Biosynthesis of beta-endorphin, beta-lipotropin and the putative ACTH-LPH precursor in the frog pars intermedia.

When frog pars intermedia are incubated for 3 h with radioactive methionine, the predominant labeled peptide is one with an apparent molecular weight of 33, 100. This peptide can be immunoprecipitated with antisera against β-melanotropin (β-MSH), adrenocorticotrophin (ACTH), and β-endorphin and is believed to be the common precursor of ACTH and β-lipotropin (β-LPH). Immunoprecipitation experiments have also demonstrated the presence of labeled β-LPH and β-endorphin. The labeled β-endorphin has been shown to behave identically to sheep β-endorphin on both carboxymethyl-cellulose chromatography and polyacrylamide gel electrophoresis. Frog β-endorphin has methionine as the fifth residue, as do all other β-endorphins that have been sequenced.     

Expression of variant forms of proopiomelanocortin, the common precursor to corticotropin and beta-lipotropin in the rat pars intermedia

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.     

Incorporation of canavanine into rat pars intermedia proteins inhibits the maturation of pro-opiomelanocortin, the common precursor to adrenocorticotropin and beta-lipotropin

Pro-opiomelanocortin, the common glycoprotein precursor to adrenocorticotropin and beta-lipotropin, is the most abundant protein synthesized in rat neurointermediate lobes. Dissected rat neurointermediate lobes were incubated in the presence of canavanine, an analog of arginine, to determine (a) whether canavanine could be incorporated into pro-opiomelanocortin molecules and (b) if incorporation occurs, whether there is any effect on the processing mechanism of the prohormone. Preincubation of rat neurointermediate lobes for 16 h in the presence of 10 mM canavanine results in the production of pro-opiomelanocortin molecules in which most, if not all, the arginine residues have been replaced by canavanine. Identification of canavanine-containing pro-opiomelanocortin forms was done by two-dimensional electrophoresis, tryptic and chymotryptic peptide mapping, as well as by analysis, on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the fragments resulting from a partial digestion with chymotrypsin. During pulse-chase experiments, canavanine-containing pro-opiomelanocortin molecules were found to be processed at a much slower rate than the normal precursor forms: after a 2-h chase, conversion of approximately 25% of the analog-containing prophormone was observed compared to 83% of the nonanalog-containing precursors. Moreover, the small proportion of canavanine-containing precursor molecules which had undergone cleavage during the chase yielded atypical large molecular weight peptides. These results indicate that canavanine incorporation into neurointermediate lobe proteins considerably slows down the conversion of pro-opiomelanocortin into its different end products.     

Partial N-terminal amino acid sequence of pro-opio-melanocortin (ACTH/beta-LPH precursor) from rat pars intermedia

Posterior lobes of rat pituitary (pars intermedia plus pars nervosa) were incubated with various labeled amino acids and the cell extracts analyzed by NaDodSO₄ polyacrylamide disc gel electrophoresis. Two forms of precursor proteins for betaendorphin and alpha-MSH were synthesized. Both forms have been shown to contain the fragments beta-LPH 61–69 and ACTH 1–8 and are thought to have the same peptide backbone. The two forms were simultaneously submitted to automatic Edman degradation and the following partial sequence was obtained: Trp/Arg₁-Leu₃-Phe₄-Ser₅-Ser₆-Leu₁₁-Thr_12–13-Tyr₁₄-Ser₁₅-Leu_17–18-Ala₁₉-lle₂₁-Arg_22–25- Leu_26–28-Ser₂₉. This sequence was compared with that reported by Nakanishi et al. (18). Their amino acids sequence was indirectly derived from DNA sequencing after isolation of mRNA from bovine pars intermedia. This comparison indicates the presence of a signal peptide of 26 amino acids in the sequence of beef ACTH/beta-LPH precursor.     

NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: identification, radioimmunoassay and half-life

A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the bovine corticotropin/beta-lipotropin precursor gene

The entire bovine corticotropin/beta-lipotropin precursor gene has been isolated as a set of overlapping genomic DNA fragments which extend over a length of approximately 17000 base pairs. Restriction mapping of the cloned DNA fragments and nucleotide sequence analysis of the whole mRNA-coding segments and their surrounding regions have established that the corticotropin/beta-lipotropin precursor gene is approximately 7300-base-pairs long and contains two intervening sequences; one with an approximate length of 4000 base pairs is located within the segment encoding the 5'-untranslated region of the mRNA, and the other with an approximate length of 220 base pairs interrupts the protein-coding sequence near the signal peptide region. Sequence analysis of more than 200 base pairs preceding the proximal end of the corticotropin/beta-lipotropin precursor gene has revealed a 'Hogness box' and a variant of the model sequence d(G-G-TC-C-A-A-T-C-T) as well as palindrome structures as observed in other eukaryotic genes. Furthermore, some sequence similarities in the 5'-flanking region are found between the corticotropin/beta-lipotropin precursor gene and the mouse alpha-globin and beta-globin genes, all of which are negatively regulated by glucocorticoids. At least four homologous repetitive sequences are distributed at 3000-5000-base-pair distances in the corticotropin/beta-lipotropin precursor gene region; two such sequences are located in the 5'-flanking region, and one within each intervening sequence. Blot hybridization analysis of bovine pituitary nuclear RNA has indicated that the entire corticotropin/beta-lipotropin precursor gene is transcribed into a primary hnRNA product, which is then spliced to form the mature mRNA.     

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).     

Microtubules-associated intracellular localization of the NH2-terminal cellular prion protein fragment

By utilizing double-labeled fluorescent cellular prion protein (PrPC), we revealed that the NH2-terminal and COOH-terminal PrPC fragments exhibit distinct distribution patterns in mouse neuroblastoma neuro2a (N2a) cells and HpL3-4, a hippocampal cell line established from prnp gene-ablated mice [Nature 400 (1999) 225]. Of note, the NH2-terminal PrPC fragment, which predominantly localized in the intracellular compartments, congregated in the cytosol after the treatment with a microtubule depolymerizer (nocodazole). Truncated PrPC with the amino acid residues 1-121, 1-111, and 1-91 in mouse (Mo) PrP exhibited a proper distribution profile, whereas those with amino acid residues 1-52 and 1-33 did not. These data indicate the microtubules-associated intracellular localization of the NH2-terminal PrPC fragment containing at least the 1-91 amino acid residues.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the NH2-terminal fragment with synthetic analogs of the COOH-terminal fragment

Complementation of the natural NH₂-terminal fragment (consisting of 134 amino acid residues) with synthetic analogs of COOH-terminal fragments of 52 or 47 amino acids of reduced-carbamidomethylated human somatotropin gave recombinants with full growth-promoting activity as evidenced by the tibia test. Radioimmunoassay data show that the semisynthetic recombinant hormones possess nearly full immunoreactivity as compared to the native molecule.     

Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.     

Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.     

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.     

Immunohistochemical localization of adrenocorticotropin and melanocyte stimulating hormone in pars intermedia of rat hypophysis

Summary The sites of production of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are studied by the immunoglobulin-peroxidase bridge technique, using antisera prepared against synthetic porcine 1–24 and 17–39 ACTH, and bovine MSH on the rat adenohypophysis. Presence of ACTH all over the pars intermedia (PI) is indicated by staining with antisera _p 1–24 and _p 17-3-9 ACTH. There are darkly stained ACTH cells in the PI and pars tuberalis (PT), similar to those in the pars distalis (PD). With higher dilutions of the ACTH antiserum, staining intensity disappears or reduces markedly in majority of the PI cells, whereas, the ACTH cells in the PI, PD and PT do not vary much in their staining intensity. Therefore, it is concluded that majority of the PI glandular cells (light glandular and dark cells) contain less corticotropin than the ACTH cells. From these observations, it seems to me that the major amount of corticotropin is supplied by the ACTH cells of the PD, PI and PT, and less by the light glandular and dark cells of the PI. The antiserum is ineffective after absorption, so the staining reaction appears to be specific for _p 1–24 and _b 17–39 ACTH.Presence of MSH all over the PI is indicated by staining with antisera to bovine MSH. Majority of the PI cells are highly stained even with higher dilution of the antiserum. The unstained cells in the PI seem to be ACTH cells and/or marginal cuboidal cells. The antiserum was ineffective after absorption, so the staining reaction appears to be specific for _b MSH.Control over the PD corticotropin through the median eminence portal circulation and the PI and PT control through nervous system is also discussed.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledges the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré.     

Lack of release of ACTH from the denervated rat pars intermedia in vivo.

The purpose of this study was to ascertain whether the pars intermedia of the rat adenohypophysis, isolated from direct innervation via the infundibular stem, could maintain adrenal cortical weight and plasma corticosterone levels. We compared the adrenal cortical response of rats 40 days after either complete hypophysectomy, hypophysectomy with reinsertion of only the pars distalis, or hypophysectomy with reinsertion of only the nervosa-intermedia. Adrenal weight and plasma corticosterone levels were partially maintained in the group with reinserted pars distalis. These parameters were not different from the complete hypophysectomy group in the animals with reinserted nervosa-intermedia. Thus, the pars intermedia, with its nerve supply disrupted, cannot maintain adrenal cortical function.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the natural NH2-terminal fragment with a synthetic analog of the COOH-terminal fragment

Complementation of the natural [Cys (Cam)⁵³]-HGH-(1–134) fragment with synthetic analogs of COOH-terminal fragments of human somatotropin (HGH), namely [Nle¹⁷⁰, Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) has been investigated. It was found that the synthetic fragment, [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187), gave a recombinant with about 40% HGH radioreceptor-binding activity. When compared with the natural recombinant, the semisynthetic hormone exhibited similar receptor-binding activities. The natural and semisynthetic recombinants were indistinguishable in radioimmunoassay. The α-helical content of the semisynthetic recombinant was completely restored in comparison with that of the native hormone as revealed by circular dichroism spectra. On the other hand, attempts to obtain a recombinant with the synthetic [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) were unsuccessful. The synthesis of [Nle¹⁷⁰,Ala^165,182]-HGH-(140–182) and [Nle¹⁷⁰,Ala^165,182]-HGH-(140–187) is herein described.     

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and α-melanocyte-stimulating hormone (α-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of α-MSH in the homogenate, 36% was recovered in the 11,500 g pellet and 31% sedimented between 11,500 and 105,000 g. α-MSH was not detected in the 105,000 g supernatant fluid. When the 900 g supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing α-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain α-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing α-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing α-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing α-MSH are synaptosomes.     

Immunological studies on pancreatic phospholipase A2. Antigenic characterization of the NH2-terminal region.

Rabbit antisera elicited against pure pig, horse, ox, and sheep pancreatic phospholipase A2 revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Snake venom phospholipases did not show any detectable cross-reactions with the pancreatic enzymes. Microcomplement fixation also clearly demonstrated conformational differences between porcine phospholipase A2 and its zymogen. NH2 terminally modified analogs of porcine phospholipase A2 could be clearly distinguished using the same assay. Moreover, strong evidence was obtained that Ala1-Arg6 is a part of an antigenic determinant. Radioimmune assay, using monovalent phospholipase-specific Fab fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The Fab fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase A2. They also exhibited different protective effects against active center modification.