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E Herbert

Publications and source records attributed to E Herbert.

At least 91 records · Page 5Linked to original sources
PubMed1979

Molecular weight forms of adrenocorticotropic hormone secreted by primary cultures of mouse anterior pituitary.

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.

Adrenocorticotropic Hormone↗
PubMed1979

Processing of common precursor forms of adrenocorticotropin and endorphins in cultures of mouse pituitary cells and in mouse pituitary.

The initial steps in the processing of the common precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH) in mouse pituitary cells (AtT-20) have been investigated. Three forms of the precursor have been resolved by sodium dodecyl sulfate (NaDodSO4) polyacrylamide gel electrophoresis with apparent molecular weights of 29,000, 32,000 and 34,000 (29K, 32K, and 34K ACTH-endorphin). The three precursor forms have a very similar peptide backbone, but their carbohydrate content differs. In particular, a tryptic glycopeptide has been observed in 32K ACTH-endorphin which is not present in 29K ACTH-endorphin and has been identified as a tryptic peptide containing the alpha(22--39) sequence of ACTH. Pulse chase and continuous-labeling studies with radioactive amino acids and sugars suggest that the 29K form is converted to the 32K and 34K forms of the precursor by the addition of carbohydrate. The glycopeptide and pulse chase studies suggest that 29K ACTH-endorphin can either be converted to 4.5K ACTH by proteolytic processing or to 32K ACTH-endorphin by the further addition of carbohydrate.

Adrenocorticotropic Hormone↗
PubMed1978

Steps involved in the processing of common precursor forms of adrenocorticotropin and endorphin in cultures of mouse pituitary cells.

The initial steps in the processing of the common precursor to adrenocorticotropin (ACTH) and endorphin in mouse pituitary tumor cells (AtT-20) have been investigated. Three forms of the precursor have been resolved by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with apparent molecular weights of 29 000 (29K ACTH-endorphin), 32 000 (32K ACTH-endorphin) and 34 000 (34K ACTH-endorphin). These forms have a similar peptide backbone, but their carbohydrate content differs. In particular, a tryptic glycopeptide has been observed in 32K ACTH-endorphin which is not present in 29K ACTH-endorphin and has been identified as the tryptic peptide containing the alpha(22--39) sequence of ACTH. Similar heterogeneity in carbohydrate has been observed in some of the smaller molecular weight forms of ACTH which are resolved by NaDodSO4 gel electrophoresis. Pulse chase and continuous labeling studies using radioactive amino acids and sugars suggest that the 29K ACTH-endorphin is converted to 32K and 34K ACTH-endorphin by the addition of carbohydrate. The glycopeptide and pulse chase studies suggest that 29K ACTH-endorphin is at a branch point in the processing pathways. It can either be converted to 4.5K ACTH by proteolytic processing or to 32K ACTH-endorphin by the further addition of carbohydrate. The 32K ACTH-endorphin can then be converted to 13K ACTH, the glycosylated form of 4.5K ACTH (Eipper, B.A., & Mains,, R.E. (1977) J.Biol. Chem.252, 882), by proteolytic processing. A comparison of the distribution of the different molecular weight forms of ACTH and endorphin in mouse pituitary extracts and in the mouse pituitary tumor cells reveals that the pituitary contains all of the forms of ACTH and endorphin seen in the tumor cells, including the three forms of the ACTH-endorphin precursor. However, the molecular weight distribution of the forms in the anterior lobe is very different from that in the intermediate lobe of mouse pituitary.

Adrenocorticotropic Hormone↗
PubMed1978

Coordinate control of corticotropin, beta-lipotropin, and beta-endorphin release in mouse pituitary cell cultures.

Hypothalamic extract stimulates the release of corticotropin (ACTH) and endorphins 2.5- to 30-fold in mouse pituitary tumor cell cultures (AtT-20/D(16v) line) and primary cell cultures from mouse anterior pituitary. ACTH and endorphin activities were measured by radioimmunoassay and immunoprecipitation. Pretreatment of tumor cell cultures with 1 muM dexamethasone reduced the stimulatory effect of the extract on release of ACTH and endorphins. Pretreatment of primary cell cultures with 10(-6) M dexamethasone reduced the stimulatory effect of both vasopressin and the extract on the release of ACTH and endorphins. Release of ACTH and endorphin was coupled in both kinds of cultures in the basal, stimulated, and inhibited states. The molecular weight forms of ACTH and endorphin in tumor cell culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Radioimmunoassay and immunoprecipitation show that the 13,000-dalton and 4500-dalton forms of ACTH were present in about equal amounts in medium from cultures incubated with or without hypothalamic extract for 15 min, 30 min, or 2 hr. Smaller amounts of the high molecular weight forms of ACTH (20,000- to 23,000-dalton and 31,000-dalton ACTH) were observed in the culture medium at these times. The predominant forms of endorphin released after 20 min or 3 hr of incubation had molecular weights of 31,000, 11,700 (beta-lipotropic hormone-size material) and 3500 (beta-endorphin-size material). No degradation of the forms of endorphin released into the culture medium was observed after incubating the culture medium for 1.5 hr in the absence of cells. The proportions of the different forms of endorphin and ACTH present in the culture medium resembles that seen in cell extracts.

Adrenocorticotropic Hormone↗
PubMed1978

Reversal of dexamethasone inhibition of adrenocorticotropin release in a mouse pituitary tumor cell line either by growing cells in the absence of dexamethasone or by addition of hypothalamic extract.

Release of ACTH by a mouse pituitary tumor cell line )AtT-20/D-16v) is inhibited by 10(-8)-10(-6) M 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-trihydroxy-1,4-pregnadiene-3,20-dione (dexamethasone). Dexamethasone does not inhibit cell growth in this concentration range. Cortisol and corticosterone are almost as potent as dexamethasone in inhibiting ACTH release, whereas 17 beta-estradiol and testosterone have no effect. In rapidly growing cultures of tumor cells removal of dexamethasone leads to complete reversal of the inhibitory effect of the steroid on ACTH accumulation in culture medium within 4-5 days (3.5-4 generation times). The extent of reversal of the dexamethasone effect in slowly growing cultures (generation time 96-150 h) and in rapidly growing cultures (24-30 h) is proportional to the amount of growth that takes place in the absence of dexamethasone. Addition of hypothalamic extract to dexamethasone-treated cultures and to untreated cultures stimulates the release of ACTH 4- to 8-fold. The response occurs within 15 min after the addition of the extract and is dependent on the dose of the extract.

Adrenocorticotropic Hormone↗
PubMed1977

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.

Adrenocorticotropic Hormone↗
PubMed1977

Characterization of a common precursor to corticotropin and beta-lipotropin: identification of beta-lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system.

Radioactive proteins synthesized in an mRNA-dependent reticulocyte cell-free system under the direction of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either alpha(1-24) corticotropin or beta-endorphin [beta(61-91) lipotropin]. Each immunoprecipitate was fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and shown to contain only one labeled protein with an apparent molecular weight of 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin and beta-lipotropin molecules isolated from the gels demonstrated that the two proteins had the same lysine, methionine, and tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromatographic mobilities as marker tryptic peptides from bovine beta-melanotropin and porcine beta-endorphin. The identification of these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The beta-lipotropin tryptic peptides were also shown to be located carboxy terminal to the corticotropin tryptic peptides.

Adrenocorticotropic Hormone↗
PubMed1976

Nepsilon-acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acids.

Unfractionated Escherichia coli tRNA has been aminoacylated with lysine and preferentially acetylated at the epsilon-amino nitrogen of lysine by reaction with N-acetoxysuccinimide. After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were Nepsilon-acetyl-Lys-tRNA. Post-ribosomal supernatant enzymes would not deacylate Nepsilon-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which had been exposed to the acetylation reaction conditions. Poly(rA) stimulated the binding of Nepsilon-acetyl-Lys-tRNA to E. coli ribosomes. At the ribosome and tRNA concentrations used, Nepsilon-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound one-half as well as Lys-tRNA. Both Lys-tRNA and Nepsilon-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 30 mM Mg2+. When Lys-tRNAE. coli or Nepsilon-acetyl-Lys-tRNAE. coli were added to rabbit reticulocyte cell-free protein synthesizing incubations, the incorporation of either amino acid into protein was complete within 5 min. The final incorporation level of the analogue was 82% that of the unmodified lysine. After protein synthesized in the presence of Nepsilon-acetyl-[14C]Lys-tRNA had been digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as Nepsilon-acetyllysine. Gel filtration of the post-ribosomal supernatant revealed that most of the Nepsilon-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin.

Animals↗
PubMed1975

Ribosomal subunit localization of hemoglobin messenger RNA.

When rabbit reticulocyte polyribosomes are treated with 0.5 M KCl, they dissociate into subunits and release a protein fraction which is required for peptide chain initiation in a cell-free system using KCl-treated subunits as the source of ribosomes. Three independent methods were used to determine the fate of mRNA after KCl treatment of the subunits. These three methods (sucrose gradient analysis of RNA after dissociating it from protein with sodium dodecylsulfate, acrylamide gel electrophoresis of RNA and electron microscopic analysis of subunits) all showed the 8--9-S mRNA to be associated with the small subunit, but not the large subunit. Furthermore, no mRNA was found to be associated with either "native" ribosomal subunit in a reticulocyte lysate.

Animals↗
PubMed1972

Coding properties of reticulocyte lysine transfer RNA's in hemoglobin synthesis.

Two isoacceptor transfer RNA's for lysine were found in rabbit reticulocytes. The codon recognition properties of these isoacceptors were studied in hemoglobin synthesis in a cell-free systemn. The two isoacceptors transferred lysine into different sites in hemoglobin, but showed no preference for one chain over the other. Codon cross recognition was less than 4 percent.

Adenosine↗
PubMed1972

Acetylcholine and lobster sensory neurones.

Experiments are presented in support of the hypothesis that acetylcholine functions as a sensory transmitter in the lobster nervous system.1. Several different peripheral sensory structures incorporate radioactive choline into acetylcholine. The preparation most enriched in sensory as opposed to other nervous elements (the antennular sense organs of the distal outer flagellum) does not incorporate significant amounts of glutamate, tyrosine or tryptophan into any of the other major transmitter candidates.2. There is a parallel between the distribution of the enzyme choline acetyltransferase and the proportion of sensory fibres in nervous tissue from many parts of the lobster nervous system.3. Isolated sensory axons contain at least 500 times as much choline acetyltransferase per cm of axon as do efferent excitatory and inhibitory fibres.4. Abdominal ganglia and root stumps show a decline in the rate of incorporation of choline into acetylcholine 2 to 8 weeks after severing the first and second roots bilaterally (leaving the connectives and third roots intact). Extracts of the root stumps exhibit a significantly lower level of choline acetyltransferase 2 weeks after this operation.5. Curare and atropine partially block an identified sensory synapse in the lobster abdominal ganglion.

Acetylcholine↗