Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes

Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.9-kb-containing PNMT promoter constructs is stimulated approximately twofold by nicotine and muscarine. Depolarizing concentrations of K+ produce fourfold increases in expression. These responses are not detected with constructs containing the proximal 0.3-kb promoter, indicating that the regions between -273 and -877 bp convey neural responsiveness for the PNMT gene in bovine chromaffin cells. Electrophoretic mobility shift assays (EMSAs) with oligonucleotides encoding these regions of the PNMT promoter revealed distinctions in migration of nuclear protein complexes formed following treatment of chromaffin cells with nicotine, muscarine, or 50 mM K+. Thus, the PNMT promoter between 0.3 and 0.9 kb contains sequences capable of responding to cholinergic and depolarization stimuli. Moreover, these treatments influence the interactions of specific nuclear proteins with this region of the PNMT promoter.     

Molecular cloning and cell-free expression of mouse antithrombin III.

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.     

Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data

Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.     

Characterization of recombinant bovine phenylethanolamine N-methyltransferase expressed in a mouse C127 cell line.

Bovine phenylethanolamine N-methyltransferase (PNMT) cDNA was inserted into a bovine papilloma virus-based expression vector and used to transfect a mouse C127 cell line. The resultant recombinant bovine PNMT was characterized biochemically and immunochemically. Recombinant bovine PNMT activity, like the native bovine enzyme, was enhanced by phosphate ion in a concentration-dependent manner. Their molecular weights were shown to be identical by Western blot analysis. Antibodies raised against native bovine adrenal PNMT equally immunoprecipitated the activity of the recombinant and native enzymes. In addition, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine PNMT. These antibodies immunostained the recombinant enzyme protein in transfected cells and in their neurite-like processes. In addition, in situ hybridization with the bovine PNMT cDNA probe resulted in a labelling pattern similar to the immunostaining. The recombinant bovine PNMT as the native bovine enzyme exist in multiple-charge forms, but only one form is predominant. Taken together, our results suggest that recombinant bovine PNMT, expressed from bovine PNMT cDNA in a mouse cell line is enzymatically active and shares many common features with native bovine adrenal PNMT.     

Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.     

cDNA cloning and chromosome assignment of the gene for human brain 14-3-3 protein eta chain.

We present the nucleotide sequence of a cDNA clone of mRNA encoding human 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 1,730-nucleotide sequence of the cloned cDNA contains 191 bp of a 5'-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3'-noncoding region containing three canonical polyadenylation signals. The 14-3-3 protein eta chain cDNA encoded a polypeptide of 246 amino acids with a predicted molecular weight 28,196. The predicted amino acid sequence of human 14-3-3 protein eta was highly homologous to that of previously reported bovine and rat 14-3-3 proteins with only two amino acid differences. The sequence carries structural features as putative regions responsible for activation of tyrosine and tryptophan hydroxylases and for inhibition of Ca2+/phospholipid-dependent protein kinase C. Northern blot analysis demonstrated widespread expression of the 14-3-3 protein eta chain in cultured cell lines derived from various human tumors. These findings suggest the conservative functions of the 14-3-3 protein among species. Spot blot hybridization analysis with flow-sorted chromosomes showed that the human 14-3-3 protein eta chain gene is assigned to chromosome 22.     

Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.     

The 5′-flanking region of the human dopamine β-hydroxylase gene promotes neuron subtype-specific gene expression in the central nervous system of transgenic mice

Dopamine β-hydroxylase (DBH, EC 1.14.17.1) catalyze the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeriic gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631–1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5′-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.     

Molecular cloning and characterization of a novel phospholemman-like protein from rat hippocampus

A cDNA encoding a putative ion channel protein was isolated from a rat hippocampus library. This gene, termed phosphohippolin (Php), contains 318 bp open reading frame encoding a single transmembrane protein with a 5' signal peptide region. The deduced amino acid sequence shares 48.1% homology with phospholemman (Plm). Expression sequence tag database (dEST) search identified a mouse (AA521976) and human (AA209241) Php gene homologues. The tissue distribution studies of Php mRNA showed its abundant expression in rat brain and kidney, and in the brain, high expression was observed in hippocampus and cerebellum.     

Isolation and characterization of a cDNA coding for a novel human 17.3K myelin basic protein (MBP) variant

Human fetal spinal cord poly A (+) mRNA was found to direct the synthesis of three major myelin basic protein (MBP) variants with molecular weights of 17K, 18.5K, and 21.5K when translated in reticulocyte lysates. In order to investigate the structural relationships between these MBP variants and their corresponding mouse variants, human fetal spinal cord and mouse brain cDNA libraries were constructed and screened for MBP cDNAs. A number of MBP cDNA clones were isolated and characterized. One of these, PP535 contained the entire coding region of the mouse 14K MBP; and another mouse cDNA clone, PP1.85, was almost full-length and coded for either the 21.5K MBP or the 18.5K MBP. A human clone (KK36), 1,173 nucleotides in length, contained the entire coding region of an MBP variant with a molecular weight of 17,342. The structure of this clone within its coding region is significantly different from the corresponding mouse 17K MBP cDNA. It is missing two sequences found in the mouse 17K MBP cDNA (exons 2 and 5); and it contains a sequence (exon 6) that is missing from the mouse 17K MBP cDNA. Thus, this human 17.3K cDNA codes for a "17K" human MBP variant that is quite different from the corresponding mouse variant and is identical to the human 18.5K MBP except for a deletion of a peptide consisting of 11 amino acids that includes the single tryptophan residue of the 18.5K MBP. An analysis of the structure of this 17.3K human MBP cDNA suggests that the major pathway for splicing the primary human MBP gene product may be different from that in the mouse.     

Mouse protein Z-dependent protease inhibitor cDNA

Protein Z-dependent protease inhibitor (ZPI) is plasma proteinase inhibitor in the serpin superfamily that produces rapid inhibition of factor Xa in the presence of phospholipids, Ca++ and protein Z (PZ). Mouse ZPI cDNA was isolated and cloned from mouse liver RNA using RT-PCR. The cDNA contains 100 nucleotides 5' of a translation initiation codon and an open reading frame of 1344 nucleotides followed by a 163 nucleotide 3' untranslated sequence with a poly (A) tail. The cDNA predicts a signal peptide containing 21 amino acids and a mature protein of 427 residues with 8 potential sites for N-linked glycosylation. The oligonucleotide and predicted amino acid sequences of mouse ZPI are 72% and 81% homologous with those of human ZPI. Like human ZPI, mouse ZPI contains tyrosine-serine (P1-P1') at its reactive center in contrast to the rat molecule which contains tyrosine-cysteine. By Northern analysis, mouse ZPI mRNA is 1.6 kb in size and, similar to both human and rat, it is detectable in liver, but not in heart, brain, spleen, lung, kidney, skeletal muscle or testes.     

Molecular cloning and nucleotide sequence of rat brain argininosuccinate lyase cDNA with an extremely long 5'-untranslated sequence: evidence for the identity of the brain and liver enzymes

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme present in the brain of ureotelic animals. Using as a probe rat liver argininosuccinate lyase cDNA, already isolated and sequenced (Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. and Mori, M., J. Biochem., 103 (1988) 177-181), we screened a rat brain cDNA library constructed in the lambda gt11 expression vector and obtained a single cDNA clone. This cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,390), a 5'-untranslated sequence of 967 bp and a 3'-untranslated sequence of 74 bp. The length of the 5'-non-coding region of the cDNA seems to be one of the longest among the cDNAs heretofore isolated. A comparison of the brain cDNA sequence (2424 bp) with the corresponding region of the liver cDNA (1574 bp) revealed differences in 5 nucleotides. The brain clone contained A----G and C----G base differences from the hepatic sequence, resulting in amino acid changes from Tyr and Arg in the liver clone, to Cys and Gly in the brain clone, respectively. The other 3 nucleotide differences are silent with respect to the amino acid sequence of the protein. Therefore, the amino acid sequence of the brain argininosuccinate lyase, as deduced from the nucleotide sequence of its cDNA clone, was identical with that of the liver protein, except for two amino acid residues. These minor changes may reflect a microheterogeneity of the argininosuccinate lyase gene. The brain and liver enzymes seem to be encoded by the same structural gene.