Beta-endorphin-like and adrenocorticotropin-like materials in heart tissues of the rat, gerbil, hamster and guinea pig

1. Heart tissues of several rodent species including the rat, gerbil (Meriones unguiculatus), hamster (Mesocricetus auratus) and guinea pig (Cavia porcellus) were extracted with an acetone-water-HCl mixture. An acid acetone powder was obtained by adding a copious volume of acetone to the extract. 2. Rat heart acid acetone powder was subjected to ion exchange chromatography on CM-cellulose. Gerbil heart acid acetone powder was subjected to salt fractionation, gel filtration on Sephadex G-10 and then ion exchange chromatography on CM-cellulose. Hamster and guinea pig heart acid acetone powders were subjected to gel filtration on Sephadex G-25. 3. The fractions were assayed for the ability to stimulate corticosterone production in isolated rat adrenal decapsular (zona fasciculata, zona reticularis and medulla) cells, to displace D-ala2-D-leu5-(tyrosyl-3,5-3H) enkephalin from binding to rat brain membranes, and to inhibit 125I-human beta-endorphin from binding to its antibodies. 4. The widespread occurrence of beta-endorphin-like immunoreactivity among the rat heart CM-cellulose fractions may reflect different species of beta-endorphin. The fraction with the highest beta-endorphin-like immunoreactivity and opiate receptor binding activity was strongly adsorbed on CM-cellulose. 5. In hamster and guinea pig hearts, beta-endorphin-like immunoreactivity and opiate receptor binding activity were distributed among high molecular weight and low molecular weight fractions. 6. In gerbil hearts, opiate receptor binding activity was present in fractions unretarded on Sephadex G-10 (i.e. with a molecular weight greater than 700) as well as in the retarded fractions (i.e. with a molecular weight smaller than 700).(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of corticotropin-like and opiate-like hormones in rat and bovine placental tissues

Acid acetone powder of rat placentas was fractionated on Sephadex G-25 into a void volume peak (R-1) and three retarded peaks (R-2, R-3 and R-4). R-3 contained opiate-like activity and R-4 corticotropin-like activity, suggesting that separate corticotropin-like and opiate-like activities with molecular weight smaller than 5000 were present in rat placentas. Acid acetone powder of bovine placentas contained opiate-like activity which was unretarded on Sephadex G-25. Acid acetone powder of rat brains but not those of lungs, livers or kidneys possessed opiate receptor binding and steroidogenic activities, indicating that the activities in placentas were not due to enzymatically generated artifacts or to peptides contained in blood trapped in the organs.     

Substances with alpha-melanotropin-like immunoreactivity in bovine brains.

1. Bovine cerebral hemispheres were extracted with an acidic medium (acetone-water-hydrochloric acid mixture, 40:5:1 by volume, pH 1.8). The precipitate which formed upon addition of a copious volume of cold acetone to the extract was designated acid acetone powder (AAP). 2. The AAP was then subjected to ion exchange chromatography on carboxymethyl (CM)-cellulose, gel filtration on Sephadex G100 and Sephadex G25, second ion exchange chromatography on CM-cellulose and high performance liquid chromatography. The absorbance of all fractions was measured at 280 nm and their alpha-melanotropin-(alpha-MSH)-like immunoreactivity was monitored with radioimmunoassay. 3. It was found that alpha-MSH-like immunoreactivity and bioactivity (lipolytic activity) was due to low molecular weight materials as evidenced by their retardation on Sephadex G-100 and Sephadex G-25. The immunoreactivity was distributed among fractions adsorbed and fractions unadsorbed on CM-cellulose and also among high performance liquid chromatographic fractions signifying the presence of multiple alpha-MSH-like molecules.     

Adrenocorticotropin- and beta-endorphin-like substances in brains of the freshwater snake Ptyas mucosa

Snake (Ptyas mucosa) brains (400 g) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate (5.6 g) that formed upon addition of five volumes of acetone to the extract, designated acid-acetone powder, was subjected to gel filtration on Sephadex G-25. A large unretarded peak (SB-1) with molecular weight greater than 5000 and a small retarded peak (SB-2) with molecular weight smaller than 5000 were obtained. They were then separately subjected to ion-exchange chromatography on CM-cellulose. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal cells to produce corticosterone. Opiate activity was detected in the fractions by their ability to inhibit the binding of (D-Ala2,D-Leu5)-[tyrosyl-3,5-3H]enkephalin to rat brain membranes and their cross-reactivity in a beta-endorphin radioimmunoassay. Adrenocorticotropic and opiate activities were found to be concentrated in fractions strongly adsorbed on CM-cellulose, which were eluted by combined pH and ammonium acetate concentration gradients. There appeared to be a separation between adrenocorticotropic and opiate activities, suggesting that they were due to separate molecular entities.     

The eel pancreas contains opiate-like but not corticotropin-like materials

Eel pancreatic acetone powder was extracted with an acetone-water-HCL mixture. An acid acetone powder was formed by addition of a copious volume of cold acetone to the extract. The powder was subsequently subjected to gel filtration on Sephadex G25. Opiate receptor binding activity was present mainly in fractions retarded on Sephadex G25 (MW less than 5,000) although some activity was present in the unretarded fraction (MW greater than 5,000). Steroidogenic activity was absent from all fractions.     

Materials with opiate receptor binding activity in bovine testis and ovine pancreas

Two groups of opiate-like materials, one with a molecular weight equal to or greater than 5000 daltons and another with a molecular weight smaller than 5000 daltons as judged by gel filtration on Sephadex G-25, were detected in bovine testes. The existence of opiate-like materials with a molecular weight smaller than 5000 daltons was demonstrated in ovine pancreas. The pancreatic fraction most strongly adsorbed on CM-cellulose possessed the highest opiate receptor binding activity. Bovine testis contained corticotropin-like material(s) which stimulated corticosterone production by isolated rat adrenal cells.     

Hormones with adrenocorticotropic and opiate-like activities from the carp (Cyprinus carpio) pituitary

Carp (Cyprinus carpio) pituitary acetone powder was extracted with a mixture of water, hydrochloric acid and acetone. An acid acetone powder was formed by adding the pituitary extract into a large volume of chilled acetone and subsequently recovering the precipitate. The powder was subjected to ion exchange chromatography on CM cellulose. Fractions adsorbed on the ion exchanger exhibited ACTH-like activity as evidenced in the ability to stimulate lipolysis in isolated hamster adipocytes and corticosterone production in isolated rat adrenal decapsular cells and also in cross-reactivity in an ACTH-specific radioimmunoassay. A portion of the ACTH-like bioactivity and immunoactivity was unadsorbed on the ion exchanger. Opiate-like activity in opiate receptor binding assay, employing [3H]D-ala2-D-leu5 enkephalin or [3H]naloxone as ligand, also resided in fractions adsorbed on CM cellulose. The data indicate a separation of ACTH-like and opiate-like activities, and the presence of opiate-like molecules with different affinities of binding to mu and delta opiate receptors.     

beta-Endorphin-like and adrenocorticotropin-like hormones in the pituitary of the snake Ptyas mucosa

Snake (Ptyas mucosa) pituitaries were heated, extracted with an acidic medium, and the extract subsequently chromatographed on carboxymethyl cellulose (CMC). Fractions were assayed for their abilities to displace D-ala2-D-leu5-[tyrosyl, 3,53H] enkephalin from binding to rat brain membranes and 125I-beta-endorphin from binding to its antibody, and to stimulate corticosterone production by isolated rat adrenal cells. The fraction unadsorbed on CMC (having the least basic character) had the lowest opiate receptor binding activity and beta-endorphin-like immunoreactivity. The highest steroidogenic activity, opiate receptor binding activity and beta-endorphin-like immunoreactivity were concentrated in a strongly adsorbed fraction. Snake pituitaries were also extracted with Tris-HCl buffer (pH 7.8). The extract from the second procedure was subsequently chromatographed on ConA-Sepharose, ultrafiltered and dialyzed to obtain a nonglycopeptide fraction with a molecular weight between 1,000 and 10,000. This fraction also exhibited steroidogenic and opiate receptor binding activities.     

Adrenocorticotropin- and opiate-like hormones from pituitaries of the sockeye salmon Oncorhynchus nerka

The pituitaries of vitellogenic sockeye salmon (Oncorhynchus nerka) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate which formed upon the addition of a copious volume of acetone to the extract, designated acid acetone powder, was subjected to salt fractionation and desalting, followed by ion-exchange chromatography on CM-cellulose. An unadsorbed fraction (S-1) and four adsorbed fractions (S-2, S-3, S-4 and S-5) were obtained. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal decapsular cells to produce corticosterone and by their immunoreactivities in an adrenocorticotropin-specific radioimmunoassay. The steroidogenic activities of all fractions, except S-4, were blocked by corticotropin inhibiting peptide. Opiate activity was detected in the fractions by their ability to inhibit the binding of either [3H]naloxone or (D-ala2, D-leu5)-[3H]enkephalin to rat brain membranes. There was a discrepancy in the potencies of the five fractions in the two opiate radioreceptor assays, indicating the presence of opiate peptides with different affinities of binding to the micron- and delta-opiate receptors of the rat brain. There was a separation between adrenocorticotropic and opiate receptor binding activities, suggesting that the activities were due to separate molecular entities.     

Corticotropin-like material in pigeon brains

1. Pigeon brain acetone powder was extracted with a mixture of acetone, water and hydrochloric acid (40:21:1 v/v/v). The extract was added to a large volume of chilled acetone. 2. The resultant precipitate was washed with cold acetone and then chromatographed on Sephadex G-25. The void volume peak constituted fraction A. The trailing peak immediately following fraction A was divided into two fractions designated B and C. 3. Fraction B (mol. wt less than 5,000) stimulated corticosterone production in isolated rat adrenal cells while the other fractions were devoid of similar ACTH-like bioactivity. 4. None of the fractions showed activity in the opiate radioreceptor assay or hot plate test, implying the absence or presence of only trace amounts of opiate-like materials. Other possibilities that cannot be ruled out include the presence of molecules with substitutions in the sequence by amino acids that result in little or no activity.     

Isolation, characterization and opiate activity of beta-endorphin from the pituitary gland of the ostrich, Struthio camelus.

1. Avian beta-endorphin was purified from adenohypophyseal glands of the ostrich Struthio camelus by a procedure involving acid/acetone extraction, NaCl fractionation, CM-cellulose chromatography, Sephadex G-50 chromatography and paper electrophoresis (pH 6.7). 2. The 31-amino acid peptide behaved as a single substance during polyacrylamide-gel electrophoresis and isoelectric focusing, the isoelectric point being 8.84. 3. Ostrich beta-endorphin exhibited significant opiate activity in the guinea-pig ileum preparation.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

Thyrotropin-releasing hormone in the gastrointestinal tract.

TRH immunoreactivity has been shown to occur throughout the rat gastrointestinal tract. This immunoreactivity demonstrates parallelism with TRH, is destroyed by fresh human serum, and co-chromatographs with TRH on a Sephadex G-10 column and on a SP Sephadex C-25 column. In addition pancreatic extracts showed bioactivity in a mouse bioassay for TRH.     

Hepatic and renal desamidinases in swine]

By cellulose acetate electrophoresis we have confirmed the presence of two desamidinasic fractions in aqueous extracts of acetone powder from pig liver. Pig kidney extracts have been partially purified by thermal denaturation and chromatography on Sephadex G-200 and D.E.A.E. Sephadex A-50. Michaelis constants of the two eluted arginasic fractions have been determined.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Labeling of beta-endorphin and beta-lipotropin with iodine 125

5 micrograms of human beta-endorphin were labelled with 2 mCi 125I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 microCi/ug. Kept at + 4 degrees C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a 125I beta-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-beta-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 micrograms of human beta-lipotropin were labelled with 0.5 mCi 125I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 microCi/micrograms was obtained. It remained utilizable for 30 days when kept at + 4 degrees C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled beta-LPH was 32% for an anti-beta-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Endorphins in human cerebrospinal fluid

Opiate activity in CSF samples drawn from patients with suspected intracranial hydrodynamic dysfunction has been fractionated on Sephadex G-10 and separated by column electrophoresis in agarose suspension. From the Sephadex G-10 chromatography two receptor active fractions (FI and FII) were recovered. Both FI and FII were further resolved by the electrophoresis. FI separated into at least four components and FII into two components. The study also includes a comparison of the endorphin concentrations in CSF (samples drawn from healthy volunteers) measured by receptorassay with those detected by radioimmunoassay of beta-endorphin, [Met]enkephalin and dynorphin, respectively. The data obtained indicated negligible quantities of the radioimmunoassayable endorphins in the total CSF opiate activity.