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.     

Highly sensitive assay for phenylethanolamine N-methyl-transferase activity in rat brain by high-performance liquid chromatography with electrochemical detection

This paper describes a new and highly sensitive assay for phenylethanolamine N-methyl-transferase (PNMT) activity with noradrenaline as substrate in various rat brain regions by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% of contaminating adrenaline, which was removed to reduce the blank value. Enzymatically formed adrenaline was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid, separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. 3,4-Dihydroxybenzylamine was added to the incubation mixture as an internal standard after the reaction. This assay was very sensitive and 0.5 pmol of adrenaline formed enzymatically could be detected. This assay method was applied to measure PNMT activity in various rat brain regions. The highest activity was observed in the hypothalamus, pons plus medulla oblongata, septum, lower brain stem, and cerebral cortex; the lowest activity was in the striatum, hippocampus, cerebellum, and limbic brain.     

ARGINASES OF MOUSE BRAIN AND LIVER

The arginase present in mouse brain and liver was studied in order to determine if the activity in both tissues was due to the same enzyme. Mouse liver contains only one arginase enzyme whereas mouse brain contains two. One of the arginases in the brain is distinct from the liver enzyme as determined by fractionation on DEAE-cellulose, CM-cellulose and disc gel electrophoresis. The second enzyme from brain tissue has the same properties as the liver enzyme when subjected to these same fractionation techniques. However this arginase can be distinguished from the liver enzyme by its K_m for arginine heat lability and MnCl₂ activation curve. Thus both arginases in brain differ from the liver enzyme. No interconversion of the brain enzymes was detected, and the molecular weight of all the arginases appears to be the same. The observation of multiple distinct brain and liver arginases in mouse brain and liver was confirmed with bovine tissues.     

Isolation and polymerization of brain actin

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar to actin, is the major constituent. A method is described for isolating the “brain actin” to a purity of 90–95%. The isolation method begins with an extraction of bovine brain in low ionic strength buffer with ATP and sucrose. The extract is treated with NH₄SO₄, MgCl, and KCl and incubated at 37°C. A precipitate is formed which contains primarily tubulin and brain actin. Resolubilization of the brain actin is achieved with a low ionic strength buffer solution with sucrose and ATP. Further purification is accomplished by a cycle of polymerization—depolymerization. This “brain actin” shares with muscle actin the following properties: (1) Similar molecular weight and molecular charge as determined by SDS polyacrylamide gel and ordinary disc electrophoresis; (2) Polymerization to a filamentous form under the same conditions; (3) Contains 3-methylhistidine; (4) Vinblastine sulfate will induce filament formation.     

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.     

Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O₂) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl₂ or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal ?893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (?282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

N-acetyl-aspartate amidohydrolase: purification and properties.

Abstract— The enzyme in rat brain responsible for the de-acetylation of N-acetyl-aspartic acid has been partially purified. In contrast to the enzyme from hog kidney which is stable at 70°C, it rapidly denatures above 57°C. The rat brain enzyme has the same K_m for its substrate and the same solubility in ammonium sulphate solution as the hog kidney enzyme. Results of migration on starch gel electro-phoreses and isoelectric focusing indicate a pI for the amidohydrolase of 5.1. A variety of potential substrates, modulators, and inhibitors have been examined.     

Kinetic and pH studies on human phenylethanolamine N-methyltransferase

Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-l-methionine (AdoMet) is converted to S-adenosyl-l-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the log V pH-profile was not well defined, plots of log V/K versus pH for AdoMet and phenylethanolamine, as well as the pK_i versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.     

Properties of L-glutamate decarboxylase from brains of adult and newborn mice.

Abstract— l -Glutamate 1-carboxy-lyase (EC 4.1.1.15) (GAD) and 4-aminobutyrate-2-oxo-glutarate aminotransferase (EC 2.6.1.19) (GABA-T) have been purified from mouse brain (Wu et al. 1973; Schousboe et al., 1973) and their properties have been extensively studied (Wu & Roberts , 1974; Schousboe et al., 1974). The above enzymes were prepared from a water lysate of crude mitochondrial fraction, which accounted for only 25–30% of total GAD or GABA-T activities in brain. A procedure has been developed which liberates approx 85% of total GAD and GABA-T activities into supernatant. Two distinct, well-separated peaks with GAD activity and a single peak with GABA-T activity were observed when a concentrated extract from brain of adult or newborn mice was chromatographed on Sephadex G-200 or Bio-Gel A–1.5 m. The first peak appeared in the void volume and is. therefore. an entity of high molecular weight. The second peak gave elution characteristics which were identical to those of the enzyme that had been purified previously (mol wt = 85,000). These two GAD peaks were also clearly separated on polyacrylamide gel electrophoresis. The GAD activities in the two peaks showed similar pH profiles (optimum, 6.5). K_m values (1–2 mM), immunodiffusion patterns and inhibitions by anti-GAD IgG prepared against GAD purified from synaptosome-containing crude mitochondrial fraction (60–80%). The physiological implications of high molecular weight and low molecular weight forms of GAD are discussed.     

Isolation and characterisation of cathepsin-B from bovine pancreas

A simple procedure for the isolation of cathepsin-B from bovine pancreas employing ammonium sulphate fractionation, DEAE cellulose chromatography and Sephadex G-200 gel filtration is described. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight as determined by gel filtration of the enzyme was 26,850. ItsK _m andV _max values were 12.8 mM and 0.303 Μmol/min/mg, respectively. TheK _i for iodoacetamide was 0.16 mM.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

Purifcation and properties of multiple forms of brain acetylcholinesterase (EC 3.1.1.7)

—Approximately 70 per cent of the total AChE of bovine brain tissue was solubilized by repeated homogenization and centrifugation in 0.32 m sucrose containing EDTA. After ammonium sulphate fractionation, application of the enzyme preparation to an agarose affinity gel column effected a 700-fold purification. Subsequent molecular filtration separated three active forms of AChE with molecular weights of 130,000, 270,000 and 390,000 with an average specific activity of 575 mmol of acetylthiocholine hydrolysed/mg of protein/h. The complete procedure represented an approximate 23,000-fold purification of the enzyme from that in the original tissue homogenate. The three forms of AChE exhibited certain differences in properties, including apparent K_m values, pH optima and sensitivity to inhibitory agents. Ancillary studies on less purified enzyme preparations by use of polyacrylamide gel electrophoresis and isoelectric focusing techniques also suggested that brain AChE exists in multiple forms.     

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Effects of Rheological Properties and Molecular Weight Distribution on the Spinnability of Soy Protein

Experiments were carried out to elucidate the correlation between gel chromatographic profiles and rheological properties of dope with respect to the spinnability of soy protein. It was found that dope prepared with 20% protein concentrate and 1.2% sodium hydroxide showed a good transition zone from rubber-like elasticity to rubber-like flow, as frequency (log ω) in limitation was increased from ?1 to 1, and also the dope showed good spinnability when prepared in the above manner. Good spinnable dopes showed three main fractions in the gel chromatographic profiles on using a Sepharose 4B gel column. The first peak was of a high molecular weight fraction (M_w > 1,000,000) eluted at the void volume, the second peak was of low molecular weight fraction (M_w < 200,000; the peak was around 10,000) eluted at the end of the gel chromatogram, and the third part was a gentle upward slope between the two peaks (1,000,000 > M_w > 200,000). It was clear that the spinnabilities and rheological properties of dope depended on differences in composition of the three main fractions mentioned.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

Properties of the cell envelope and a cell-envelope protein of Pseudomonas facilis

The molecular weight of the protein moiety of a phospholipoprotein complex isolated from Pseudomonas facilis has been examined with a variety of sodium dodecylsulfate-polyacrylamide gel electrophoretic systems. A molecular weight of 35 000 was determined for the protein in all analyses. A 35 000-dalton protein was present in the EDTA extract of P. facilis and in the cytoplasmic and outer membrane fractions, but not in the lipopolysaccharide and peptidoglycan. Prior inoculation of mice with the phospholipoprotein complex led to a 7.5- to 15-fold increase in the LD₅₀ when mice were subsequently inoculated with Salmonella typhimurium; this pathogen has a cell-surface protein which cross-reacts immunologically with antibody to the P. facilis phospholipoprotein complex.Abbreviations KDO 2-keto-3-deoxyoctanoate - LD₅₀ the dosage of Salmonella typhimurium at which there is 50% survival in mice - LPS lipopolysaccharide - PLP phospholipoprotein - P_PLP the protein moiety of PLP - SDS sodium dodecylsulfate - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TMS trimethylsilyl     

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134?kDa for G6PD, 107?kDa for 6PGD and 121?kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63?kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13?mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.