TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1-3 h in a low O₂ atmosphere. Basal TH levels were 5-6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty-eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O₂ in N₂ for two 30-min periods (20-min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O₂ in N₂ for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.     

外源性TH基因在帕金森氏病鼠脑内的表达——行为和TH免疫组化的研究

用成年大鼠７５只,给右侧黑质区注射６－羟基多巴胺（６－ＯＨＤＡ）,损毁黑质多巴胺能神经元,制备偏侧帕金森氏病（ＰＤ）鼠模型。四周后,注射阿朴吗啡（ＡＰＯ）诱发大鼠向左侧旋转。旋转数为每分钟７次以上的３５只ＰＤ鼠作实验用。其中实验组１５只,对照组２０只。向实验组ＰＤ鼠右侧纹状体多点植入含大鼠酪氨酸羟化酶ｃＤＮＡ（ＴＨｃＤＮＡ）的真核表达载体ｐＳＶＫ３－ＴＨ和脂质体Ｌｉｐｏｆｅｃｔｉｎ混合的基因转染复     

酪氨酸羟化酶基因载体－pCMVTH 质粒的构建及在大鼠骨骼肌内的表达

为用转基因方法治疗巴金森氏病大鼠模型,本研究采用分子克隆技术,将合成多巴胺的关键酶－酪氨酸羟化酶（ＴＨ）的基因,克隆进入以巨细胞病毒ＣＭＶ为启动子的载体质粒内,经限制性内切酶定位分析证实该重组的ＤＮＡ质粒的可靠性。携带ＴＨ基因的ＰＣＭＶＴＨ质粒以ＬＩＰＯ－ＦＥＣＴＩＮ介导,在培养的原代骨骼肌细胞中高效表达。本研究为进一步用转基因的细胞植入脑内以治疗巴金森氏病打下一定基础。     

REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN MOUSE NEUROBLASTOMA CLONE N1E-115

Abstract— Mouse neuroblastoma (clone N1E-115) cells in the logarithmic growth phase were incubated for 12 days. From early log phase to late stationary phase, the specific activity of tyrosine hydroxylase (EC 1.14.3a) increased greater than 30-fold. The increase in tyrosine hydroxylase per cell and per dish was 12- and 2700-fold, respectively. When cell division was stopped by removing serum or by adding 0.1 m m -5-fluorodeoxyuridine and 0.1 m m -uridine, the enzyme activity was also found to increase. These results show that tyrosine hydroxylase is regulated in neuroblastoma clone N1E-115.     

INHIBITION OF GUINEA-PIG BRAIN TYROSINE HYDROXYLASE BY CATECHOLS AND BIOPTERIN

—The inhibition by catechols and biopterin of tyrosine hydroxylase from guineapig caudate nuclei has been examined. Inhibitory constants of 10–20 μm were obtained for dopamine and noradrena-line and 150–250 μm for l -DOPA and dihydroxyphenylacetic acid. When examined under similar conditions homovanillic acid was found not to be inhibitory. Using an acetone dried powder as the source of tyrosine hydroxylase no change in K_m or V_max was observed when cyclic AMP or Ca²⁺ were added to the medium. Enzyme mechanisms and a possible explanation of the mechanisms controlling catechol synthesis are discussed.     

ALLOSTERIC ACTIVATION OF HYPOTHALAMIC TYROSINE HYDROXYLASE BY IONS AND SULPHATED MUCOPOLYSACCHARIDES

Abstract— The kinetics of canine hypothalamic tyrosine hydroxylase were studied in the presence of various ions and sulphated mucopolysaccharides. Enzymic activity was dependent on ionic strength, a specific sulphate effect and the presence of the highly sulphated mucopolysaccharide, heparin. Whereas both sulphate and heparin activated tyrosine hydroxylase by increasing V_max heparin, but not sulphate, also increased the affinity of the enzyme for the synthetic cofactor, 2-amino-4-hydroxy-6,7-dirnethyl-5,6,7,8-tetrahydropteridine, by nearly an order of magnitude. Other rnucopolysaccharides, such as chondroitin sulphate and hyaluronic acid, were not effective as activators of tyrosine hydroxylase. The allosteric activation of tyrosine hydroxylase by heparin may serve to 'sensitize' the enzyme to low levels of its end product, norepinephrine.     

PROPERTIES AND REGIONAL DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN THE CHICKEN BRAIN

Tryptophan hydroxylase in young chicken brain had a pH optimum of 7.5–8, depending on the buffer used. It had apparent K_m values for tryptophan and tetrahydrobiopterin of 49 μM and 32 μM respectively. The enzyme in chicken brain, but not rat brain, was cold-shock labile but was stable for up to 4 days at — 20°C. Lability was observed both in tissues and homogenates of these tissues subjected to cold shock, but the extent of loss of activity varied between brain regions. Supernatant fractions did not lose activity after cold shock. The highest level of tryptophan hydroxylase was found in the rostral region of the chicken brainstem. High levels were also found in the caudal region of the brainstem, the midbrain, thalamus, caudate and cerebral cortex. The cerebellum and optic chiasma contained only traces of activity.     

THE DE NOVO SYNTHESIS OF TYROSINE HYDROXYLASE IN RAT SUPERIOR CERVICAL GANGLIA IN VITRO: THE EFFECT OF NERVE GROWTH FACTOR

Abstract— An immunoprecipitation technique has been employed to measure the rate of synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia and the effect of nerve growth factor on that rate. Ganglia which have been maintained in culture for 16 h without nerve growth factor synthesize tyrosine hydroxylase; the hydroxylase comprises approx 0.2% of the newly synthesized soluble protein. While the total amount of tyrosine hydroxylase synthesized de novo increases in the presence of physiological levels of nerve growth factor, the differential rate of tyrosine hydroxylase synthesis is essentially unchanged. At higher levels of nerve growth factor (3–10 μg/ml) there is a small increase in the differential rate of tyrosine hydroxylase synthesis. The major action of nerve growth factor appears to be on the survival of the tissue, but a small effect on the induction of tyrosine hydroxylase is evident at high levels of nerve growth factor.     

ASPARAGINASE IN PERIPHERAL NERVES OF THE GUINEA PIG IN WALLERIAN DEGENERATION AND EXPERIMENTAL ALLERGIC NEURITIS

Abstract— Asparaginase ( 1 -asparagine amidohydrolase EC 3 , 5.1.1.) activity in peripheral nerves in Wallerian degeneration and in experimental allergic neuritis was studied in the guinea pig. After mechanical damage to the sciatic nerve, asparaginase enzymic activity increased markedly in the regenerating (central) stump at the end of the first week, followed by a decrease in activity almost to control values in the course of 4 weeks, whereas in the degenerating (peripheral) stump, after an increase in activity at the end of the first week the enzyme activity continued to increase reaching 250 per cent of the control values at the end of the fourth week. An increase in asparaginase activity was also observed in experimental allergic neuritis.     

COMPARISON OF THE BEHAVIOUR OF A SOLUBLE AND A MEMBRANE-BOUND ENZYME IN TRANSECTED PERIPHERAL NERVES

Phosphoglucoisomerase (PGI), a soluble enzyme, and AChE, a membrane-bound enzyme were studied in transected peroneal nerves of dog and in isolated segments of these nerves. Although activities of both enzymes increased at the ends of transected nerves, marked differences in their behaviour were observed. The increment in AChE activity was much sharper than that of PGI and continued to grow with time whereas the increase in PGI developed fully within the initial hours after transection and did not change thereafter. In an isolated nerve segment AChE accumulated at both ends with a concomitant decrease in the middle part, whereas changes in PGI activity appeared only in the terminal parts, the rest of the nerve remaining at the normal level. The terminal increase of PGI did not, contrary to that of AChE, depend on the length of the isolated segment. The changes in PGI activity may be features of a local peritraumatic reaction whereas those of AChE indicate involvement of the whole segment along which the enzyme containing organelles are transported.     

周围神经43kD蛋白免疫化学研究

目的：制图周围神经43kD蛋白单克隆抗体,并检测该蛋白在正常及损伤坐骨神经中的表达,方法：实验用SDS－聚丙烯酰胺胺凝胶电泳系统,从周围神经中分离回收43kD蛋白作为抗原,免疫BALB／c小鼠,通过杂交瘤技术和点膜印迹法检测,获得分泌识别43kD蛋白的单克隆抗体的杂交瘤细胞株,以Westernblot方法检测单克隆抗体的特异性,并检测43kD蛋白在正常坐骨神经及损伤坐骨神经远侧端中的表达。结果：经检测获得了识别43kD蛋白的单克隆抗体,Westernblot显示在正常大鼠坐骨神经与损伤后2周的坐骨神经远侧端组织电泳图谱43kD处均出现特异的阳性反应条带,在损伤神经中43kD蛋白阳性反应产物着色较深。结论：43kD蛋白具有独特的免疫化学特性,在正常与损伤坐骨神经中的有表达,在损伤坐骨神经中表达更强。     

SOLUBLE AND PARTICLE-BOUND ACETYLCHOLINESTERASE AND ITS ISOENZYMES IN PERIPHERAL NERVES

Abstract— The distribution of AChE (EC 3.1.1.7) in soluble and particulate fractions of the peripheral nerves of dogs, cats, rabbits and frogs was examined. About 20–30% of the total AChE activity was found in the supernatant fluid after centrifugation (100,000 g for 90 min) of iso-osmotic sucrose homogenates. The effect of different media on the extent of solubilization of the enzyme was studied and Triton X-100 (0.2%) was found to be the most effective. The electrophoretic pattern of AChE in peripheral nerves was also investigated. The 2–3 types of AChE observed previously were found in both particulate and soluble fractions, but the proportions of these forms were different. The most slowly migrating form of AChE is the most firmly bound to nerve membranes. A very small but consistent proportion (3%) of AChE escaped into the medium from surviving dog nerves kept in aerated Ringer solution. It was calculated that the possible contribution of blood AChE contained in the nerve is negligible. Electrophoretograms of AChE released during incubation into Ringer solution were similar in pattern to those found for the soluble fraction.     

THE EFFECTS OF REPEATED-LYSERGIC ACID DIETHYLAMIDE INJECTIONS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN REGIONS

Daily injections of 100 μg/kg of d -lysergic acid diethylamide (LSD) for 14 days produced a significant decrease in the dopamine level in rat brain corpus striatum which was still apparent 15 days after the last LSD treatment. Further LSD injections did not change the amount of dopamine depletion. In cerebral cortex, 14 days of LSD injections produced a significant decrease in the norepinephrine level and a significant increase in tyrosine hydroxylase activity. The elevated tyrosine hydroxylase activity was still present 15 days after the final LSD injection but only in those animals receiving daily vehicle injections during this period. Pre-treatment of rats with daily saline injections for 2 weeks before the 2 week period of LSD treatment prevented both the reduced norepinephrine content and elevated tyrosine hydroxylase activity usually found 24 h after the last LSD injection.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES

Abstract— The redistribution of rapidly migrating [³H]leucine-labelled proteins and [³H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies, rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve, rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 16 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.     

MINIMUM DURATION OF TRANS-SYNAPTIC STIMULATION REQUIRED FOR THE INDUCTION OF TYROSINE HYDROXYLASE BY RESERPINE IN THE RAT SUPERIOR CERVICAL GANGLION

—The period during which trans-synaptic stimulation is required by the rat superior cervical ganglion for induction of tyrosine hydroxylase by reserpine has been studied. Ganglia were decentralized on one side at various times before or after an injection of reserpine. The tyrosine hydroxylase activity of the denervated and control ganglia was assayed 72 h after drug treatment. When decentralization was performed 8 h after an injection of reserpine the increase in tyrosine hydroxylase activity was blocked in the denervated ganglia. Decentralization 12 h after reserpine treatment or later had no effect on the enzyme induction. The actual increase in tyrosine hydroxylase activity occurred between 24 and 48 h after injection of reserpine.     

PROPERTIES OF PARTIALLY PURIFIED PIG BRAIN STEM TRYPTOPHAN HYDROXYLASE

—Tryptophan hydroxylase form pig brain has been purified using a method which involved sonic disintegration of a whole homogenate, ammonium sulphate fractionation, hydroxylapatite fractionation, column chromatography on Sephadex G-100 or G-200 and finally electrophoresis on poly-acrylamide gel. The enzyme was stabilized during purification by tryptophan and dithiothreitol. The partially purified enzyme has a molecular weight of 55,000-60,000 as measured by gel-filtration. The K_m of the soluble partially purified enzyme was 0-4 mm , which differed significantly from that of the particulate enzyme (0·02mm ). Enzyme activity was not stimulated by ferrous ion. However, it was inhibited by the chelating agents 8-hydroxyquinoline, O-phenanthroline and EDTA. In contrast to dopamine, high concentration of tryptophan (10 mm ), 5-hydroxytryptamine, tryptamine and tyramine at 0-5 mm concentration did not inhibit the enzyme in the presence of dimethyltetrahydropterin (DMPH4). A number of monoamine oxidase inhibitors, phenelzine, pheniprazine and chlorgyline at 1 mm strongly inhibit the formation of 5-hydroxytryptamine. Evidence is presented for the presence of an endogenous inhibitor of tryptophan hydroxylase.