玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

斑玉蕈酸性磷酸酯酶的酶学性质研究

以斑玉蕈为材料分别从菌盖和菌柄中提取一种酸性磷酸酯酶（ACPase,EC.3.1.3.2）,进一步用硫酸铵沉淀分离,Sephadex G-200柱纯化,从菌盖中分离到3个酶组分,从菌柄中分离到4个酶组分,分别对菌盖和菌柄的酶Ⅰ和酶Ⅰ′进行聚丙烯酰胺凝胶（PAGE）电泳纯度鉴定,均呈现单一酶蛋白带。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定酶Ⅰ和酶Ⅰ′的相对分子量均为65kDa,SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤测定分析,酶Ⅰ和酶Ⅰ′均为单亚基蛋白。紫外吸收光谱（UV）测     

Diamine oxidase from millet catalyzes the oxidation of 1, 3-diaminopropane

Diamine oxidase was purified sixty-fold from millet shoots. The partially purified enzyme of 150 kDa oxidized 1, 3-diaminopropane (1, 3-DAP) to 3-aminopropionaldehyde. The K_m values were 9.1×10⁻⁵M for 1, 3-DAP and 6.3×10⁻⁴M for putrescine. Extracts of shoots of prosomillet, maize and barley also contained an activity that oxidized 1, 3-DAP.     

Tea Leaf Polyphenol Oxidase

The effect of polyamide on the extraction of polyphenol oxidase in the leaves and the localization of the enzyme in tea leaf cell were investigated.

In the homogenate treated with polyamide, a major part of the enzyme activity appeared in the centrifugal precipitates in the range of l,400×g to 15,000×g, and polyphenol content decreased. Therefore, it was thought that the enzyme in tea leaves existed primarily on the particle as small as mitochondria, but that it coagulated easily in the homogenate containing a high concentration of polyphenol, and precipitated by centrifugation at l,400×g.

And also from the comparison of the ratio of the enzyme activity to chlorophyll concentration in each fraction separated by centrifugation, it was assumed that a major part of the enzyme did not exist in the chloroplast, but was bound on the precipitable particle in the range of l,400×g to 15,000×g.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Palmityl-CoA synthetase activity in the muscle of dystrophic mice.

Palmityl-CoA synthetase activity (acid CoA ligase (AMP), E C 6.2.1.3.) was determined using the radioassay method. The rate of formation of palmityl-CoA under the optimal conditions established was 20 nmoles per mg protein per min for mitochondria and 5.8 nmoles for the 9000 × g supernatant. The activity of palmityl-CoA synthetase in mitochondria from skeletal muscle of dystrophic mice was not significantly different from that obtained in normal littermate controls, whereas the activity of this enzyme in the 9000 × g supernatant fraction from dystrophic muscle preparation was found to be significantly higher than for the corresponding controls. It is concluded that the previously observed decrease in palmitate-1-¹⁴C oxidation in dystrophic muscle mitochondria was not due to a defect in the activation of palmitic acid.     

Changes in Hypocholesterolemic Activity in Rats by Various Konnyaku Powder Treatments

Galactosylsucroses contained in soybeans are not digestible. Thus we wished to detect α-galactosidase (EC 3.2.1.22) in intestinal bacteria. The strain of E. coli in the title was found to produce considerably this enzyme adaptively. We could prepare rather pure solution of the enzyme from the sonicate of the strain. It was purified about 142-fold. It showed optimum pH and temperature at 6.8 and 37°C, respectively, with the substrate p-nitrophenyl-α-d-galactoside (PNPG). Dilute enzyme solutions were very unstable even at 0–5°C. However, concentrated solutions were considerably stable. The Michaelis constant (m) was 1.07 × 10^?4, 2.33 × 10^?3, and 3.65 × 10^?2 for PNPG, melibiose, and raffinose, respectively. The maximum velocity (mole/min/mg protein) was 2.72 × 10^?5, 2.67 × 10^?5, and 2.04×l0^?5, respectively for the same three substrates. This enzyme had a weak transferase action.     

17-Hydroxyprogesterone metabolism in the monkey fetal adrenal: C17–20Lyase and 21-hydroxylase activities

C^17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey ($\text{Macaca mulatta}$) fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [¹⁴C]17-hydroxyprogesterone to [¹⁴C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, K_m and V_max. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10^?3 μmoles NADPH oxidized/min. × mg^?1 protein with an apparent K_m of 3.6 × 10^?7 M and a V_max of 2.2 × 10^?3 μmoles/min. × mg^?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis.     

Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10^?3, 1.24 × 10^?3 and 4.17 × 10^?3 h^?1 for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.     

Crystallization and Characterization of Polyamine Oxidase from Penicillium chrysogenum

Polyamine oxidase was purified and crystallized with an overall yield of 35% from mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 column chromatographies. The crystalline enzyme was homogeneous, as judged by disc gel electrophoresis and ultracentrifugation. The sedimentation coefficient (s_{20, w}⁰) of the enzyme was determined to be 6.9S, and diffusion coefficient (D_{20, w}) to be 4.2 × 10^?7 cm² sec^?1. The enzyme showed a molecular weight of about 160,000 by gel filtration method and ultracentrifugal analysis, and it was composed of two identical subunits. The enzyme was a flavoprotein with absorption maxima at 275, 375 and 450 nm. The prosthetic group was identified to be FAD. The enzyme oxidized spermine, and slightly oxidized spermidine. Diamines and monoamines were not oxidized.     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. II. Coagulation of activated sludge suspensions

Chitosan samples manufactured under different conditions were compared for effectiveness of coagulating an activated sludge suspension grown on vegetable canning wastes. Computer analysis of data from Buchner funnel filterability tests resulted in quadratic polynomial equations describing the response curves for volume of filtrate versus dosage, expressed as g/liter chitosan/100 g sludge suspended solids (SSS). The quotient of the filtrate volume and dosage at the inflection points of the equations obtained for 10 test samples and 1 commercial chitosan sample were compared to evaluate the response (effectiveness) per unit amount for each chitosan product. The product made by a standard procedure (deproteinated with 3% NaOH at 100°C for 1 hr, demineralized with 1N HCL at ambient temperature for 30 min, and deacetylated with 50% NaOH at 145–150°C under N₂ for 5 or 15 min) gave the best performance as a coagulating agent for this activated sludge system. Other products, including the commercial preparation, required higher dosages to achieve the same effectiveness. Products deacetylated in the presence of sir rather than nitrogen decreased waste treatment effectiveness, which approximated the trends of reduced viscosity and molecular-weight distribution. The products containing minerals were less effective than products from which minerals had been removed prior to deacetylation, but they were more effective than the enzyme treated sample and the commercial product. In general, although chitosan products obtained after 15 min deacetylation were more effective than those receiving 5 min deacetylation, effectiveness did not correlate linearly with viscosity and molecular-weight distribution trends. However, chitosan products deacetylated for 15 min did show that the higher-molecular-weight products (0.65–1.1 × 10⁶) were more effective coagulating agents for activated sludge than the manufactured product having the lowest molecular weight (0.47 × 10⁶) and the commercial reference sample (0.56 × 10⁶). Thus, higher values for molecular weight were predictive of greater effectiveness for coagulation of activated sludge suspensions.     

Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on β-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (α₂β₂) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the α-subunit (42.3-kDa subunit) and the β-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the β-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and β-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, β-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 μM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).     

INHIBITION OF ACETYLCHOLINESTERASE IN VITRO BY PENTYLENETETRAZOL1

Abstract—The effect of pentylenetetrazol (PTZ) on acetylcholinesterase (E.C.3.1.1.7) was studied in vitro. The kinetics of the reaction were studied on AChE in crude homogenates of rat brain and in purified preparations from Electrophorus electricus. The K_m for rat brain AChE was 1·22 × 10^-4m, with a V_max of 1·37 μmol/g/min whereas the K₄ for competitive inhibition of the enzyme by PTZ was 4·7 × 10^-3m. The commercially purified enzyme exhibited a K_m of 1·73 × 10^-4m and a K_i of 1·00 × 10^-3m.     

Enticing nonhuman primates to forage for their standard biscuit ration

Ordinary feeder-boxes for macaques were converted into food puzzles by remounting them onto the square mesh (22 × 22 mm) of the front of the cages, away from original access holes. Feeding a standard ration of bar-shaped biscuits (40 × 24 × 16 mm; Purina Monkey Chow #5038; 236 g per animal), 8 adult pair-housed male rhesus macaques spent, on average, 61.0±15.6% of the first 30 min retrieving biscuits from food puzzles, but only 0.5±0.1% from feeder-boxes. Their total amount of time engaged in gathering food was, on average, 141 times higher at food puzzles (42.2±7.2 min) than at feeder-boxes (0.3±0.1 min). It was concluded that using feeder-boxes as food puzzles, baited with the standard biscuit ration, offers a cost- and work-effective way to promote foraging activities in captive nonhuman primates. © 1993 Wiley-Liss, Inc.     

The Separation and Isolation of Plasma Membranes and Mesosomal Vesicles from Staphylococcus Aureus

We have separated and Isolated the plasma membranes and mesosomal vesicles of Staphylococcus aureus ATCC 6538P. Cells were grown aerobically in Difco synthetic AOAC broth, washed and resuspended in hypertonic buffer (3.45 M NaC1) containing 0.02 M MgSO₄. Cell wall was removed by treatment with lytic enzyme from S. aureus, strain LS. The protoplasts were collected by centrifugation at 10,000 × g for 1 hour, resuspended in hypotonic buffer containing 0.02 M MgSO₄ and lysed. The resultant plasma membranes were washed and centrifuged on a 60tr>75Z sucrose density gradient at 55,000 × g for 15 hours. Gradient patterns showed two bands of membranes. Crude mesosomes were obtained from the 10,000 × g supernatant fractions by centrifugation at 100,000 × g for 2 hours. The reddish-brown gelatinous pellet, which consisted of mesosomal vesicles and a few ribosomes, was washed and centrifuged on a 60 to 85% sucrose density gradient at 100,000 × g for 15 hours. Gradient patterns produced two bands of mesosomal vesicles. The homogeneity of the plasma membranes and mesosomal vesicles was determined by electron microscopy and chemical analyses.     

A Method for Preparing 16α-Hy-droxypregnenolone-3-succinate by Streptomyces roseochromogenes

A choline dehydrogenase, which was present in the particulate fraction of the cell-free extract of Pseudomonas aeruginosa A-16, oxidized choline to betaine aldehyde without any dissociable coenzymes, while the enzyme, which was treated with Triton X-100, oxidized choline only with a supplement of phenazine methosulfate. The difference spectrum showed the presence of cytochrome-like components in the particulate. Km values for choline and phenazine methosulfate were 1.7 × 10^?3 m and 1.4 × 10^?4 m, respectively. The dehydrogenase was inhibited by SH-reagents such as p-chloromercuribenzoate and iodoacetic acid. Of a variety of substrates tested, only choline caused the enzymatic reduction of phenazine methosulfate. The estimation of choline was tried using the enzyme.     

Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase.

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.     

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.     

Solubilization of glucagon and epinephrine sensitive adenylate cyclase from rat liver plasma membranes

Hormonally sensitive adenylate cyclase has been solubilized from rat liver plasma membranes using Triton X-305 in Tris buffers containing mercaptoethanol and MgCl₂. The solubilized enzyme was stimulated 5 fold by NaF, 7 fold by glucagon and 20 fold by epinephrine. Criteria for solubilization included lack of sedimentation at 100,000 × g for one hour, the absence of particulate material in the 100,000 × g supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cyclase activity in Sephadex G 200 gels. The molecular weight of the solubilized, hormonally sensitive enzyme was approximately 200,000 in the presence of Triton X-305.