Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase

An increase of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4-dihydroxyphenylalanine (L-DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L-DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brainstem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6-hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.     

Subcellular Distribution and Developmental Expression of Cholesterol Ester Hydrolases in Fetal Rat Brain Cultures

Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19- to 21-day-old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two- to three-fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Respiration-dependent proton translocation and the mechanism of protonmotive force generation in Nitrobacter winogradskyi

Abstract Proton translocation associated with electron flow to oxygen has been observed with cells of Nitrobacter winogradskyi in the presence of either potassium ferrocyanide or isoascorbate plus N , N , N ', N ' tetramethyl- p -phenylenediamine. The data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa ₃, in this organism as the mechanism for generating a protonmotive force. The failure of previous work with Nitrobacter [4] to detect proton translocation linked to oxidation of nitrite, the physiological substrate, is discussed.     

光对水稻(Oryza sativa)幼苗乙醇酸氧化酶活性的影响

水稻黄化幼苗用白光照射3～5小时后可诱导出乙醇酸氧化酶活性。绿色水稻幼苗在黑暗中乙醇酸氧化酶活性逐渐下降以至消失,再给以照光又恢复酶活性。亚胺环己酮对光的诱导及再诱导均有抑制作用。在黑暗中真空渗入乙醇酸于黄化幼苗可诱导出乙醇酸氧化酶活性,渗入乙醇酸加FMN能使酶活性迅速增强。弱的白光以及红、绿,蓝光均有诱导作用。因此认为光的诱导作用是使黄化幼苗形成乙醇酸——作为诱导物,以诱导乙醇酸氧化酶的新合成。光也可以加速叶组织内FMN的合成,从而提高乙醇酸氧化酶的活性。     

棉纤维细胞伸长生长与过氧化物酶和IAA氧化酶的关系

棉纤维细胞于开花当天从棉胚珠表皮上发生,随即开始伸长生长,星S型生长曲线。棉纤维细胞的可溶性蛋白、过氧化物酶活性和IAA氧化酶活性同伸长生长的关系不大;而离子型结合的细胞壁蛋白质含量、过氧化物酶活性和IAA氧化酶活性同棉纤维细胞的伸长生长关系较大,表现在棉纤维细胞快速伸长期活性较低,而在伸长生长停止时出现活性高峰,同棉纤维细胞的伸长生长有负相关现象。     

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 µm diameter fraction believed to be composed largely of basal cells, and a 15 µm diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 µm cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 ± 2.7% ciliated cells with 6.5 ± 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 ± 2.1%) and Clara cells (27.0 ± 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 ± 0.09 × 10⁶ cells/ trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 µm cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 ± 66 pmol/ min/ mg protein or 51.2 ± 20.5 pmol/ min/ 10⁶ cells for 7-ethoxycoumarin deethylase and 31.7 ± 15.4 pmol/ min/ mg protein or 10.5 ± 4.8 pmol/ min/ 10⁶ cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.Abbreviations CD cell digest - DNase deoxyribonuclease I - E-1 first elutriator fraction - E-2 second elutriator fraction - E-3 third elutriator fraction - 7-Ec 7-ethoxycoumarin - FCS fetal calf serum - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - HpBS HEPES-buffered salt solution - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PEG Carbowax polyethylene glycol 6000     

Purification and properties of indole 2,3-dioxygenase from maize leaves

An indole 2,3-dioxygenase was purified ca 38-fold from maize leaves. The enzyme had an MW of about 98000, an optimum pH of 5.0 and the energy of activation was 9.1 kcal/mol. The K_max for indole was 1.4 × 10^?4 M. The enzyme was inhibited by diethyldithiocarbamate, salicylaldoxime and sodium dithionite. The inhibition by diethyldithiocarbamate was specifically reversed by Cu²⁺. The dialysed enzyme was stimulated by Cu²⁺. Four atoms of oxygen were utilized in the disappearance of 1 mole of indole. Inhibition of the enzyme by -SH compounds and -SH group inhibitors, and their partial removal by Cu²⁺ only, suggested the involvement of -SH groups in binding of Cu²⁺ at the catalytic site.     

Carbon monoxide sensitivity of cytochrome oxidase in wheat leaves in relation to dwarfing genes (Rht) in wheat cultivars

Summary Leaves of young seedlings of a number of tall cultivars of wheat, lacking the dwarfing Rht genes, readily responded to a brief 2 min exposure to CO, as assessed by in vivo aerobic assay of nitrate reductase. This test depends on the inhibition of cytochrome c oxidase by CO, which in turn renders cytosolic NADH available for the reduction of nitrate to nitrite in vivo. Semi-dwarf cultivars of wheat (Rht present) did not respond to CO in this way. Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃, during in situ respiration of leaves from tall and semi-dwarf plants which are likely to be under genetic control.     

Isolation and characterization of human heart cytochromec oxidase

Cytochromec oxidase was isolated from human hearts and separated by SDS gel electrophoresis. The identity of polypeptide bands with known subunits was demonstrated by immunoblotting with monospecific antisera to rat liver cytochromec oxidase subunits. The polarographically determined kinetics of cytochromec oxidation were similar to those reported for the bovine heart enzyme.