Inhibitor of diamine oxidase in cotyledons of groundnut seedlings

The apparent absence of diamine oxidase in extracts of cotyledon of germinating groundnut seeds is due to the presence of a natural inhibitor. The inhibitor was associated with the lipid in the upper layer obtained on centrifugation of the cotyledon extract. It was non-dialysable, thermolabile and was inactivated by TCA and Triton X-100. The inhibitor was detected in the cotyledon extracts from early stages of seed development and was present up to 20 days after germination.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu     

Purification and characterization of diamine oxidase from rice embryos

Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10⁴ and the enzyme contained two identical subunits each with a MW of 6.12 × 10⁴. The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K_m of 10^?5 M. Each enzyme molecule contained four molecules of FAD.     

Diamine oxidase from Lens esculenta seedlings: Purification and properties

A diamine oxidase (DAO) (EC 1.4.3.6) has been purified to homogeneity from lentil seedlings. The purified protein has a MW of 154 000 and is composed of two apparently identical subunits. It contains two CU²⁺ atoms and one carbonyl-like group per mol. The purified enzyme is pink-red in concentrated solution and shows a broad, well-defined, absorption band in the visible region centered at 498 nm. The ESR spectrum is typical of Cu²⁺ in a tetragonal symmetry. The enzyme oxidizes only aliphatic diamines and spermidine with formation of the corresponding aldehydes, hydrogen peroxide and ammonia. Putrescine and cadaverine are oxidized most rapidly and the oxidation rate decreases when longer diamines are tested.     

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.     

Diamine oxidase of Lathyrus sativus seedlings

A diamine oxidase has been purified (ca 40-fold) from 5-day old etiolated seedlings of L. sativus by MnCl₂ treatment, (NH₄)₂SO₄ and Me₂CO fractionations, positive adsorption on alumina Cγ-gel followed by column chromatography on DEAE-Sephadex. This cytosol enzyme oxidatively deaminates a number of aliphatic and aryl alkylamines but not histamine. NSD-1055, semicarbazide and other carbonyl reagents, α,α'bipyridyl, 1,10-phenanthroline and 8-hydroxyquinoline inhibit the enzyme. Pargyline, SKF trans-385, atabrine were without effect on the enzyme.     

Stereochemistry of reactions catalysed by l-lysine decarboxylase and diamine oxidase

The decarboxylation of l-lysine to yield cadaverine, catalysed by l-lysine decarboxylase (EC 4.1.1.18) takes place with retention of configuration. In the course of the dehydrogenation of cadaverine by diamine oxidase (EC 1.4.3.6) from pea seedlings, the pro-S-hydrogen is labilized.     

The oxidation of monodansyldiamines by pea seedling diamine oxidase

In the oxidation of a homologous series of monodansyldiamines by pea seedling diamine oxidase, monodansylcadaverine was the best substrate. Monodansyldiaminohexane was oxidized at 74% of the rate with monodansylcadaverine, and monodansylputrescine and monodansyldiaminopropane were oxidized only very slowly. The optimum pH for the oxidation of monodansylcadaverine was 8.5, and the K_m 2.4 × 10^?4 M. Under optimum conditions, putrescine was oxidized eleven times faster than monodansylcadaverine. Oxidation of monodansylcadaverine by diamine oxidase, and the exhaustive dansylation of lysine in equivalent amounts ultimately showed equal fluorescence in the dansyl-5-aminovaleraldehyde formed, indicating stoichiometric conversion to this product in both reactions.     

Effect of ethrel and chloroethanol on pea diamine oxidase activity

The activity of cotyledon and embryo diamine oxidase was reduced by feeding ethrel and chloroethanol to the seedlings. The inhibitory effect of 2,4-D on the activity of enzyme in the cotyledon which may be mediated through ethylene was reversed by exposure of seeds to red light.     

Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase （GAPDH, EC 1.2.1.12） from human erythrocytes. The comparison between this rapid method （one step） and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of -150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide （NAD＋）. Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively. The kinetic parameters were also calculated： the Vmax was 4.3 U/mg and the Km values against G3P and NAD＋ were 20.7 and 17.8 μM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.     

Isolation and characterization of catechol oxidase from Solanum melongena

Catechol oxidase was distributed in soluble and particulate fractions of Solanum melongena. The purified preparation appears to be homogeneous by polyacrylamide gel electrophoresis. The enzyme shows two pH maxima—with catechol, 6.5 and 7.5; and with dopa, 6.5 and 7.9. The latent form of the enzyme does not occur in S. melongena. The preparation resembles the enzyme from other sources in substrate specificity towards various mono- and diphenols, having a higher affinity for catechol than dopa; this tendency increases on purification. The cresolase activity decreases with purification and a lag period with p-cresol is observed. The oxidation of mono- and diphenols is inhibited by ascorbic acid, sulphydryl compounds and chelating agents.     

Purification and properties of glycollate oxidase from Pisum sativum leaves

A rapid and efficient method for the isolation of glycollate oxidase from pea leaves is described. The method utilizes the unusually high isoelectric point (pH 9·6) which has been determined for the enzyme using isoelectric focusing. The enzyme is apparently homogeneous by polyacrylamide gel electrophoresis and has a MW of ca 100000. Some properties of the enzyme are described.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

免疫亲和层析法分离纯化含次黄嘌呤核苷mRNA

采用免疫亲和层析法分离纯化含次黄嘌呤核苷的mRNA(I-mRNA）。将Poly-I与BSA交联,并用Poly-I-BSA交联体免疫家兔,获取抗次黄呤核苷抗血清,斑点印迹法检测到该抗体滴度高、选择性强,将抗次黄嘌呤核苷抗体与蛋白A Sapharose交联,并制备抗体亲和层析柱。将小鼠肺mRNA上样于层析柱,淋洗层析柱后,用洗脱液洗脱I－mRNA,并以斑点印迹法在洗脱液中检测到很强的I－mRNA阳性信号;淋洗液中I－mRNA阳性信号的强度则随淋洗液体积的增加而减弱。以上结果表明,应用本方法可以有效分离得到含次黄嘌呤核苷的mRNA。     

Regulation of diamine oxidase activity in germinating pea seeds

Diamine oxidase present in the cotyledons of germinating pea seeds is induced by putrescine, spermidine and ornithine. Auxins inhibit enzyme synthesis in cotyledons only in the presence of embryo. Cycloheximide inhibits the synthesis of the cotyledon enzyme but has no effect on the embryo enzyme. 5-Fluorouracil inhibits the synthesis of both cotyledon and embryo enzymes.     

cDNA sequences of variant forms of human placenta diamine oxidase

Genes for two forms of human placenta diamine oxidase(dao) were cloned from a cDNA library and sequenced. One gene,pdao 1, is identical in length to human kidneydao but differs from it by two bases in the coding region and differs slightly in the 3′- and 5′-noncoding regions. The second gene,pdao2, is nearly identical to these genes in the coding region, except that it has an extra 57-nucleotide coding segment near the 3′ end of this region. This segment corresponds to the contiguous sequence of the 3′ end of intron 3 of human kidneydao. pdao2 also differs significantly frompdao1 and human kidneydao in a 13-base sequence in the 5′-noncoding region. It is proposed thatpdao1 and human kidneydao are polymorphic forms of the same allele. Whetherpdao2 is a polymorph of these two is not certain, because of the significant differences in the coding and noncoding regions.Pdao2 may represent a different allele. This work was supported by a Department of Veterans Affairs Merit Review Grant, Program Project Grant HL 16251 from the Heart, Lung and Blood Institute of the NIH, and an Academic Senate Grant from the University of California, San Francisco.     

Identification of cadaverine in Pisum sativum

Cadaverine has been identified in normal young peas and in saline grown (necrotic) peas by GLC and MS. Its concentration was at least of 5 gm/g fresh plant in both cases. Analysis of the alkaloidal extracts failed to reveal any lupinine, anabasine or other pyridine alkaloids in normal or necrotic, salt grown, peas.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

La diamine oxydase dans les jeunes plantes de Glycine max

Diamine oxidase activity against putrescine and agmatine is investigated in young decotylized Glycine max plants growing, in the light, under sterile conditions on a liquid medium in the presence of ammonium chloride or nitrates. Optimal conditions for the enzyme activity are determined. Both amines are oxidised more slowly in plants cultivated on ammonium chloride than in plants grown on nitrates.     

苹果多酚氧化酶的纯化与表征

运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.