CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

THE DISTRIBUTION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE IN THE HYPOTHALAMUS

Abstract— Quantitative measurements of capacities of microgram-sized samples of tissue to hydrolyze adenosine 3',5'-phosphate (cyclic AMP) and guanosine 3',5'-phosphate (cyclic GMP) have been made in 32 discrete regions of the rabbit hypothalamus. The capacities to hydrolyze the two cyclic nucleotides were found to be distributed over a 3-fold range and to vary in parallel from region to region. Notable exceptions, however, were observed. A positive association was found between the capacity to hydrolyze cyclic AMP and the reported density of catecholaminergic innervation. No clear relationship was observed between the distributions of the cyclic nucleotide phosphodiesterase activities and that of other enzymes and neurotransmitters.     

SOME PHYSICAL PROPERTIES OF BOVINE ADRENAL MEDULLARY DOPAMINE β-HYDROXYLASE

(1) Dopamine, β-hydroxylase (EC 1.14.2.1) was purified from bovine adrenal medullae according to the method of Foldes , Jeffrey , Preston and Austin (1972). (2) The kinetics, pH optimum and the effect of Cu²⁺ ions on the purified enzyme were found to resemble those of the enzyme isolated by more involved procedures. (3) The sedimentation coefficient (s₂₀) of the homogeneous enzyme in 10 mM-phosphate buffer, pH 7·2, containing 0·1 M-NaCI was found to be 10·24 ± 0·12 (S.E.M. of 10 determinations). (4) The effect of pH on the mol. wt. of the enzyme was investigated and no large deviation was found from the native mol. wt. of 290,000 in the pH range 3·9 to 11·1. (5) The amino acid analysis of dopamine β-hydroxylase is presented, and is contrasted to that of chromogranin A purified from the same chromaffin granule lysate. (6) Treatment with either 8 M-urea or 0·1% (w/v) sodium dodecyl sulphate was found to dissociate the enzyme into three similar, non-active subunits, each of mol. wt. of the order of 100,000.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

浙江蝮蛇蛇毒蛋白水解酶(蛋白酶a)的酶学性质及生物学活性研究

应用离子交换及凝胶柱层析,从浙江蝮蛇(Agkistrodon halys Pallas)蛇毒中分离纯化出一种具有酪蛋白水解活性的蛋白酶a。经研究表明,以酪蛋白为底物,该蛋白酶a作用的最适pH值为9.5,最适温度为45℃,Km值为1.33×10~(-5)mol/L,酶活性受EDTA抑制。蛋白酶a不水解TAME,BAEE等精氨酸脂。实验表明,蛋白酶a具有纤维蛋白溶解作用而无出血毒性,EDTA和半胱氨酸可抑制其纤溶活性。     

豆乳凝固醇产生菌UV—10发酵条件及其酶学性质的研究

豆乳凝固醇产生菌Bacillus sp.UV-10的最适产酶条件,初始pH6.4,温度26℃,培养时间19h,需要较大的通气量,酶的最适作用pH和温度分别为5．8和70℃,在最适条件下酶活力可达1.84u/mL,pH6.0-7.0稳定性较好,60℃下1h残余酶活60％,Ca^2 ,Fe^2 ,Mg^2 ,Na^2 对其有较强的激活作用,而Zn^2 ,Al^3 则有抑制作用。     

豆乳凝固酶产生菌UV-10发酵条件及其酶学性质的研究

豆乳凝固酶产生菌Bacillussp .UV 1 0的最适产酶条件 :初始pH6 4,温度 2 6℃ ,培养时间 1 9h ,需要较大的通气量。酶的最适作用pH和温度分别为 5 8和 70℃。在最适条件下酶活力可达 1 84u/mL。pH6 0～ 7 0稳定性较好。 6 0℃下 1h残余酶活 6 0 %。Ca²⁺,Fe²⁺,Mg²⁺,Na⁺对其有较强的激活作用 ,而Zn²⁺,Al相似文献   

PHOTOSYNTHETIC NITRITE REDUCTASE II. FURTHER PURIFICATION AND BIOCHEMICAL PROPERTIES OF THE ENZYME

1. Further purification of photosynthetic nitrite reductase (PNiR),which catalyzes the transfer of hydrogen (or electron) fromthe photolytic system to nitrite, is reported in this paper.Chromatography on DEAE- cellulose and Sephadex gel-filtrationwere effective for the purification of PNiR.
2. PNiR could befractionated into two components. It was inferredfrom the dataobtained that one of these components is identicalwith PPNR,and the other one may probably be a hitherto unreportedflavinenzyme containing FMN as prosthetic group.
3. The propertiesof these two components of PNiR were described,and the interrelationshipbetween these catalysts and possibleintermediary carriers ofthis electron transfer system was discussed.

¹Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.This investigation was supported by a Grant in Aid for FundamentalScientific Research from the Ministry of Education (No. 407130-1961)and a Grant in Aid for Organized Scientific Research from theMinistry of Education (No. 95037-1960), which are gratefullyacknowledged here. The authors also wish to acknowledge thatthe progress of this study was facilitated by a Grant from theKAISEI-KAI. A preliminary report on this work was read beforethe 25th Annual Meeting of the Botanical Society of Japan (1960,Osaka).     

竹荪的菌丝培养及其抗菌性的初步研究

对长裙竹荪（Ｄｉｃｔｙｏｐｈｏｒａ　ｉｎｄｕｓｉａｔａ）等３种的菌丝培养进行了研究并初步测定菌丝的抗菌谱。培养液的 ｐＨ和液体培养基粘度是竹荪在液体中生长的关键因素,在 ｐＨ４.７和添加 ０.５％ＣＭＣ－Ｎａ的合适培养条件下竹荪菌丝在液体中能够生长,菌丝得率达到１％。还探讨了其他几种竹荪菌丝的培养方法,为竹荪菌丝的大量生产提供了依据。另外,在竹荪抗菌性实验中发现,竹荪对许多易造成食品腐败的细菌和酵母具有抗性,但没有发现其对霉菌有抗性。     

AM真菌和镰刀菌对西瓜根系几种酶活性的影响*

在温室盆栽条件下研究了丛枝菌根(Arbuscular Mycorrhiza, AM)真菌Glomus versiforme和西瓜枯萎镰刀菌Fusarium oxysporum f.sp. niveum对西瓜根系中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶和几丁质酶活性的影响。结果表明,接种AM真菌的西瓜根系中4种酶的活性均高于对照,先接种G. versiforme,后接种F. oxysporum f.sp. niveum处理的4种酶的活性均高于只接种F. oxysporum f.sp. niveum 的处理,且酶的活性峰值出现较早。表明接种G. versiforme 能预先诱导这4种酶的产生,提高其活性,从而提高西瓜对F. oxysporum f.sp. niveum侵染的抗性。接种G. versiforme的感枯萎病西瓜品种“郑杂5号”酶的增加幅度大于抗病品种“京欣1号”的接种处理,说明G. versiforme对提高感病西瓜品种酶活性的作用更大。     

AM真菌和镰刀菌对西瓜根系几种酶活性的影响

在温室盆栽条件下研究了丛枝菌根(Arbuscular Mycorrhiza, AM)真菌Glomus versiforme和西瓜枯萎镰刀菌Fusarium oxysporum f.sp. niveum对西瓜根系中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶和几丁质酶活性的影响。结果表明,接种AM真菌的西瓜根系中4种酶的活性均高于对照,先接种G. versiforme,后接种F. oxysporum f.sp. niveum处理的4种酶的活性均高于只接种F. oxysporum f.sp. niveum 的处理,且酶的活性峰值出现较早。表明接种G. versiforme 能预先诱导这4种酶的产生,提高其活性,从而提高西瓜对F. oxysporum f.sp. niveum侵染的抗性。接种G. versiforme的感枯萎病西瓜品种“郑杂5号”酶的增加幅度大于抗病品种“京欣1号”的接种处理,说明G. versiforme对提高感病西瓜品种酶活性的作用更大。     

盐度对花鳗鲡和太平洋双色鳗鲡幼鳗生长性能及消化酶活力的影响

文章研究了不同盐度对花鳗鲡(Anguilla marmorata)幼鳗和太平洋双色鳗鲡(A. bicolor pacifica)幼鳗生长性能及消化酶活力的影响。将花鳗鲡幼鳗(9.760.36) g和太平洋双色鳗鲡幼鳗(11.820.04) g分别在淡水(盐度0)与盐度5、10、18水体中养殖30d, 测量每组实验鱼总重后检测胃、肠道和肝脏蛋白酶、淀粉酶和脂肪酶的活力。结果表明, 花鳗鲡和太平洋双色鳗鲡在各盐度处理中存活率均为100%, 未出现死亡。两种鳗鲡在淡水中生长良好, 特定生长率最高, 而饵料系数最低。盐度对花鳗鲡幼鳗和太平洋双色鳗鲡幼鳗消化酶活力的影响存在差异, 其中花鳗鲡胃、肠道和肝脏蛋白酶活力在各盐度处理中均无显著变化(P0.05), 淀粉酶和脂肪酶活力均随盐度的增加而下降; 太平洋双色鳗鲡胃蛋白酶活力在盐度10时最大, 肝蛋白酶活力在盐度18时最大, 而淀粉酶和脂肪酶活力在各盐度处理组无显著变化(P0.05)。这表明盐度对花鳗鲡胃、肠道和肝脏的淀粉酶和脂肪酶活力具有抑制作用, 对太平洋双色鳗鲡的蛋白酶活力有一定的激活作用。在相同盐度条件下, 不同消化器官中同种消化酶活力存在差异, 各盐度的两种鳗鲡肠道中淀粉酶和脂肪酶的活力均显著高于肝脏和胃(P0.05), 胃中蛋白酶活力高于肝脏和肠道, 但不显著(P0.05)。研究发现两种鳗鲡体内脂肪酶活力相对较高, 表明其对脂肪具有较强的消化能力。建议在配制花鳗鲡幼鳗和太平洋双色鳗鲡幼鳗饲料时, 适当提高粗脂肪比例, 有助于促进对营养物质的消化吸收, 提高养殖效益。       

SOME PROPERTIES OF THREE VIRUSES ISOLATED FROM A DISEASED PLANT OF SOLANUM JASMINOIDES PAXT. FROM INDIA

Three mechanically transmissible viruses were isolated from a diseased Solanum jasminoides plant obtained from India. One is a strain of potato virus Y , which in some potato varieties produces symptoms resembling those caused by potato virus C , but unlike potato virus C it is readily transmitted by Myzus persicae. The second, named tobacco wilt virus, is also transmitted by M. persicae but much less readily, whereas the third, named datura necrosis virus, is not. All three have a wide host range, but neither tobacco wilt nor datura necrosis viruses infects potato plants. All three have long flexuous particles and similar general properties.
Simultaneous infection with datura necrosis virus usually decreases the concentration reached by potato virus Y in tobacco plants but not in Nicotiana glutinosa.     

日光照周期对大菱鲆幼鱼摄食、消化酶活力与血清激素含量的影响

研究了4种光照周期[24L﹕0D(D1)、16L﹕8D(D2)、8L﹕16D(D3)和0L﹕24D(D4)]对体重(30.5±2.0) g大菱鲆Scophthalmus maximus L.幼鱼20h (8:00am—4:00am)内摄食、消化酶活力、血清激素含量的影响。结果显示: (1)实验鱼的摄食率随光照时间的缩短而降低; D1组实验鱼每隔8h出现摄食高峰, 其他组均在8:00am及4:00pm出现摄食高峰。(2)D1、D2和D3组在12:00am和8:00pm出现肠道蛋白酶及淀粉酶活力峰值, 脂肪酶活力显著高于D4组(P<0.05)。(3)各组8:00am至8:00pm生长激素(GH)含量无显著变化, D1组4:00am时显著高于其他组(P<0.05), D4组0:00am及4:00am显著低于其他组(P<0.05); D1、D2和D3组初次摄食8h内皮质醇(COR)含量无显著变化, 8h后先升高后降低, D4组COR含量先升高后降低, 8:00pm时达到最高; D1和D2组0:00am时去甲肾上腺素(NE)含量显著高于其他组(P<0.05), D4组8:00pm时显著低于其他组(P<0.05); D2组8:00am及12:00am时三碘甲状原氨酸(T3)含量显著高于其他组(P<0.05), D4组8:00pm时显著低于其他组, 0:00am时显著高于其他组(P<0.05), D2和D3组4:00am显著低于D1和D4组(P<0.05), 各组T3最高值均出现在8:00pm。在实验条件下, 光照周期影响了大菱鲆幼鱼摄食、消化酶活力及血清激素含量。在此光照强度下, 大菱鲆养殖中以8—16h光照周期、日投喂2次为宜。     

ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA : II. NAD- and NADP-Dependent Glutamic Dehydrogenases and Nicotinamide Adenine Dinucleotidase

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged.     

POLYOL DEHYDROGENASE: PURIFICATION. EVIDENCE FOR MULTIPLE FORMS AND SOME PROPERTIES OF THE DOMINANT VARIANT OF THE HORSE LIVER ENZYME

Purification of horse-liver polyoi dehydrogenase (PDH) on DE52 anion-exchange cellulose reveals the presence of three fractions with enzyme activity. These appear in the breakthrough volume (PDH-3) and the salt gradient (PDH-1, -2) respectively. The major band of activity (< 90%) is found in the PDH-2 fraction. A reexamination of sheep-liver polyol dehydrogenase also reveals the presence of three bands of activity, with the dominant fraction (PDH-3) corresponding to the preparation described by Smith (Biochem. J., 83, 135–144, (1962))³. The interaction between horse-liver (and sheep-liver) PDH and Blue Sepharose CL-6B is found to be endothermic. This property is utilized in the final purification step. Horse-liver PDH-2 has a molecular/subunit weight of 85, 000/28, 000, a Stokes' radius of 3.8 nm, and an isoelectric point of 7.4.