Arachin polymorphism in groundnut (arachis hypogaea l.)

Arachin, the major seed storage protein of groundnut, showed polymorphism. The polymorphic forms were due to differences in molecular size, net charge and polypeptide composition of the native protein. Purified arachin at low ionic strength resolved into monomeric and dimeric forms both on sucrose density gradient centrifugation and cellulose acetate membrane (CAM) electrophoresis. The dimers had more net negative charge compared with the monomers. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of arachin from the cultivar Spanish improved (SP) under non-reducing conditions showed three major components of M, 70.7, 63.8 and 60.9 k. Arachin from Trombay Groundnut 1 (TG-1) showed three components of M, 70.7, 63.8 and 59.5 k while in TG-18, a derivative of a cross between SP and TG-1, there were only two components of M, 70.7 and 63.8 k. Data from two dimensional gel electrophoresis in the presence and absence of 2-mercaptoethanol shows that each of the above components in turn consists of subunit pairs that are held together by disulphide linkages. The M,s of the major polypeptides for the three arachins is as follows: SP, 47.5, 45.1, 42.6 and 21.4 k; TG-1, 47.5, 45.1, 41.2 and 21.4 k; TG-18, 47.5,45.1 and 21.4 k. Two dimensional gel electrophoresis (IEF and SDS-PAGE) indicates that the arachin subunits consist of two major groups—the acidic polypeptides (three in SP, TG-1 and two in TG-18) and the basic polypeptides (three in SP, TG-1 and two in TG-18). The acidic polypeptides did not show charge variation while the basic polypeptides were charge heterogeneous. Absence of both an acidic polypeptide along with a basic polypeptide in TG-18 suggests that the acidic and basic polypeptides are probably products of the same gene and arise as a result of post-translational cleavage. Antibodies raised against purified arachin from SP reacted with arachin from TG-1 and TG-18 showing similar antigenic determinants. The acidic polypeptides show considerable homology in their structure as revealed by peptide mapping patterns.     

Appearance of storage lipid (triglycerides) in somatic embryos of peanut (arachis hypogaea L.)

Summary We estimated the level of triglycerides (triacylglycerol) at intervals of 2 wk during somatic embryogenesis in peanut. The initial triglyceride content in the leaflet explants was depleted during the formation of embryogenic tissue. It increased with the onset of somatic embryogenesis. Concentration of triglyceride in a fully developed embryo increased further if incubated in the same dehydrated medium for a longer period of time. Transferring these embryos to fresh medium led to germination of somatic embryos with a depletion of storage lipids.     

Water transport in the xylem as related to calcium uptake by groundnuts (arachis hypogaea L.)

Summary Recent investigations have shown that the gynophore and young fruit of the groundnut must themselves absorb the calcium necessary for their growth. Since this is not the case for other minerals, experiments were conducted to find the reason for this inadequate transfer of calcium from stem to developing fruit.The course of the transpiration stream was followed by cutting off part of the root system to obtain penetration of a dye solution into the tissues. It was shown that even in a very dry soil, no water moves along the xylem vessels towards the expanding tip of the peg.An explanation is offered for the extra-ordinary behaviour of calcium in view of the fact that whilst the xylem is considered to be the main path of conduction for this element the stream of water necessary for its transport is absent.     

Purification, kinetic properties and physicochemical characterization of a novel acid phosphatase (AP) from germinating peanut (Arachis hypogaea) seed

Acid phosphatase activity was detected in peanut (Arachis hypogaea) cotyledons during germination. Four (4) to six (6) days of germination was the meantime corresponding to maximum hydrolytic activity of this enzyme. The understanding of the role of acid phosphatase activity during germination led to purify this enzyme by successive chromatography separations on DEAE-Sepharose CL-6B, Sephacryl S-100 HR and Phenyl-Sepharose HP to apparent homogeneity from germinated peanut cotyledon five days old. This enzyme designated peanut cotyledon acid phosphatase (AP) had native molecular weight of 24 kDa by gel permeation. SDS-PAGE of the purified acid phosphatase resolved a single protein band that migrated to approximately 21.5 kDa. Thus, this acid phosphatase likely functions as a monomer. The enzyme had optimum pH (5.0) and temperature (55 degrees C), and appeared to be stable in the presence of anionic, cationic and non-ionic detergents. Substrate specificity indicated that the purified acid phosphatase hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP and ATP were the compounds with highest rate of hydrolysis for the enzyme. Moreover, the purified acid phosphatase exhibited phytase activity. These results showed that this enzyme played a peculiar role during germination, notably in reducing the rate of phytic acid, an antinutritional substance contained in peanut seed.     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

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     

Large-Scale isolation of acid phosphatase from human erythrocytes

A laboratory process for the isolation of type B acid phosphatase from human erythrocytes has been scaled up 80 times. The enzyme was purified 1300-fold by adsorption on calcium phosphate gel, ammonium sulfate precipitation, and gel filtration. The yield was 27%. Results for the type A enzyme are also presented.     

花生根瘤菌类菌体氢氧化体系的研究

花生根瘤菌类菌体含有吸H₂酶,以O₂或亚甲蓝为电子受体均表现吸H₂活性。类菌体还原减氧化的吸收差示光谱在424、500、520、550、560、585及595 nm处呈现吸收峰,表明细胞色素c、b、和o参与H₂的氧化过程。CN-明显影响520、550、560 nm处的吸收峰,意味着有些细胞色素c和b对CN^-敏感,在H₂代谢中充当末端氧化酶的作用。抗霉素A、HONO、CN^-、N₃^-和DBMIB均强烈抑制吸H₂活性,但鱼藤酮不抑制吸H₂活性。说明细胞色素b、c、a和泛配参与H₂氧化的电子传递,而NADH脱氢酶不参与。花生根瘤菌类菌体的H₂氧化系统是个复杂具分支的电子传递体系。     

A phosphotyrosyl-protein phosphatase activity associated with acid phosphatase from human prostate gland

Using [32P]P-Tyr-IgG and [32P]P-Tyr-casein phosphorylated by pp60v-src as substrates, studies on the phosphotyrosyl-protein phosphatase activity in human prostate gland indicate that it is associated with prostatic acid phosphatase. Evidence to support this conclusion include the following: (a) these two enzymatic activities co-purify to apparent homogeneity; (b) they co-migrated on polyacrylamide gel electrophoresis, ion-exchange and gel filtration chromatographies; (c) the exhibit identical thermostability; and (d) the phosphotyrosyl-protein phosphatase activity is sensitive to inhibition by p-nitrophenyl phosphate and by several classical inhibitors of prostatic acid phosphatase including L(+)-tartrate, molybdate, vanadate and NaF. The purified enzyme exhibits high specificity towards phosphotyrosyl-proteins with little activity towards several phosphoseryl-proteins and phosphothreonyl-proteins examined. The present findings indicate that prostatic acid phosphatase may function in vivo as a phosphotyrosyl-protein phosphatase.     

Transfer of iron from uteroferrin (purple acid phosphatase) to transferrin related to acid phosphatase activity

There is continuing controversy as to whether iron can be exchanged from the purple phosphatase, uteroferrin (Uf), to fetal transferrin (Tf) and whether this process might be of physiological relevance during pregnancy in the pig. Here, iron transfer from Uf to apoTf at pH 7.1 was followed by measuring the loss of acid phosphatase activity from native Uf as a function of incubation conditions and time. In the presence of apoTf and 1 mM ascorbate (but not in the presence of either agent alone), 50% of enzyme activity was lost in about 12 h. Loss of activity was accompanied by bleaching of Uf purple color and the appearance of the characteristic visual absorption spectrum of Fe-Tf. Citrate could replace ascorbate in the reaction. Loss of Uf iron did not occur at pH 5.3, at which pH Tf cannot bind Fe. [59Fe]Uf was prepared and shown to be identical in its enzymatic and physical properties with unmodified Uf. Transfer of 59Fe from Uf to apo-Tf was promoted by conditions identical to those which led to loss of purple color and acid phosphatase activity. However, the results suggested that only one of the two iron atoms at the bi-iron center on Uf was readily lost, and that exchange of the second iron occurred more slowly. Loss of iron made Uf more susceptible to denaturation. A third technique, quantitation of the g' = 4.3 signal of iron specifically bound to Tf by EPR, was also tested as a means assaying accumulation of Fe-Tf, but the method was too insensitive to measure the kinetics of iron transfer at physiological protein concentrations. We conclude that iron can be transferred directly from Uf to apoTf in the presence of low molecular weight chelators, and that the process is likely to be of physiological significance.     

N-氨甲酰基-D-氨基酸酰胺水解酶的固定化工艺

以TJS环氧基树脂作为载体对N-氨甲酰基-D-氨基酸酰胺水解酶进行固定化,最佳工艺条件为：1g树脂载体大约对应133U酶液,蛋白质量浓度0.35mg/mL,固定时间15h,温度28℃,pH7.5,固定化酶活达到58.5U。蛋白固定率可达97.4%,酶活回收率达到49.3%,得到的固定化酶使用半衰期达到26批。