to react with senile cardiac amyloid, pancreatic islet amyloid, or medullary thyroid amyloid.     

LABELING WITH 14C AMINO ACIDS OF ALBUMIN-LIKE PROTEIN BY RAT LIVER RIBONUCLEOPROTEIN PARTICLES

Ribonucleoprotein particles were prepared by treatment of rat liver microsomes with detergents and high concentrations of KCl. They were active in incorporating ¹⁴C amino acids into protein when incubated with cell sap together with ATP, GTP, and a system to regenerate the triphosphates. The albumin of the incubation mixture, soluble at 105,000 g, and that of the fraction released by ultrasonication of the particles were studied by immunoelectrophoresis in agar gel. When the ribonucleoprotein particles were incubated with cell sap the immunological precipitation lines formed with antiserum to rat serum albumin were highly radioactive as tested by autoradiography. After zone electrophoresis on cellulose acetate, two immunologically reactive albumins were obtained which differed in their electrophoretic mobility from rat serum albumin. Labeled albumin, when purified on DEAE-cellulose columns, retained its radioactivity as tested by autoradiography following immunoelectrophoresis. On cellulose acetate this purified albumin showed an electrophoretic mobility higher than that of rat serum albumin.     

Immunological characterization of cytochrome H-450 in rat tissues

Rabbit antiserum produced against rat liver cytochrome H-450 was specific for cytochrome H-450. The antiserum did not react with hemolysate, microsomal and mitochondrial fractions of liver, and tissue extracts from heart, lung skeletal muscle, and testis of rat. With the monospecific antiserum, a rocket immunoelectrophoretic assay method was developed for the quantitation of the antigen with a sensitivity of 25 ng. By using rocket immunoelectrophoresis, the total amounts of the antigen found in liver, kidney, and brain of 20 rats were 33.6, 3.6, and 1.3 mg, respectively. It appears that the antigens in liver, kidney, and brain are immunologically identical. From immunological studies with subcellular fractions of rat liver, the antigen was found only in the postmicrosomal fraction. This indicates that the antigen is not a precursor or a proteolytic product of known cytochromes in mitochondria or microsomes. Therefore, cytochrome H-450 is a unique cytosolic protein found in brain, kidney, and liver.     

Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents

In the course of a study of tumor antigens we prepared an absorbed antiserum to a breast tumor that reacted strongly with breast tumor but not with normal breast. The antigen was purified by adsorption to an antibody immunoadsorbent prepared from this antiserum, elution with 2 m potassium thiocyanate at neutral pH, and passage through an immunoadsorbent containing antibodies to human serum. The purified antigen was identified as ferritin by electrophoretic, chemical, and immunological criteria. Isoelectric focusing in acrylamide gels revealed that tumor ferritin contained six bands seen in normal liver ferritin plus a variable number of acidic components not detected in normal liver ferritin. The acidic components were concentrated by chromatography on a DEAE-cellulose column. Similar acidic components described previously in ferritins isolated from cultured human tumor cells, hepatomas, and fetal liver have been designated as “carcino-fetal” ferritins.     

Culture shock. Selective uptake and rapid release of a novel serum protein by endothelial cells in vitro

A novel protein has been purified from fetal calf serum and from serum-free bovine aortic endothelial cell conditioned culture medium. This protein consists of a single polypeptide chain of reduced Mr 70,000 (70K protein) and was separated from bovine serum albumin and other proteins by ion-exchange chromatography and immunoabsorption on Sepharose-coupled anti-70K protein antiserum. The 70K protein was shown to be structurally and immunologically distinct from bovine serum albumin, alpha-fetoprotein, and vitronectin by one- and two-dimensional peptide mapping, amino acid analysis, and enzyme-linked immunosorbent assay and/or immunoblotting. The 70K protein was located in endothelial cell cytoplasmic granules of irregular size and distribution. Metabolic radiolabeling studies showed that the 70K protein was not a biosynthetic product of these cells; its cytoplasmic location was due to a selective uptake from the fetal calf serum in which the cells were initially grown. After subconfluent cultures of endothelial cells were shifted to serum-free medium, nearly 80% of the total 70K protein that was measurable in the medium was released between 0 and 20 min. Moreover, sparse, rapidly proliferating cells released approximately 18-fold more 70K protein within 2 min as compared to dense, nonproliferating cultures. The concentration of 70K protein in fetal calf serum was estimated to be 400-600 micrograms/ml. Proliferating bovine aortic endothelial cells, 24 h after plating at an intermediate density, released approximately 250 pg of 70K protein/cell within the first 20 min after exposure to serum-free conditions. The data provide evidence for a novel protein in serum which is selectively internalized by endothelial cells in vitro and which in turn is released rapidly under conditions such as osmotic imbalance due to serum removal, or during periods of cellular proliferation, conditions which we term "culture shock."     

Rete testis fluid (RTF) proteins: purification and characterization of RTF albumin

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.