Double-antibody Immunoprecipitation Research Articles

Mini-RIA: adaptation of conventional 125I-labeled radioimmunoassay to a 96-tube format

Radioimmunoassay (RIA) employing iodinated ligands represents a popular measurement method for small molecules due to its excellent sensitivity and specificity. Yet performing RIAs of large numbers of tubes remains a tedious laboratory chore due to the need to individually handle tubes multiple times. We here present a method in which conventional 125I-labeled RIA ([ 125I] RIA) is adapted to a microtiter plate format, termed mini-RIA. Tubes are handled in batch for centrifugation or during the separation of antibody-bound ligand from free ligand. A simple draining device for batch decantation of free ligand from 96 minitubes is used. Optimal conditions for the mini-RIA were established using two workup methods—double-antibody immunoprecipitation and direct polyethylene glycol precipitation. Use of the mini-RIA method was found to result in a considerable savings in assay times; in addition, the sensitivity of the mini-RIA was improved over conventional RIA. The mini-RIA is particularly useful for assay of large numbers of samples derived from chromatographic methods, since aliquots can be transferred directly from the fraction collector into the minitubes using multiple channel pipettors. Because the method is flexible with regard to assay workup, we predict that most conventional [ 125I] RIAs can be adapted to the mini-RIA format.

Synthesis of gap junction proteins and collagenases in the preimplantation rat uterus.

Synthesis of gap junction proteins (GJPs) and of collagenases in the rat uterus has been studied under two physiological conditions: various stages of the estrus cycle, and the early pregnancy period. The synthesis has been studied by incubating uterine horns in a short-term tissue culture medium containing radioactively-labeled amino acids, followed by a double antibody immunoprecipitation of the labeled proteins. After exposure of the media to either anti-collagenase IgG(s) or anti-GJPs IgG(s), the final immunoprecipitation was achieved with the use of goat anti-rabbit IgG. Collagenase(s) synthesis was found to reach the peak, during the estrus cycle, at the proestrus stage, while GJP synthesis reached the maximum during the estrus stage. In the preimplantation, pregnant, rat uterus the syntheses of both the proteins reached the respective peak activities on day 4 of pregnancy, about 24 h before the expected time of ovum implantation. A study of the literature reveals that this time coincides with a spurt in exposure of the progesterone dominated uterus to estradiol.

Isoform, kinetic and immunochemical characterization of rodent liver α- l-Fucosidases

1. 1. α- l-Fucosidase from hamster and six inbred mouse strains contains two to three unique basic isoelectric forms (above pI 7.0) in addition to the usual acidic and neutral isoforms from pI 4–7. Rat liver α- l-Fucosidase contains multiple isoforms between pI values of 4.0 and 7.3 whereas guinea pig liver α- l-Fucosidase exhibits a single, broad isoform at pI 5.3. 2. 2. All the α- l-fucosidases have similar K M values (0.05–0.12 mM) for 4-methylumbelliferyl-α- l-fucopyranoside, but pH-activity curves which are significantly different in optima and per cent of optimal activity in the acid region. 3. 3. Double-antibody immunoprecipitation experiments indicate that rodent liver α- l-fucosidase cross react to varying extents with polyclonal antibody against human liver α- l-fucosidase. 4. 4. Hamster, guinea pig and mouse liver α- l-fucosidase exhibit significantly less binding than human and rat liver fucosidases to the agarose- E-aminocaproylfucosamine affinity resin.

Hormonal and non-hormonal protein biosynthesis in the pancreatic beta cell of the intact rat after prolonged hyperglycaemia.

We examined the relative changes in the rates of biosynthesis of (pro)insulin and of non-hormonal beta cell proteins in rats with pronounced hyperglycaemia for up to several days. Labelling of pancreatic cells in vivo eliminated certain pitfalls that we encountered when isolated pancreatic islets from these rats were labelled in vitro. Rats were infused with glucose or buffer solutions for 24 and 72 h. Glucose-infused rats had sustained hyperglycaemia throughout the infusion periods. L[4,5-3H]leucine or L[2,3-3H]tryptophan (an amino acid absent from proinsulin) was injected iv 30 min before the rats were killed. Pancreatic islets were isolated by enzymatic digestion of the pancrease. Pancreatic islets from the rats injected with [3H]leucine were processed for measurement of [3H]proinsulin and [3H]insulin by a double antibody immunoprecipitation procedure. Islets from rats injected with [3H]tryptophan were processed for autoradiography, in order to assess the incorporation of label into non-hormonal sedentary beta cell proteins. Incorporation of [3H]leucine into proinsulin and insulin per beta cell was estimated to be about 2-2.5 (24 h infusion) and 3.5-4 (72 h infusion) times greater in the hyperglycaemic than in normoglycaemic rats. Incorporation of [3H]tryptophan into non-hormonal beta cell proteins showed similar increments in the hyperglycaemic rats. Contrary to our expectation these results indicate that glucose does not exert a significant preferential effect on insulin biosynthesis even after sustained stimulation of the beta cells. Instead, glucose seems to increase equally the incorporation of labelled amino acids into proinsulin and into non-hormonal, beta cell proteins.

Cleavage of newly translated NADPH-cytochrome P-450 reductase in a rabbit reticulocyte lysate cell-free system in the absence of exogenous membranes

In a rabbit reticulocyte lysate cell-free system and using double antibody immunoprecipitation method, newly translated rat liver NADPH-cytochrome P-450 reductase (E.C. 1.6.2.4) was shown to be cleaved in the absence of exogenous membranes. Reductase having a M r=78,000 was shown to be converted to a M r=67,000 upon incubation with either lysate or antisera. Peptide maps of 78,000 dalton reductase and 67,000 dalton protein were identical except for three additional peptides present in the 78,000 dalton reductase map. The cleavage activity associated with the antisera could be prevented by using the IgG fraction, while that associated with the lysate was inhibited by using the protease inhibitors leupeptin, pepstatin or bestatin.

Role of intracellular degradation of epidermal growth factor in mitogenesis

Addition of leupeptin, methylamine and the antitubulin agent nocodazole did not affect the initial rate of association of 125I-labelled epidermal growth factor ( 125I-EGF) to Swiss mouse 3T3 fibroblast cells in vitro, but continued incubation with these drugs (up to 24 h) led to an increase in cell-associated radioactivity in a time- and dose-dependent fashion. Combinations of these drugs caused additive increments in cell-associated and internalized radioactivity. Throughout the incubation period, 81–89% of the cell-associated 125I-EGF was internalized. Upon incubation of 125I-EGF with 3T3 cells in the presence or absence of the three inhibitors of degradation for periods of up to 24 h, and after removal of the surface-bound material, the internalized 125I-EGF was extracted and 42–53% was found to be biochemically intact (by acid precipitation) and 56–65% was antigenically similar to native EGF (using double antibody immunoprecipitation in an EGF radioimmunoassay). The extracted internalized 125I-EGF was capable of binding to fresh 3T3 cells. Despite causing a similar increase in intact internalized 125I-EGF, leupeptin did not interfere with and nocodazole alone or in combination with leupeptin markedly enhanced EGF-stimulated DNA synthesis, whereas methylamine inhibited mitogenesis. These data indicate a dissociation between EGF degradation and DNA synthesis, and are not consistent with the hypothesis that intracellular degradation of EGF is necessary for its mitogenic effects.

Estrogen synthetase stimulation by dibutyryl cyclic adenosine 3',5'-monophosphate in human choriocarcinoma cell culture: the relative biosynthetic rate of the nicotinamide adenine dinucleotide phosphate (reduced form)-cytochrome c reductase component.

Human trophoblast cells, derived from choriocarcinoma (JAr line), in continuous culture with 1 mM (Bu)2cAMP plus 1 mM theophylline (dbT) in the culture medium secrete increased amounts of estrogen compared with cells grown without dbT, and after 72 h with dbT, they demonstrate a 6- to 8-fold increase in the specific activity of estrogen synthetase (aromatase; a cytochrome P-450 monooxygenase enzyme system) and an increased cytochrome P-450 concentration. Furthermore the dbT stimulation of aromatase requires protein and RNA synthesis, suggesting that an increase in the biosynthetic rate of at least one of the aromatase component proteins is involved in the mechanism for dbT stimulation of aromatase. This hypothesis was tested for the NADPH-cytochrome c reductase component of aromatase after 25 h growth with dbT, while aromatase activity is increasing linearly, by pulse labeling control and dbT-grown cells for 4 h with a 3H- or 14C-labeled amino acid mixture and by measuring the relative rate of amino acid incorporation into this aromatase component. A 15–20% stimulation in the biosynthetic rate of NADPH-cytochrome c reductase in dbT-grown cells relative to the biosynthetic rate in control cells was detected using a double-antibody immunoprecipitation procedure with rabbit antiserum made against purified human placental microsomal NADPH-cytochrome c reductase as the primary antibody, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This 15–20% stimulation of the biosynthetic rate of reductase 24 h after addition of dbT cannot account for the approximately 2.5-fold higher rate of aromatase activity increase in dbT-grown cells relative to control cells, unless the NADPH-cytochrome c reductase component is compartmentalized so that less than 10% of the total cellular reductase functions in aromatization. Since we know of no evidence for this kind of partitioning, we conclude that some other protein, either the aromatase cytochrome P-450 or an “activator” protein, is responsible for the aromatase stimulation caused by dbT.

Radioimmunoassay of apolipoprotein A-i. application of a non-ionic detergent (Tween-20) and solid-phase staphylococcus.

We describe two techniques for radioimmunoassay of apolipoprotein A-I (apoA-I) in human plasma, each involving use of a non-ionic detergent, Tween-20, to expose antigenic sites, and one involving "IgG SORB" (a suspension of killed staphylococci) as a solid-phase separator. Tween-20 (3.75 g/L) decreased nonspecific binding and unmasked the antigenic sites on the apoA-I molecule in plasma to the same extent as did a tedious delipidation procedure, without altering the binding affinity between apoA-I and apoA-I antibodies as determined by Scatchard analysis (Ka congruent to 2.83 X 10(8) L/mol). The widely accepted double-antibody immunoprecipitation technique for separating bound and unbound 125I-labeled apoA-I is time-consuming, owing to extended periods of incubation and centrifugation IgG SORB effectively separates bound from unbound 125I-labeled apoA-I and the reaction is complete within 10 min. On comparing concentrations of apoA-I in human plasma by the conventional second-antibody (y) and solid-phase IgG SORB methods (x), we found results by the two techniques to be reasonably identical (r = 0.98, y = 1.2x -- 0.17). The mean concentrations of apoA-I in plasma from 65 normal and five hyperlipidemic patients were 1.33 (SD 0.32) and 0.78 (SD 0.35) g/L, respectively, and apoA-I and high-density lipoprotein cholesterol were significantly correlated (r = 0.72, p less than 0.001).

A rapid immunoadsorbent radioimmunoassay for anti-acetylcholine receptor antibody

Antibody to the human nicotinic acetylcholine receptor has been demonstrated in the disorder myasthenia gravis and is the pathologic factor producing the characteristic symptoms of the disorder. A rapid, quantitative and sensitive radioimmunoassay using human acetylcholine receptor, affinity labeled with iodinated alpha-bungarotoxin, and utilizing the immunoadsorbent protein A-bearing Staphylococcal aureus is described. The assay has proven as effective and more efficient than double antibody immunoprecipitation assays in diagnostic screening, evaluation of various forms of therapy, determining kinetics of receptor and antibody interaction, and the production of antireceptor antibody by peripheral blood lymphocytes in culture.

711 REGIONAL CHROMOSOMAL ASSIGNMENT OF THE STRUCTURAL GENE FOR HUMAN ACID β-GLUCOSIDASE

Human acid β-glucosidase (GBA), the lysosomal enzyme deficient in Gaucher disease, has been mapped to chromosome 1 using somatic cell hybridization techniques. Antibody against purified human GBA was raised in Balb/C mice and a sensitive double antibody immunoprecipitation assay was developed to detect human GBA activity in mouse RAG X human fibroblast hybrids. The mouse anti-human GBA antibody was specific for human GBA and no cross-reactivity between mouse β-glucosidase or several other human enzymes, including neutral β-glucosidase could be detected. Fifty-two primary, secondary and tertiary hybrid lines, derived from three separate fusion experiments, were analyzed for human GBA and enzyme markers for each human chromosome. There was 100% correlation between the presence of human GBA and the presence of human chromosome 1 as determined by cytogenetic and chromosome 1 enzymatic marker (phosphoglucomutase 1 and fumurate hydratase) analyses. All other human chromosomes segregated independently of human GBA. The structural locus for human GBA was further defined to the region 1p11 → lqter by using a RAG X human fibroblast line which carried a mouse-human rearrangement involving human chromosome 1.

Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.

Occurrence and Significance of Antibody to Liver-Specific Membrane Lipoprotein by Double-Antibody Immunoprecipitation Method in Sera of Patients with Acute and Chronic Liver Diseases

A double-antibody immunoprecipitation method was developed for detecting antibody to liver-specific membrane lipoprotein (anti-LSP) in sera of patients with various liver diseases and primary nonhepatic autoimmune diseases. Liver-specific membrane lipoprotein prepared from normal rat livers was labeled with 125I (chloramine-T) and monospecific antibody raised in rabbits. Cross-reactivity and absorption studies demonstrated that the assay used was highly specific. The frequency and titer of anti-LSP were similar for HBsAg-positive and -negative patients with both acute and chronic liver diseases. Patients with chronic active hepatitis had the highest frequenzy (25 of 44 cases, 57%) when compared with those with chronic persistent hepatitis (5 of 23 cases, 22%) and nonalcoholic cirrhosis (8 of 21 cases, 38%). Of the anti-LSP positive cases, the mean titer in patients with chronic active hepatitis tended to be the highest. In patients recovered from acute viral hepatitis, anti-LSP was transiently positive (7 of 20 cases, 35%) in the acute phase. In those who progressed to chronic hepatitis, a late rise as well as an early rise occurred in 6 of 10 patients before the diagnosis was made. Two of 6 patients with primary biliary cirrhosis had anti-LSP, but none of 41 patients with other nonviral liver diseases and none of 60 patients with primary nonhepatic autoimmune diseases. These data indicate that an autoimmune reaction directed against LSP can be initiated during the acute phase of viral hepatitis and it may persist in chronic hepatitis in both HBsAg-positive and -negative cases.

Filtration and Immunoprecipitation in the Elimination of DNA Polymerase Activity Associated with Bacterial Contamination of Sera Positive for Hepatitis B e Antigen and Its Corresponding Antibody

Samples of serum inoculated with Escherichia coli and serum that became contaminated with bacteria after exposure to a laboratory atmosphere demonstrated elevated DNA polymerase activity. The levels of activity were well within the range of values found in hepatitis B e antigen (HBeAg)-positive samples. The bacterial polymerase activity was markedly reduced by a single passage of serum samples through a 0.22-micron Millipore filter prior to analysis. Repeated filtration did not result in a substantial further decrease in polymerase activity. In sera that were heavily contamined with E. coli, however, filtration was not successful in reducing bacteria-associated polymerase activity to a base-line uncontaminated level. In such instances double antibody immunoprecipitation proved effective in elimination of bacterial activity. When bacterial contamination of serum samples is a possibility, specimens should be subjected to either Millipore filtration or immunoprecipitation prior to analysis, particularly when correlation of DNA polymerase activity with HBeAg and its corresponding antibody is attempted.

Synthesis of fibronectin by cultured human endothelial cells.

Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

Synthesis of cold-insoluble globulin by cultured calf endothelial cells.

Radiolabeled amino acids were incorporated into nondialyzable protein by cultures of endothelial cells derived from calf aorta. Antibody prepared against purified bovine plasma cold-insoluble globulin (CIG) formed a strong precipitin line upon immunodiffusion against 3H-labeled proteins from endothelial cell culture media. This precipitin line formed a line of identity with the precipitin line formed by anti-CIG and purified CIG. Double-antibody immunoprecipitation of 3H-labeled protein showed that 36.3% of the radioactivity was specifically precipitated by the anti-CIG antibody. Gel filtration and polyacrylamide gel electrophoresis of the immunoprecipitate indicated that it contained a single protein species whose molecular weight was consistent with that of CIG isolated from plasma. When cultured cells were examined by indirect immunofluorescence microscopy, the endothelial cells stained specifically with anti-CIG antibody. These data indicate that cultured endothelial cells derived from calf aorta synthesize and secrete a protein antigenically similar to CIG.

Intestinal apoproteins during fat absorption.

To compare the roles of apolipoprotein (Apo) A-I, B, and E (or arginine-rich apoprotein, ARP) in the intracellular production of intestinal chylomicrons (and/or VLDL), these apoproteins were localized in rat intestinal mucosa by the light microscope method of indirect immunofluorescence. In addition, tissue levels of ApoA-I and ApoB were measured during fat absorption by radioimmunoassay. Antisera were produced using ApoA-I isolated from rat plasma high density lipoprotein, and ApoB and ARP from plasma VLDL by column chromatography. The apoproteins yielded single bands on polyacrylamide disc gel electrophoresis in urea and in sodium dodecyl sulfate. Anti-apoprotein antisera were produced in rabbits. These antisera appeared to be monospecific on double-antibody immunoprecipitation of 125I-labeled apoproteins. In fasted animals granular staining of ApoA-I was noted in the supranuclear (Golgi) regions of epithelial cells in the top third of the villus. At 30 min, when fat droplets were seen in the supranuclear cytoplasm of the cells along the top two-thirds of the villus, intense ApoA-I staining surrounded droplets in the cytoplasm. At later times when epithelial cells and lamina propria both contained fat droplets, bright ApoA-I stain surrounded many droplets in the supranuclear cytoplasm of cells and in the lamina propria. Over the same period of time, tissue levels of ApoA-I rose 10-fold. The distribution and time-course of ApoB staining was nearly identical with that of ApoA-I. Concomitantly, tissue ApoB levels doubled. By contrast, in fasting rat intestine, staining of ARP was sparse, punctate, and confined to the lower quarter of the villus. After fat feeding, stained droplets were seen only in the lamina propria near the base of the villus even though abundant ARP was found in cells along most of this length of the villus. Stain was never seen to surround any droplets inside cells. Thus, ApoA-I and ApoB appeared to participate in the intracellular assemply of lipoproteins in gut, whereas ARP did not, although ARP was found within mucosal cells. Liver and intestine differed in their stainable contents of ApoA-I and ARP. Whereas intestine stained heavily for ApoA-I and lightly for ARP, liver stained heavily for ARP and lightly for ApoA-I. Both organs stained for ApoB. These findings suggest that there may be some quantitative "specialization" of the two organs which secrete lipoproteins.

Insulin kinetics and distribution in rainbow trout ( Salmo gairdneri)

The plasma disappearance of intravenously-injected 125I-cod insulin and a mixture of 131I-beef insulin and 125I-beef monoiodoinsulin, studied in rainbow trout ( Salmo gairdneri), was found to be multiexponential, exhibiting both a rapid and a slow component. Biological half-disappearance times ( T 1 4 ) and metabolic clearance rates (MCR) varied significantly with three different assay methods for 125I-cod insulin, while the “apparent” distribution spaces (DS) were similar for all methods. The immunoassay technique with anti-fish insulin serum provided a significantly greater discrimination between total radioactivity and 125I-cod insulin radioactivity than either precipitation with 20% trichloracetic acid (TCA) or charcoal adsorption. The labelled beef insulins were assayed by double-antibody immunoprecipitation using anti-beef insulin serum. The MCR for 125I-beef monoiodoinsulin (1.78 ± 0.20 ml/kg/min) was significantly greater than for either 131I-beef insulin (0.58 ± 0.06 ml/kg/min) or immunoassayable 125I-cod insulin (0.74 ± 0.06 ml/kg/min). The results support the current view that monoiodoinsulin is a more valid tracer for insulin kinetic studies than randomly iodinated forms. A tissue distribution study of 125I-cod insulin showed the presence of TCA-precipitable radioactivity in all tissues and fluids studied, but the kidney was found to be the major accumulation site.

Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.

mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement.

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [(14)C]glycine and [(14)C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99-100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3alpha-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. ;non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.

Common precursor to corticotropins and endorphins

Double-antibody immunoprecipitation procedures with antisera to endorphins and to corticotropin (ACTH) were used to study the biosynthesis of these peptides in a mouse pituitary tumor cell line. Cultures were incubated with a (3)H-labeled amino acid, and aliquots of culture medium were immunoprecipitated. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of [(3)H]phenylalanine-labeled immunoprecipitates prepared with endorphin antisera resolved three forms of endorphin with apparent molecular weights of 31,000, 11,700, and 3500; immunoprecipitates prepared with the ACTH antiserum contained four forms of ACTH with apparent molecular weights of 31,000, 23,000, 13,000 and <4500. Sequential immunoprecipitation of culture medium with the ACTH antiserum and then with the endorphin antiserum (or the reverse order) indicated that both antisera precipitated the same 31,000 dalton molecule. Purified pools of the different forms of ACTH and endorphin were prepared by immunoprecipitation and gel filtration. The tryptic peptides found in [(3)H]phenylalanine- or [(3)H]tryptophan-labeled 31,000 dalton ACTH were identical to the tryptic peptides found in digests of 31,000 dalton endorphin labeled with the same amino acid. A tryptic peptide similar to the lipotropin tryptic peptide [betaLPH(61-69)] that contains the opiate-active methionine-enkephalin sequence could be identified in 31,000 dalton ACTH and in all the different forms of endorphin. Most of the peptide cleaved from 31,000 dalton ACTH when it is converted to 23,000 dalton ACTH could be precipitated by endorphin antisera; this 11,700 dalton endorphin molecule is similar to the pituitary hormone betaLPH in size and structure. The 3500 dalton endorphin is similar to beta-endorphin in size and structure. The culture medium from the AtT-20 mouse pituitary tumor cells contained approximately equimolar amounts of ACTH-related peptides and endorphin-related peptides.