Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics,screening, and stability

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.     

Phenylethanolamine N-methyltransferase kinetics: bovine versus recombinant human enzyme.

Epinephrine (Epi) acts as a neurotransmitter in the brain, but its function therein is not well understood. Phenylethanolamine N-methyltransferase (PNMT) catalyzes the final step in the biosynthesis of Epi and is thus a pharmacological target to investigate the function of Epi in the central nervous system. The kinetic differences between bovine adrenal PNMT and human brain PNMT for a number of substrates and inhibitors are examined and the results reported.     

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 asN-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 andS-adenosyl-l-methionine were used as substrates, the apparentK _m values found with PNMT from rat adrenals and rat brain were similar.Preliminary reports were presented at XXV Convención Anual AsoVAC, Caracas, Venezuela, October 1975, and at XII Congreso Latinoamericano de Ciencias Fisiológicas, Bogotá, Colombia, November 1975.     

Increase in Rat Adrenal Phenylethanolamine N-Methyltransferase mRNA Level Caused by Immobilization Stress Depends on Intact Pituitary - Adrenocortical Axis

Abstract: The effects of a single and of repeated immobilization stress on the expression of the final enzyme involved in epinephrine biosynthesis, phenylethanolamine N -methyltransferase (PNMT), are described. A single immobilization (whether lasting 5 or 120 min) caused a severalfold increase of the adrenal PNMT mRNA level as measured 2 h after the beginning of the procedure. This elevation was of a transient nature, peaked 3–6 h after the 2-h immobilization, and returned to control values by 12 h after the stress. When the animals were immobilized for 2 h/day for seven consecutive days, an increase in content of PNMT mRNA of a similar magnitude was observed, which persisted for at least 2 days after the seventh immobilization. The immobilization-induced increase was completely abolished in hypophysectomized animals, whereas adrenal denervation failed to prevent it. These data suggest that the immobilization-induced increase in adrenal PNMT mRNA level depends primarily on pituitary-adrenocortical regulation.     

A radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[methyl-3H] adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.     

An (R)-specific N-methyltransferase involved in human morphine biosynthesis

The biosynthesis of morphine, a stereochemically complex alkaloid, has been shown to occur in plants and animals. A search in the human genome for methyltransferases capable of catalyzing the N-methylation of benzylisoquinoline alkaloids, as biosynthetic precursors of morphine, yielded two enzymes, PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49). Introduction of an N-terminal poly-histidine tag enabled purification of both proteins by immobilized metal affinity chromatography. Recombinant PNMT and NMT were characterized for their catalytic activity towards four benzylisoquinolines: tetrahydropapaveroline (THP), 6-O-methyl-THP, 4′-O-methyl-THP and norreticuline. Human PNMT accepted none of the offered alkaloids and was only active with its established substrate, phenylethanolamine. The second enzyme, human NMT, converted all four benzylisoquinolines, however, with a strict preference for (R)-configured morphine precursors. Determination of kinetic parameters of NMT for the four (R)-configured benzylisoquinoline alkaloids by LC–MS/MS revealed (R)-norreticuline to be the best substrate with an even higher catalytic activity as compared to the previously reported natural substrate tryptamine. In addition, isolation of the morphine precursor salutaridine from urine of mice injected (i.p.) with (R)-THP provides new evidence that the initial steps of morphine biosynthesis in mammals occur stereochemically and sequentially differently than in plants and suggests an involvement of the herein characterized (R)-specific NMT.     

Differential Regulation of Phenylethanolamine N-Methyltransferase Expression in Two Distinct Subpopulations of Bovine Chromaffin Cells

Abstract: Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were ～20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.     

Comparative studies of PC12 and mouse pheochromocytoma-derived rodent cell lines as models for the study of neuroendocrine systems

Summary We have compared PC12 cell lines derived from different laboratories and the newly developed mouse pheochromocytoma (MPC) cell line. Morphologically, there were distinct differences in size, shape, adherence, and clumping behavior, which varied in response to different culture media, growth substrates, and nerve growth factor. Quantitative messenger ribonucleic acid (mRNA) analysis showed significant variability in the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase (TH) phenylethanolamine N-methyltransferase (PNMT), the noradrenaline transporter (NAT), and neuron-specific enolase (NSE) between all lines examined. Of most significance were the increased levels of PNMT mRNA in the MPC cells, which were to 15-fold greater than in the PC12 cell lines grown under the same conditions in Dulbecco modified Eagle medium (P<0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P≤0.05). Immunohistochemistry for TH, PNMT. and NAT was generally consistent with mRNA analysis, with the MPC cells demonstrating strong immunoreactivity, for PNMT. The MPC cells showed the highest levels of desipramine-sensitive [³H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P≤0.05), despite relatively low levels of NAT mRNA. These results indicate that PC12 cell lines should be carefully chosen for optimal utility in the study of chromaffin cell or sympathetic neuron biology and that cell features will be influenced by type of media and substrate chosen. Furthermore, they confirm that the new MPC cell line is likely a useful model for the study of adrenergic mechanisms or studies involving NAT.     

Effects of a 3-alkyl-, 4-hydroxy- and/or 8-aromatic-substituent on the phenylethanolamine N-methyltransferase inhibitor potency and alpha2-adrenoceptor affinity of 2,3,4,5-tetrahydro-1H-2-benzazepines

2,3,4,5-Tetrahydro-1H-2-benzazepine (THBA; 1) is nearly 100-fold more selective an inhibitor of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) versus the alpha2-adrenoceptor than is 1,2,3,4-tetrahydroisoquinoline (THIQ; 2) (1: PNMT K(i)= 3.3 microM, alpha2-adrenoceptor K(i) = 11 microM, selectivity [alpha2 K(i)/PNMT K(i)] = 3.3; 2: PNMT K(i) = 9.7 microM, alpha2 K(i) = 0.35 microM, selectivity=0.036;). Since the PNMT inhibitory activity and selectivity of THIQ were enhanced by the introduction of a hydrophilic electron-withdrawing 7-substituent and a 3-alkyl-substituent, a similar study was conducted on THBA. 8-Nitro-THBA (3) was found to be as potent an inhibitor of PNMT as its THIQ analogue (21) and to be more selective due to its reduced alpha2-adrenoceptor affinity (3: PNMT K(i) = 0.39 microM, alpha2 K(i) = 66 microM, selectivity = 170; 21: PNMT K(i) = 0.41 microM, alpha2 K(i) = 4.3 microM, selectivity = 10). Introduction of a 3-alkyl substituent on the THBA nucleus decreased both the alpha2-adrenoceptor affinity and the PNMT inhibitory activity, suggesting an area of steric bulk intolerance at both sites. 4-Hydroxy-THBA (15), which can be considered a conformationally-restricted analogue of 3-hydroxymethyl-THIQ (30), exhibited poorer PNMT inhibitory activity and less selectivity than 30 (15: PNMT K(i) = 58 microM, alpha2 K(i) = 100 microM, selectivity = 1.7; 30: PNMT K(i) = 1.1 microM, alpha2 K(i) = 6.6 microM, selectivity = 6.0). While the addition of an 8-nitro group to 15 increased the selectivity of 16 as compared to its THIQ analogue (31), it was not as potent at PNMT nor as selective as 8-nitro-THBA (3) (16, PNMT K(i) = 5.3 microM, alpha2 K(i) = 680 microM, selectivity = 130; 31: PNMT K(i) = 0.29 microM, alpha2 K(i) = 19 microM, selectivity = 66). Compound 3 is the most selective (PNMT/alpha2) and one of the more potent at PNMT compounds yet reported in the benzazepine series, and should have sufficient lipophilicity to penetrate the blood-brain barrier (CLogP = 1.8).     

Label-free high-throughput functional lytic assays

Refractive index-sensitive resonant waveguide grating biosensors are used to assay the label-free enzymatic degradation of biomolecules. These assays provide a robust means of screening for functional lytic modulators. The biomolecular substrates in this study were covalently immobilized through amine groups. Using the Corning Epic System, the digestion signatures for multiple protein substrates on the biosensors are measured. Label-free digestion profiles for these proteins were substrate specific. Similarly, the authors find that the label-free digestion is protease specific. Enzyme-substrate pairs were used to evaluate high- throughput biosensors as tools for screening functional modulators. The lytic inhibitor properties for several proteases and dextranase are determined. The authors find that the IC50 values for the protease inhibitors agree with the reported values for several known inhibitors. The Z' values, using biosensor-based functional lytic screens, were routinely greater than 0.5, making this label-free application feasible for high-throughput screening.     

A bead-based approach for large-scale identification of in vitro kinase substrates

Deciphering the kinase-substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.     

Phenylethanolamine N-methyltransferase inhibition: re-evaluation of kinetic data

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we demonstrate that a routinely used assay for screening PNMT inhibitors is not appropriate for those inhibitors having K(i) values less than 1 microM. A revised assay has been developed that shows some inhibitors bind two orders of magnitude more tightly than previously reported.     

Synthesis of functional proteins using Escherichia coli extract entrapped in calcium alginate microbeads

In this report, we describe the construction and analysis of a cell-free protein synthesis system immobilized in calcium alginate microbeads. When incubated in a feeding solution that contained amino acids and other low-molecular-weight substrates, the microbeads transcribed and translated coimmobilized DNA into functional proteins. Protein synthesis continued for more than 15 h with the diffusional supply of substrates and removal of by-products. In addition, functional proteins were generated from PCR-amplified genes as efficiently as from plasmid, suggesting that these cell-like microbeads could be used for functional screening of genomic libraries.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.     

Effects of hypophysectomy and immobilization stress on S-adenosylmethionine levels in rat adrenal glands

Rat adrenal S-adenosylmethionine (SAM) levels and phenylethanolamine-N-methyltransferase (PNMT) activity were measured under conditions of hypophysectomy and stress. A new dual-label radioenzymatic assay for SAM is presented which eliminates problems found to exist with previous methods. Strain-specific differences in both PNMT and SAM were found, as well as sex differences in SAM levels. Immobilization stress resulted in an increase in adrenal SAM and PNMT activity, while hypophysectomy decreased both. The distribution of SAM between cortex and medulla did not change with either hypophysectomy or stress. Hypophysectomized Fisher rats were found to be capable of increasing PNMT activity in the absence of increased SAM levels.     

Gene expression of the phenylethanolamine N-methyltransferase is differently modulated in cardiac atria and ventricles

Phenylethanolamine N-methyltransferase (PNMT) is a final enzyme in catecholamine synthesizing cascade that converts noradrenaline to adrenaline. Although most profuse in adrenal medulla, PNMT is expressed also in the heart, particularly in cardiac atria and ventricles. In atria, the PNMT mRNA is much more abundant compared to ventricles. In present study we aimed to find out whether there is a difference in modulation of the PNMT gene expression in cardiac atria and ventricles. We used three methodological approaches: cold as a model of mild stress, hypoxia as a model of cardiac ischemic injury, and transgenic rats (TGR) with incorporated mouse renin gene (mREN-2)27, to determine involvement of renin-angiotensin pathway in the PNMT gene expression. We have found that PNMT gene expression was modulated differently in cardiac atria and ventricles. In atria, PNMT mRNA levels were increased by hypoxia, while cold stress decreased PNMT mRNA levels. In ventricles, no significant changes were observed by cold or hypoxia. On the other hand, angiotensin II elevated PNMT gene expression in ventricles, but not in atria. These results suggest that PNMT gene expression is modulated differently in cardiac atria and ventricles and might result in different physiological consequences.     

Cold-induced increases in phenylethanolamine N-methyltransferase (PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland

Previously, we reported that cold stress induces a rapid increase in adrenomedullary PNMT mRNA levels, followed by concomitant increases in PNMT immunoreactivity (10). In the present study, the extracellular signals mediating this adaptive response to stress were investigated using northern analysis and RNA slot-blot hybridization. Although adrenal denervation significantly diminished cold-induced increments in adrenomedullary PNMT mRNA levels, it did not completely abolish the cold stress response. In contrast to these results, splanchnectomy completely inhibited cold-induced increments in TH mRNAs in the same tissue samples. These findings indicate that the effects of cold exposure on PNMT mRNA levels are mediated by both neural and non-neural mechanisms, and that adrenal PNMT and TH are differentially regulated in response to cold stress. Surprisingly, the neural component of the PNMT stress response could not be attenuated by peripheral administration of chlorisondamine, a powerful nicotinic ganglionic blocking agent. In contrast, chlorisondamine was effective in inhibiting sympathetic neural activity, as judged by the drug's ability to completely block increases in blood pressure, heart rate, and plasma catecholamines resulting from spinal cord stimulation in pithed rats. The administration of atropine, a muscarinic receptor antagonist, also failed to inhibit cold-induced alterations in adrenal PNMT mRNA. These results suggest that the trans-synaptic induction of adrenal PNMT mRNA involves a non-cholinergic component, and that cold-induced increases in PNMT mRNA are not coupled to acetylcholine-mediated adrenal catecholamine release.     

Properties and applications of Penicillium funiculosum cellulase immobilized on a soluble polymer

Summary The cellobiase and xylanase activities of Penicillium funiculosum were immobilized on a soluble polymer poly(vinyl alcohol) (PVA). The kinetic parameters and the adsorption characteristics of the bound and free enzymes were compared. The Km value of the immobilized preparation was the same as the free enzyme. The hydrolysis of different cellulosic substrates by the bound enzyme is investigated.