Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.     

Novel Opioid Peptide Amidorphin: Characterization and Distribution of Amidorphin-Like Immunoreactivity in Bovine, Ovine, and Porcine Brain, Pituitary, and Adrenal Medulla

We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species, authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.     

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.     

Distribution of the messenger RNA coding for the common precursor of corticotropin and beta-lipotropin within the bovine pituitary.

The distribution of the mRNA coding for the common precursor of corticotropin and beta-lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell-free protein-synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/beta-lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/beta-lipotropin mRNA in the intermediate lobe is about 20-fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2-fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/beta-lipotropin mRNA amounts to almost one-third of the products encoded by total translatable mRNA. These results indicate that corticotropin/beta-lipotropin mRNA represents a major mRNA species in intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and beta-lipotropin.     

The structure and expression of the preproenkephalin gene.

Enkephalins are pentapeptides with opioid activity which are found in a wide variety of tissues. Studies of enkephalin-containing peptides from the adrenal gland have established that the mature pentapeptides are derived by proteolytic processing of a precursor protein. We have shown that human adrenal medullary tumours contain mRNA which can be translated in vitro to yield a single major enkephalin precursor. The sequence of cloned cDNA shows that the preproenkephalin mRNA encodes four copies of met-enkephalin, two copies of met-enkephalin extended sequences and one copy of leu-enkephalin; each copy is flanked by paired basic amino acids which are presumably recognised by the processing protease. We have used the cloned human cDNA as a hybridisation probe to detect the corresponding mRNAs in rat adrenal gland and, in smaller amounts, in rat brain. We have been unable to detect in brain any other cross-hybridising mRNAs which might encode other putative precursor proteins.     

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.     

The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.     

A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues

We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.     

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs

cDNA clones for a fifth polypeptide of rat brain calmodulin-dependent protein kinase II were isolated and sequenced. The cDNA sequence encoded a polypeptide, designated delta, consisting of 533 amino acid residues with a molecular weight of 60,080. Comparison of amino acid sequences of this and alpha, beta, beta', and gamma polypeptides of calmodulin-dependent protein kinase II reveals marked homology among them. The mRNAs for delta were expressed in rat brain tissues with different regional specificities. The distribution of alpha, beta/beta', gamma, and delta mRNAs in cerebrum, skeletal muscle, diaphragm, heart, small intestine, uterus, aorta, liver, kidney, lung, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.9-kilobase (kb) RNA species hybridizable with a probe for gamma was found in all the tissues examined, and 4.0-4.2-kb RNA species hybridizable with a probe for delta were found in all the tissues examined except for liver, while a 4.8-kb RNA species hybridizable with a probe for alpha and a 4.2-kb RNA species hybridizable with a probe for beta were present in brain but not in the other tissues. With the alpha probe, however, a 4.1- and 2.6-kb RNA species were both detected in skeletal muscle and diaphragm. With the beta probe, a 4.3-kb RNA in skeletal muscle and diaphragm, 2.9-kb RNA in small intestine, and 4.0-kb RNA in testis were detected. With the delta probe, a 3.5-kb RNA in heart and 1.8-kb RNA in testis were detected. Thus, gamma and delta mRNAs were expressed in various tissues, while alpha and beta/beta' mRNAs were primarily, if not exclusively, expressed in brain.     

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.     

Isolation and sequence analysis of cDNA for rat carboxypeptidase E [EC 3.4.17.10], a neuropeptide processing enzyme

Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.     

Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta-subunit of rat follicle stimulating hormone

To provide a hybridization probe for analysis of the regulation of rat gonadotropin subunit mRNA levels, an effort was made to isolate a cloned cDNA for the beta-subunit of rat FSH (FSH beta). Using a cloned bovine FSH beta cDNA as a hybridization probe, a rat pituitary lambda gt10 cDNA library was screened and a single, strongly hybridizing clone identified. The 874 base pair cDNA insert from this clone contains the complete sequence of rat FSH beta including an amino-terminal precursor segment. Hybridization of this cloned cDNA to rat pituitary RNA demonstrated the presence of an approximately 2.0 kilobase RNA species containing FSH beta sequences. Cloned rat cDNA was also used to demonstrate that estrogen treatment of ovariectomized female rats results in decreases in mRNA concentrations for FSH beta and the beta-subunit of LH with somewhat smaller decreases in alpha-subunit mRNA concentrations. Little or no change was detected in the mRNA for the beta-subunit of TSH.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

Purification and characterization of a gamma-melanotropin precursor from frozen human pituitary glands.

A new melanocyte-stimulating peptide has been isolated from acid extracts of frozen human pituitary glands by salt/ethanol fractionation, Sephadex G-75 gel filtration and DEAE- and cM-cellulose ion-exchange chromatography. The peptide is glycosylated, has an N-terminal tryptophan residue and an apparent mol.wt. of 16000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its amino acid analysis closely resembles residues Trp-105 to Gln-29 predicted for the common precursor protein of bovine corticotropin and beta-lipotropin by Nakanishi, Inoue, Kita, Nakamura, Chang, Cohen & Numa [(1979) Nature (London) 278, 423-427]. This fragment is expected to have melanotropin activity due to the tetrapeptide -His-Phe-Arg-Trp- (residues -51 to -48) of the predicted sequence of the common precursor. It was found to have a molar potency of 1 X 10(-5) relative to alpha-melanotropin in the frog skin bioassay. These characteristics are consistent with the isolated melanotropin peptide being a non-corticotropin, non-lipotropin peptide of the human common precursor protein of corticotropin and lipotropin. The peptide neither potentiates the adrenal weight-maintenance activity of corticotropin-(1-24)-tetracosapeptide when administered to hypophysectomized rats, nor stimulates release of non-esterified fatty acids from isolated rat epididymal cells. A second N-terminal-tryptophan glycopeptide was also isolated, which had an amino-acid composition similar to that predicted for the bovine common precursor protein, residues Trp-105 to Gly-35.