摘要:

AbstractTwo rabbit arylamineN‐acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, ρ‐aminosalicylic acid, and ρ‐aminobenzoic acid. In the pineal gland, serotonin is metabolized toN‐acetylserotonin by an unknownN‐acetyltransferase. Similarity of the liver enzymes and the pineal gland arylalkylamineN‐acetyltransferase (AA‐NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as ρ‐phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed theN‐acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressedrNAT1andrNAT2genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross‐hybridization ofrNAT1orrNAT2to the gene for the pineal gland AA‐NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step ofN

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08266.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractWe show that human and bovine dopamine β‐hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholin‐esterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4and Ga4AChE forms, we propose to call the nonamphiphilic tetramer Dna4and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II ‐Da4I‐S. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide. and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08267.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractA translational inhibitor that is activated byN‐ethylmaleimide treatment can be found in the postmicrosomal fraction prepared from the brain of adult rats, but it is almost undetectable in the same fraction prepared from suckling animals. The inhibitor is thermolabile and remains in the supernatant fraction after precipitation at pH 5. During the purification procedure, the inhibitor in its unactivated state binds to the anion exchanger (diethylaminoethyl‐cellulose) but not to the cation exchanger (phosphocellulose). Treatment withN‐ethylmaleimide increases inhibitor affinity for the cation exchanger, and this chromatographic step purifies the inhibitor by 143‐fold. Both the thermolabile nature and the behavior of the inhibitory activity during the different steps of the purification procedure suggest that this activity is most probably due to a protein. Although the addition of initiation factor 2 reverses the inhibition of protein synthesis in the presence of ATP and Mg2+, the inhibitor does not phosphorylate any of the initiation factor subunits “in vitro,” which indicates that it does not contain any intrinsic protein kinase activity. However, its presence in both a crude and a purified preparation of a kinase of the α subunit of a brain eukaryotic initiation factor increases the phosphorylation of the α subunit of the initiation factor. The mechanism of action of this inhibitor

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08268.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractThe objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 μMcholine, washed, resuspended, and then incubated for 10 min in choline‐free buffer containing 4.74 (Krebs‐Ringer bicarbonate, KRB) or 25 mMKC1. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 μMcholine did not lead to a further increase, but preincubation with 25 or 50 μMcholine increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 μMcholine. When tissues were subsequently incubated for 10 min with either KRB or 25 μMKC1, ACh release from slices preincubated with 50 μMcholine was greater than from slices preincubated with 0, 10, or 25 μMcholine. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mMKC1, the ACh content of slices preincubated with 0 or 10 μMcholine decreased significantly, whereas that of slices preincubated with 25 or 50 μMcholine did not. Although ACh synthesis by slices incubated with KRB did not differ significantly among groups, synthesis by slices incubated with 25 mM KC1 was significantly greater for tissues preincubated with 50 μMcholine than for those preincubated with 0, 10, or 25 μMcholine. Preincubation in the absence of choline decreased choline levels by 43%, whereas preincubation with 50 μMcholine increased levels by 55%; choline levels were unaltered by preincubation with 10 μMcholine. Following incubation with 25 mMKCl, the choline content of slices preincubated with 50 μMcholine decreased by 0.59 nmol, whereas the content of slices preincubated with 10 μMcholine decreased by 0.33 nmol, suggesting that the former used more of its tissue choline to support ACh synthesis; choline levels did not change in slices preincubated in the absence of choline. These results suggest that prior exposure of hippocampal slices to choline provides precursor that is used during a subsequent incubation with 25 mMKCl to support th

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08269.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractThe prenatal and postnatal human ontogeny of the central benzodiazepine receptor was investigated in six different brain regions between week 24 postconception and age 14 years. Binding studies, which were performed with [3H]flunitrazepam ([3H]FNZ), revealed a steep increase in receptor density postnatally in frontal cortex and cerebellum.Bmaxvalues were higher in medulla oblongata, pons, and thalamus than in cortex and cerebellum up to week 26. After that, receptor densities declined significantly in medulla and olive. The same tendency was apparent in pons, whereas receptor density remained unchanged in thalamus. The early ontogeny of the benzodiazepine receptor was also evaluated in fluorographs ([3H]FNZ) and immunoblots using the αrsubunit‐specific monoclonal antibody (mAb) bd‐24. Specific radiolabeled proteins with molecular weights of 53K and 59K were visible in cortical membranes from gestational week 8, the earliest time investigated. During further development, the intensity of the 53K band increased without changes in the 59K band. As in other species, postmortem proteolysis in human brain led to a specifically labeled peptide of 47K. The mAb bd‐24 immunolabeled only the 53K protein and the 47K p

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08270.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract31P and1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of1H NMR‐detected glutamate by a water‐suppressed spin‐echo method, using N‐acetyl aspartate as an internal concentration reference. To quantify the1H NMR signals, the spin‐spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions,1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the1H NMR signal. However, during severe anoxia an intracellular reorganization occurs increasing the detectability of this amino acid neurotransmitt

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08271.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractIn this study we have demonstrated that noradrenaline increases the levels of prostaglandin E2and prostaglandin I2(detected as the stable metabolite 6‐keto‐prostaglandin F1α) synthesized by homogenates of superior cervical ganglia from the adult rat. This noradrenaline‐induced prostaglandin production was further characterized: (a) Selective destruction of adrenergic sympathetic postganglionic neurons in the ganglia using 6‐hydroxydopamine abolished both basal and stimulated prostaglandin production, (b) Elimination of preganglionic cholinergic sympathetic nerve terminals in the ganglia had no effect, (c) Mepacrine (a phospholipase inhibitor) and indomethacin (a cyclooxygenase inhibitor) attenuated both basal and stimulated prostaglandin production. (d) Yohimbine, but not prazosin, suppressed the noradrenaline dose‐response curve for prostaglandin production. The results of these experiments show that, in vitro, noradrenaline stimulates de novo synthesis of prostaglandin E2 and prostaglandin I2by sympathetic postganglionic neurons. This stimulation by noradrenaline appears to result from action at an α2‐adrene

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08272.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractPolyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high‐molecular‐weight structural proteins, apparently by activating calcium‐dependent proteases (cal‐pains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium‐dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine>spermidine>putrescine, with apparent affinities of 30, 300, and 6.000 μM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium‐dependent breakdown of two endogenous high‐molecular‐weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium‐dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a rote for polyamines in protein degradation as well as

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08273.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extrasynaptosomal concentration of glutamate ([Glu]cxt) under aerobic conditions was 2.3 μMand increased to 4.9 μMafter 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]cxtThe accumulation of glutamate in the external medium during anoxia was Ca2+in dependent and insensitive to a significant reduction of the Ca2+‐dependent release of the amino acid. These results indicate that a Ca2+‐independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve term

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08274.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

AbstractThe specific binding of L‐N6‐[3H]phenylisopropyladenosine (L‐[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L‐[3H]PIA (0.5–12.0 nA/) in the presence of Mg2+showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 ± 0.03 and 0.66 ± 0.05 pmol/mg of protein. In the presence of GTP, the binding of L‐[3H]PIA also showed two sites withKDvalues of 24.7 and 811.5 nMandBmaxvalues of 0.27 ± 0.09 and 0.93 ± 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L‐[3H]PIA binding by 1, 3‐dipropyl‐8‐cydopentylxanthine (DPCPX) (0.1–300 nM) performed with the same preparations revealed two DPCPX binding sites withKivalues of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respectiveKDvalues of 0.81 and 10.7 nMand respectiveBmaxvalues of 0.19 ± 0.02 and 0.74 ± 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1subtype and another with lower affinity with characteristics of the A3su

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb08275.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY