Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Comparison of the Properties of Semipurified Mitochondrial and Cytosolic Monoamine Oxidases from Rat Brain

Mitochondrial and cytosolic monoamine oxidases were purified 220- and 129-fold, respectively, from rat brain. The purification procedure involved extraction (without the use of detergents for mitochondrial monoamine oxidase), ammonium sulfate precipitation, and chromatography on Sephadex G-25 and a DEAE-cellulose column. The properties of both enzymes with kynuramine as substrate, including Km values and pH optima at different kynuramine concentrations; the Rf values on polyacrylamide gel electrophoresis; and the thermal inactivation patterns were different. 2-Mercaptoethanol, together with heat treatment, released the flavin and decreased the enzyme activity differentially for the two enzymes. The absorption spectrum showed a "Red shift" in the absorption maxima when the spectra of the non-Triton-treated purified preparations were compared with those of the Triton-treated ones, thus possibly revealing that the mitochondrial and the cytosolic monoamine oxidases may be two different enzyme entities.     

Purification and characterization of hepatic lipase from Todarodes pacificus

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of 35-40 degrees C and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of Hg(2+) or Cu(2+) ion. Partial amino acid sequence of the enzyme was also determined.     

Comparative biochemical study of N-linked glycans from skin of a squid, Todarodes pacificus

For comparative biochemical interest, we analyzed the structures of N-glycans in a squid belonging to the Lophotrochozoa, one of the protostome clades. N-Glycans were prepared from squid skin by hydrazinolysis and re-N-acetylation followed by fluorescent tagging with 2-aminopyridine. The labeled N-glycans were purified, and their structures were determined by the two-dimensional HPLC mapping method combined with glycosidase digestions and mass spectrometry. We found that high mannose-type glycans, paucimannose-type glycans and complex-type glycans with a type-1 structure (Galbeta1-3GlcNAc) were dominant in squid skin. The complex-type glycans detected in the squid were similar to those in vertebrates, but have not yet been found in the Ecdysozoa, which is another protostome clade. However, paucimannose-type glycans are commonly found in the Ecdysozoa. Thus, the N-glycan structures of the squid belonging to the Lophotrochozoa have features common to those in vertebrates and the Ecdysozoa including insects and nematodes.     

Differential Postnatal Development of Monoamine Oxidases A and B in the Blood-Brain Barrier of the Rat

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [³H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [³H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

大鼠正常肝、肝癌前病变组织谷胱甘肽S-转移酶的纯化及部分性质的研究

本文用S-己基谷胱甘肽-琼脂糖-6B亲和层析一步纯化法分别获得电泳纯大鼠正常肝GST_S及含增生结节大鼠肝GST_S。经DE_(52)阴离子交換柱将含增生结节大鼠肝GST_S分离为三个同功酶组份,依次命名为C_(DE)A1及A2,C_(DE)占上柱总GST_S活性84.8％。等电聚焦电泳测定等电点分别为7.8、6.7及6.3。经CM_(52)阳离子交換柱获得五个同功酶组份,依次命名为A_(CM),C1,C2,C3及C4,等电点分别为7.8,7.4,7.9,8.3及8.6。A_(CM)的活性占CM_(52)柱上柱总活性的10％。SDS-PAGE电泳结果和正常大鼠肝GST_S比较,含增生结节大鼠肝GST_S同样出现Ya,Yb及Yc三条区带,而后者的氨基酸组成也与正常大鼠肝GST_S相近,但是和大鼠正常肝组织比较后者GST_S活性明显升高,以阳离子同工酶的活性为主。     

Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus

We have recently demonstrated that the common squid Todarodes pacificus express acidic lipids that were reactive with a monoclonal antibody A2B5. In the present study, two A2B5-reactive acidic lipids were isolated from squid hepatopancreatic tissue and characterized for their structures by methods including glycolipid overlay analysis, product analysis after sialidase treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, the two acidic lipid were identified as GT3 and GQ1c, respectively. Another A2B5-reactive acidic lipid in the tissue was tentatively assigned to GT2 based upon its reactivity to A2B5 and chromatographic mobility on thin-layer chromatography. The composition and concentration of c-series gangliosides significantly differed among squid tissues (i.e. hepatopancreas, cerebral ganglion, eye lens, and mantle tissue). Interestingly, the percentages of c-series gangliosides within total gangliosides of hepatopancreas and cerebral ganglion were even higher than that of cod fish brain, which is known to be highly enriched with this ganglioside species. These findings strongly support the hypothesis that c-series gangliosides in squid tissues are not derived from ganglioside-containing food intake, but biosynthesized in a tissue-specific manner.     

Purification and properties of a diisopropyl-fluorophosphatase from squid Todarodes pacificus steenstrup

A diisopropyl-fluorophosphatase (DFPase) was purified from brain and ganglia of squid Todarodes pacificus steenstrup. The DFPase had a preference in hydrolysis toward diisopropylphosphorofluoridate (DFP). It also was able to hydrolyze O-1,2,2-trimethylpropyl methylphosphofluoridate (soman) and O-isopropyl methylphosphonofluoridate (sarin) at nearly equal hydrolytic rates but only 1/10 that of DFP. The hydrolytic activity toward diethyl-p-nitrophenylphosphate (paraoxon) was very low compared with DFP, so man, and sarin. The DFPase was purified 330-fold to a specific activity of 18,300 n mol/min/mg protein. Its molecular weight was 34,000 dalton determined by gel-filtration chromatography. Mn²⁺ stimulation of the DFPase was not observed when DFP and soman were the substrates, but with sarin, the rate increased onefold in the presence of 1.0 mM of Mn²⁺. Ethylenediamine tetraacetic acid disodium (EDTA-Na₂) at 0.05 M inhibited the DFPase activity about 30%. It could be concluded that this DFPase belongs to the squid-type DFPase.     

Oxidation of Analogs of l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Monoamine Oxidases A and B and the Inhibition of Monoamine Oxidases by the Oxidation Products

Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+-channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin-2-loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 microM EGTA or 100 microM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 microM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration-dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+-evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.     

黄海南部太平洋褶柔鱼种群结构与繁殖生物学

依据2008年11月—2009年9月黄海南部帆张网太平样褶柔鱼渔业调查资料,分析了太平样褶柔鱼胴长组成结构、性比、初次性成熟胴长以及胴长与体重关系等基础生物学参数的季节变化,阐述了该海域太平洋褶柔鱼种群结构动态及繁殖生物学特征。结果表明:太平洋褶柔鱼在该水域常年均有分布,其中秋、冬季以性成熟个体为主,春、夏季以幼体为主;该水域太平洋褶柔鱼雌性个体数量显著多于雄性,总性比为0.60,当胴长(LM)在190～280mm范围内时,性比(rsex)与胴长之间的关系符合:rsex=3×10-5LM1.8017;雌、雄个体的性腺成熟度均呈现随胴长增大而升高的趋势。该水域太平洋褶柔鱼个体的性腺体指数月变化显著,以秋、冬季较高,春、夏季较低,表明该水域太平洋褶柔鱼群体的繁殖季节主要集中在秋、冬季;性腺体指数与性腺成熟度(除Ⅴ期外)和胴长之间均存在正相关关系。雌性个体的初次性成熟胴长较大,为201.9mm;雄性较小,为174.3mm。不同性腺成熟度阶段,太平洋褶柔鱼胴长与体重的关系不同,在其个体生长发育后期,体重增加主要受性腺发育的影响。     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.     

Comparison of the cholinoreceptor and cholinesterase properties of the frog Rana temporaria and the squid Todarodes pacificus

Studies have been made on the interaction of several groups of quartenary ammonium salts with cholinoreceptors of m. rectus abdominis of the frog Rana temporaria, and isolated m. retractor infundibuli of the octopus Todarodes pacificus, as well as with cholinesterases of the frog brain and visual ganglia of the octopus. The derivatives of polymethylene bis(trimethylammonium) compounds, being cholinomimetic drugs for frog muscle, do not exert cholinomimetic influence on octopus muscle. The same difference with respect to their effect on frog and octopus receptors was found in anabazin derivatives. Among amide derivatives of acetylcholine, the strongest mimetic effect on cholinoreceptors of both animals was exhibited by a piperazine isolog with gauche-conformation, whereas N-methyl isolog with trans-conformation was found to be the strongest inhibitor of cholinesterases. Cholinoreceptors and cholinesterase of the octopus were less sensitive to the effect of the investigated quartenary ammonium salts than those of the frog.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

Isolation and characteristics of trypsin inhibitor from the hepatopancreas of a squid (Todarodes pacificus)

Trypsin inhibitor was purified from the hepatopancreas of squid (Todarodes pacificus). The final inhibitor preparation was nearly homogeneous by SDS-PAGE with an estimated molecular weight of approximately 6300. The squid trypsin inhibitor was acid- and heat-stable, and active against trypsins from the pyloric ceca of starfish (Asterias amurensis) and saury (Cololabis saira) and porcine pancreatic trypsin. Amino acid composition of the squid trypsin inhibitor was compared with other invertebrate trypsin inhibitors. The squid trypsin inhibitor inhibited the autolysis of walleye pollock (Theragra chalcogramma) myofibrillar proteins.     

Inhibitory effect of squid (Todarodes pacificus) paramyosin on actomyosin ATPase and on superprecipitation

1. Paramyosin from squid mantle muscle inhibited the Mg-ATPase and the superprecipitation activities of actomyosin. 2. The inhibition was detected only when paramyosin forms a cofilament with myosin. 3. ATP-induced changes in the morphology of the cofilament of myosin and paramyosin are involved in the inhibition by paramyosin.     

Wistar大鼠实验性动脉粥样硬化的研究

目的　使用普通级雄性Wistar大鼠灌胃给药 (L 蛋氨酸 )进行动脉粥样硬化模型的研究。方法　模型组分别给药 (L 蛋氨酸 ) 2 30mg 只 (A组 )和 4 5 0mg 只 (B组 )每隔 7天进行一次血脂检查及主动脉弓常规病理组织学检查。结果　服药后引起动物血脂紊乱 ,病理组织学检查服药 8周时实验A组与B组均见到主动脉弓局部内膜下水肿 ,同期B组 (高剂量组 )病理改变重于A组 (低剂量组 ) ,偶见泡沫细胞和单核细胞侵润。对照组无上述相关改变。结论　Wistar大鼠服用L 蛋氨酸一定时间后可引起血脂的紊乱及主动脉弓局部内膜下的病变