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B A Eipper

Publications and source records attributed to B A Eipper.

At least 145 records · Page 8Linked to original sources
PubMed1983

Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.

An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.

Aerobiosis↗
PubMed1983

Phosphorylation of rat and human adrenocorticotropin-related peptides: physiological regulation and studies of secretion.

Possible physiological factors that might control the extent of phosphorylation of ACTH-related peptides in the rat pituitary were investigated both in vivo and with cultured anterior or neurointermediate pituitary cells. Phosphorylated and nonphosphorylated forms of ACTH and corticotropin-like intermediate lobe peptide [CLIP or ACTH(18-39)] were separated by isoelectric focusing or high performance liquid chromatography and quantified by immunoassay. Radiolabeled ACTH- or CLIP-related peptides from cultured pituitary cells incubated in [3H]tyrosine were isolated by immunoprecipitation or immunoadsorption, fractionated as above, and detected by liquid scintillation counting. Both in the animal and in culture, the extent of phosphorylation of anterior pituitary cellular ACTH did not vary when the release of ACTH was stimulated (by metyrapone treatment, corticotropin-releasing factor, or cAMP) or inhibited (by dexamethasone). Long term cultures of rat anterior pituitary cells continued to phosphorylate ACTH in both serum-containing and serum-free medium; the extent of phosphorylation of ACTH rose from about 55% in vivo to 80% within 1 week in culture. By comparison, the extent of phosphorylation of intermediate pituitary CLIP decreased from about 75% in vivo to about 40-50% in culture. The extent of phosphorylation of the ACTH and CLIP secreted by primary pituitary cultures simply reflected the extent of phosphorylation of ACTH and CLIP in the cells, in both basal and stimulated states. Human ACTH(1-39) contains the same amino acid sequence that is phosphorylated in rat and mouse ACTH(1-39). Based on isoelectric focusing and high performance liquid chromatography of the tryptic peptides of human ACTH (with and without alkaline phosphatase treatment), about one third of the ACTH in the human pituitary is phosphorylated.

Adrenocorticotropic Hormone↗
PubMed1981

Primary sequence of two regions of mouse pro-adrenocorticotropin/endorphin.

The amino acid sequences of two previously uncharacterized regions of the mouse anterior pituitary common precursor to adrenocorticotropin (ACTH) and beta-endorphin (pro-ACTH/endorphin) were determined. Portions of the NH2-terminal region of pro-ACTH/endorphin (called the 16K fragment) and the region between ACTH and beta-endorphin (called gamma-lipotropin) were sequenced by Edman degradations of biosynthetically labeled immunoprecipitated proteins and by Edman degradations of purified 16K fragment and beta-lipotropin. With a combination of these two approaches, 29 of the first 34 residues at the NH2-terminal end of the mouse 16K fragment were determined. The NH2-terminal region of the mouse 16K fragment was found to be nearly identical with the homologous porcine and bovine molecules. The complete amino acid sequence of the NH2-terminal region of gamma-lipotropin was determined. In contrast to the highly conserved nature of the 16K fragment, mouse gamma-lipotropin was found to differ substantially from the gamma-lipotropins of other species. Although the NH2-terminal and beta-melanotropin-like regions of the mouse gamma-lipotropin are similar to the corresponding regions of other gamma-lipotropins, the intervening region of mouse gamma-lipotropin is substantially shorter than it is in other gamma-lipotropins. In addition, mouse gamma-lipotropin lacks the pair of basic amino acids that normally mark the proteolytic cleavage site used to produce beta-melanotropin from gamma-lipotropin.

Adrenocorticotropic Hormone↗
PubMed1981

Differences in the post-translational processing of beta-endorphin in rat anterior and intermediate pituitary.

The immunoactive beta-endorphin-related material in extracts of rat anterior and intermediate/posterior pituitary was separated by ion exchange chromatography on sulfopropyl-Sephadex (Zakarian, S., and Smyth, D. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5972-5976). The profile of beta-endorphin immunoactivity in rat anterior pituitary was distinctly different from that found in intermediate/posterior pituitary. In the rat anterior pituitary, most of the immunoactivity co-migrated with synthetic camel beta-endorphin(1-31); in the intermediate/posterior pituitary, only about 10% of the immunoactivity co-migrated with camel beta-endorphin(1-31). In rat anterior pituitary cell suspensions incubated with [3H]tyrosine for 6 h or 48 h, material co-migrating with camel beta-endorphin(1-31) was the major final posttranslational product related to beta-endorphin. This material was reanalyzed by chromatography on sulfopropyl-Sephadex, analyzed by gel filtration on Sephadex G-50 in 6 M guanidine HCl, and digested with pronase and trypsin. In every analysis, the major peak of [3H]tyrosine-labeled anterior pituitary beta-endorphin was indistinguishable from camel beta-endorphin(1-31).

Animals↗
PubMed1981

Further analysis of post-translational processing of beta-endorphin in rat intermediate pituitary.

Rat intermediate pituitary cells maintained in culture synthesize the same forms of beta-endorphin observed in intermediate pituitary extracts. Biosynthetically labeled intermediate pituitary beta-endorphin-sized material was fractionated by ion exchange chromatography on sulfopropyl-Sephadex and the identities of the major peaks were determined by co-chromatography with synthetic marker peptides, gel filtration, and analysis of pronase, chymotrypsin, and trypsin digests. Peaks of alpha-N-acetyl-beta-endorphin(1-27), alpha-N-acetyl-beta-endorphin(1-31), and beta-endorphin(1-31) were identified and a fourth peak (eluting from the sulfopropyl-Sephadex column at 0.18 M NaCl) was tentatively identified as alpha-N-acetyl-beta-endorphin(1-26). Analysis of beta-endorphin synthesized in the presence of [35S]methionine and [3H]histidine confirmed the absence of His in the material eluting at 0.18 M NaCl. Based on both steady labeling and pulse-chase incubations, beta-endorphin(1-31) was the first form of labeled beta-endorphin-sized material to appear in cell extracts. This molecule was quickly N-acetylated on its NH2-terminal tyrosine residue and was then more slowly converted to alpha-N-acetyl-beta-endorphin(1-27) and then to alpha-N-acetyl-beta-endorphin(1-26).

Animals↗
PubMed1981

Comparison of rat anterior and intermediate pituitary in tissue culture: corticotropin (ACTH) and beta-endorphin.

The forms of immunoreactive beta-endorphin-sized material in extracts of anterior and intermediate-posterior pituitary from the rat examined by the ion exchange chromatography method of Zakarian & Smyth. The anterior pituitary primarily contained material that co-migrated with synthetic camel beta-endorphin(1-31), whereas the intermediate-posterior pituitary contained relatively little such material. The majority of immunoactive beta-endorphin-sized peptides in the intermediate pituitary eluted at lower concentrations of NaCl than did camel beta-endorphin. Conditions were developed for the stable, long-term tissue culture of dissociated intermediate-posterior pituitary cells. Extracts of cells maintained in tissue culture for 18 h or nine days had the same content of immunoreactive beta-endorphin, 16k fragment, ACTH(18-39) (or CLIP) and ACTH(17-24). Throughout the nine days in culture, characteristic cells that could be immunostained with antibodies to various regions of pro-ACTH/endorphin were present; during the time in culture, non-reactive background cells multiplied rapidly. The major proteolytic processing of pro-ACTH/endorphin remained characteristic of intermediate pituitary tissue throughout the nine days in tissue culture, and did not become similar to the simpler pattern of proteolytic processing found in the anterior pituitary.

Adrenocorticotropic Hormone↗
PubMed1981

Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells.

The secretion of peptide products derived from pro-ACTH/endorphin was examined with several radioimmunoassays and with polyacrylamide gel analyses of immunoprecipitates of radioactively labeled peptides. In studies using a mouse pituitary tumor cell line the accumulation of each of the four molecular forms of adrenocorticotropic hormone (ACTH) in tissue culture medium was shown to be a linear function of time. No evidence for self inhibition of secretion by accumulated, secreted peptides (i.e., ultra-short feedback) was found. Furthermore, synthetic human ACTH and synthetic camel beta-endorphin did not alter secretion of peptides when added to the culture medium at levels up to 10,000 times physiological. Stimulation of the release of ACTH-, endorphin-, lipotropin-, and 16k fragment immunoreactive material by norepinephrine was fully blocked by cobalt; by this criterion, stimulated release was calcium dependent. All the smaller molecules derived from the pro-ACTH/endorphin common precursor were secreted in equimolar amounts under all circumstances tested, within the precision of these studies (+/- 11%). Norepinephrine and cobalt did not significantly alter the secretion of pro-ACTH/endorphin and ACTH biosynthetic intermediate. The stimulation of secretion by norepinephrine and inhibition of secretion by cobalt was restricted to the lower molecular weight products derived from pro-ACTH/endorphin: glycosylated and nonglycosylated ACTH(1-39); beta-lipotropin, beta-endorphin, and gamma-lipotropin; and 16k fragment.

Adrenocorticotropic Hormone↗
PubMed1980

Evidence for a peptide similar to 16K fragment in man. Its relationship to ACTH.

A radioimmunoassay directed toward the NH2-terminal region of mouse pro-ACTH/endorphin (called 16K fragment) was used to examine human samples. Culture media from two corticotropic adenomas and plasmas from 11 patients with various ACTH hypersecretory syndromes gave parallel displacement curves; displacement curves for human samples were not parallel to purified mouse 16K fragment. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis of culture medium from one adenoma, a major peak of 16K fragment immunoreactivity with an apparent molecular weight of ca. 16,000 was detected. A significant correlation (r = 0.963 ; p less than 0.001) was found between immunoreactive 16K fragment and ACTH in the patients' plasmas. These data indicate that a peptide similar to 16K fragment exists in man ; that human and mouse 16K fragment are immunologically distinguishable and that human 16K fragment appears to be secreted concomitantly with ACTH.

Addison Disease↗
PubMed1979

Characterization of mouse tumor cell beta-lipotropin.

Mouse tumor cell beta-lipotropin (beta LPH) and gamma-lipotropin (gamma LPH) were purified from mouse pituitary tumor cell culture medium by ion exchange chromatography and gel filtration. The mouse tumor cell beta LPH was identified by immunoprecipitation with several antisera to beta-endorphin, generation of opioid bioactivity upon brief treatment with trypsin, and its identity with the molecule previously shown to serve as an intermediate in the biosynthesis of beta-endorphin. Mouse tumor cell beta LPH (Mr = 8200 +/- 250) and gamma LPH (Mr = 4600 +/- 200) are significantly smaller than known mammalian beta LPH (Mr = 10,000) and gamma LPH (Mr = 6300) molecules. The beta-endorphin region of mouse tumor cell beta LPH has the same amino acid composition as ovine, bovine, and camel beta-endorphin, and species-specific differences are thus located in the gamma LPH region of the molecule. Mouse tumor cell beta LPH and gamma LPH lack a methionine residue at what had been considered to be a highly conserved site in their beta-melanotropin-like region. A species-specific radioimmunoassay for mouse tumor cell gamma LPH was developed. Rat pituitary beta LPH and gamma LPH were shown to be similar to the corresponding mouse tumor cell molecules in size and lack of methionine in their beta-melanotropin-like segment.

Amino Acids↗
PubMed1979

Partial characterization of a glycoprotein comprising the NH2-terminal region of mouse tumor cell pro-adrenocorticotropic hormone/endorphin.

The glycoprotein accounting for most of the nonadrenocorticotropic hormone (ACTH), non-beta-lipotropin (beta LPH) region of mouse tumor cell pro-ACTH/endorphin was purified from tumor cell culture medium and shown to contain 1/2 cystine residues. Preparations of the 16,000-dalton fragment-related material (referred to as 16K fragment) were heterogeneous with respect to size and charge. Despite this heterogeneity, a partial amino acid sequence for the NH2-terminal region of the molecule was determined by automated Edman degradationof the 16K fragment labeled by reduction and alkylation with [3H]iodoacetic acid or labeled biosynthetically with [3H]tryptophan. The sequence of 1/2 cystine and tryptophan residues in the mouse tumor 16K fragment can be aligned with one region of the amino acid sequence predicted from the cDNA for a bovine precursor to ACTH/beta LPH (Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N., and Numa, S. (1979) Nature 278, 423--427).

Adrenocorticotropic Hormone↗
PubMed1979

Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.

Adrenocorticotropic Hormone↗