Primary structure of bovine adrenal phenylethanolamine N-methyltransferase.

Bovine adrenal medullary phenylethanolamine N-methyltransferase (E.C. 2.1.1.28) was sequenced to determine if primary structure or post-translational processing accounts for the charged isozymes. A blocked NH2-terminus precluded amino terminal sequencing. Therefore, cyanogen bromide and tryptic peptide fragments were isolated and subjected to gas phase/gas-liquid phase sequencing. Primary structure was identified for 45% of the protein. At least two polypeptide chains exist with alternative amino acids representing conservative changes or single-base changes in amino acid codons by comparison to the cDNA sequence for the enzyme.

Directional probes of the hydrophobic component of the aromatic ring binding site of norepinephrine N-methyltransferase.

We investigated the directional nature of the bulk tolerance and hydrophobic binding in the aromatic ring binding region of the active site of norepinephrine N-methyltransferase (NMT) by comparing the substrate and inhibitor activities of m- and p-phenyl-substituted derivatives of amphetamine, phenylethanolamine, and alpha-methylbenzylamine. The para isomers of amphetamine and phenylethanolamine displayed significantly greater activities as inhibitor and substrate, respectively, than the meta isomers, which indicated that the bulk tolerance was near the para position. For benzylamines, the greatest inhibitory activity was observed for the meta isomer, demonstrating a significant difference in the binding requirements for phenylethylamines and benzylamines. These findings are consistent with a two-state model for the NMT active site that has been proposed elsewhere to account for its ability to bind both benzylamines and phenylethylamines in a fully extended side-chain conformation.

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.

Evidence for decreased transport of PNMT protein in advanced Alzheimer's disease.

Phenylethanolamine N-methyltransferase (PNMT) is the rate-limiting enzyme in the synthesis of epinephrine and a specific marker for adrenergic neurons. PNMT protein is decreased in axon terminals in brains from patients with Alzheimer's disease due to retrograde degeneration of epinephrine neurons. To determine the subcellular mechanism underlying retrograde degeneration, the distribution of PNMT between axon terminal and cell body was calculated in early and advanced Alzheimer cases compared with age-matched controls. In early Alzheimer's disease there is a decrease in PNMT in axon terminals and in total PNMT in epinephrine cell bodies and terminals compared with control values. There is no difference in the ratio of PNMT in cell body/axon terminal compared with controls. In contrast, in advanced Alzheimer's disease, PNMT activity increases by 124% in epinephrine neuronal cell bodies compared with controls. Immunochemical titration shows that this increased enzyme activity is due to an increase in PNMT protein. The cell body/axon terminal ratio of PNMT is increased 2.5-fold in advanced Alzheimer's disease compared with controls. These findings are consistent with the hypothesis that in early Alzheimer's disease there is a decreased synthesis or increased degradation of PNMT. However, in advanced Alzheimer's disease we propose that the accumulation of this enzyme in the perikarya results from a diminished transport of PNMT to axon terminals. We further postulate that epinephrine, the product of PNMT, and its further metabolites are endogenous neurotoxins. Therefore, the accumulation of PNMT in epinephrine cell bodies may contribute to the degeneration of these neurons in Alzheimer's disease.

Characterization of the isozymes of bovine adrenal medullary phenylethanolamine N-methyltransferase.

Bovine adrenal medullary phenylethanolamine N-methyltransferase (EC 2.1.1.28) has been purified to apparent homogeneity. The enzymatically active monomer has a relative molecular weight of 30,000 and can be separated into at least four active charged isozymes. These isozymes, designated PNMT-1, PNMT-2, PNMT-3 and PNMT-4, have isoelectric points of 5.1, 5.2, 5.3 and 5.4, respectively. Kinetic parameters have been determined for each isozyme. The Kms for phenylethanolamine range from 11.9 to 45.9 microM; the Kms for S-adenosylmethionine range from 1.13 to 1.47 microM; and the Kis for the competitive inhibitor, S-adenosylhomocysteine, range from 0.12 to 0.22 microM. For isozymes PNMT-1 and PNMT-4, and Kms for S-adenosylhomocysteine are not significantly different. Vmax values for all of the isozymes do not change significantly in the presence of S-adenosylhomocysteine. Treatment of the purified isozymes with various endo- and exoglycosidases does not alter electrophoretic mobility. Hence, carbohydrate substitution must be minimal. No high mannan, complex sugars or terminal N-acetylglucosamine residues are present. The absence of carbohydrate is further supported by the inability of Schiff-periodic acid to stain the protein. Limited thermolysin digests of each isozyme show distinct peptide cleavage products. In conjunction with the kinetic and glycosylation data, this suggests that the isozymes of phenylethanolamine N-methyltransferase may be primary structural variants.

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase.

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.

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.

Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines.

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.

Phenylethanolamine N-methyltransferase: notes on its purification from bovine adrenal medulla and separation from protein carboxymethyltransferase.

Standard procedures for the purification of phenylethanolamine N-methyltransferase were modified by the addition of an affinity chromatography step utilizing immobilized S-adenosyl-L-homocysteine and by use of preparative isoelectric focusing. Enzyme derived from bovine adrenal medullae was bound to S-adenosyl-L-homocysteine agarose, and could be eluted with 0.1 M NaCl. Concentrations of S-adenosyl-L-methionine as high as 10 mM were ineffective in eluting the enzyme. Preparative isoelectric focusing of bovine phenylethanolamine N-methyltransferase showed a single peak with the pI = 4.95. The potential use of immobilized S-adenosyl-L-homocysteine in the differential separation of phenylethanolamine N-methyltransferase from other methyltransferase enzymes is discussed.

Phenylethanolamine N-methyltransferase from the brain and adrenal medulla of the rat: a comparison of their properties.

Two different molecular forms of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1) have been isolated from the brain and adrenal glands of the rat as indicated by certain of their physicochemical properties, such as: molecular weight estimated on the basis of gel chromatography of Sephadex G-100; pH optima; electrophoretic mobility on acrylamide gel; and steady-state kinetic parameters. The high Km value for the brain PNMT has been assumed to be responsible for the low methylation ratio between norepinephrine and epinephrine in the CNS.

Identification and partial purification of phenylethanolamine N-methyl transferase in the developing ovine lung.

Phenylethanolamine N-methyl transferase (PNMT), the terminal enzyme in epinephrine biosynthesis, was identified in fetal and newborn ovine lung. The ovine lung PNMT demonstrated the appropriate substrate specificity and affinity (Km, 9 x 10(-6) M). Although some homology between adrenal and lung PNMT was observed on polyacrylamide gel electrophoresis, the lung PNMT differed in its migration on ion exchange chromatography and was not inhibited by pharmacologic inhibitors active against the adrenal enzyme. Activity increased from a mean of 132 pmole/(mg protein . h) x 10(-3) at 0 days gestation to 326 pmole/mg protein . h) x 10(-3) in newborn animals between 1-4 days of age (r = 0.571, P less than 0.05). The levels of N-methylating activity in extraadrenal tissues were relatively low; lesser but significant N-methylating activities were demonstrated in brown adipose tissue and myometrial tissues. Adrenal gland activity was 1000-fold greater than lung activity expressed on a per mg protein basis and 250-fold greater expressed per mg wet weight.

Different forms of adrenal phenylethanolamine N-methyltransferase: species-specific posttranslational modification.

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.

Some properties of phenylethanolamine-N-methyltransferase of rat brain.

Phenylethanolamine-N-methyltransferase (PNMT, EC 2.1.1.28) was partially purified from rat brain. Brain homogenates were subjected to ultracentrifugation, salt fractionation, and gel filtration on Sephadex G-100. To compare the rat brain PNMT with that of adrenals, the same procedure was carried out with rat adrenal homogenates. The brain enzyme was eluted from Sephadex as a single fraction with a molecular weight of 26,900, while the enzyme from adrenals under the same conditions appeared in two fractions with molecular weights of 38,700 and 108,500. The brain fraction separated on Sephadex G-100 was active on phenylethanolamine substrates and inactive on indoleamine and phenylethylamine substrates. Products of the enzyme reaction were identified by bidimensional thin-layer chromatography as N-methyl derivatives of the corresponding amines. Kinetic studies showed that the type of inhibition of PNMT from rat brain and rat adrenals by SK&F 7698 was the same as described for PNMT from rabbit adrenals. Also, when normetanephrine and S-adenosyl-L-methionine were used as substrates, the apparent Km values found with PNMT from rat adrenals and rat brain were similar.

Genes for neurotransmitter synthesis, storage and release.

We found that the catecholamine biosynthetic enzymes, tyrosine hydroxylase, dopamine B-hydroxylase and phenylethanolamine N-methyltransferase share similar protein domains in their primary structures, and therefore are coded for by a single gene or a family of genes. In a recent report, we also demonstrated that antisera directed against tyrosine hydroxylase, choline acetyltransferase and glutamate decarboxylase cause specific complement-mediated lysis of dopaminergic, cholinergic and GABA-ergic subpopulation of synaptosomes, respectively. This implies that the neurotransmitter biosynthetic enzyme and the specific nerve ending protein(s) also share similar protein domain(s). Therefore, we postulate that the specific neurotransmitter biosynthetic enzyme and a certain membrane protein of the nerve endings probably share similar gene coding sequences and that coordinate expression of these proteins may determine the phenotype of the neuron.

Recombinant human phenylethanolamine N-methyltransferase: overproduction in Escherichia coli, purification, and characterization.

The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.