Gene pho-2 codes for the multiple active forms of Pi-repressible alkaline phosphatase in the mould Neurospora crassa

The mycelial Pi-repressible alkaline phosphatase of the wild-type strain 74A of Neurospora crassa was separated into at least ten isoforms by isoelectric focusing. The components visualized by activity with sodium -naphthyl phosphate as the substrate were predominantly acidic proteins with isoelectric points ranging from pH 4.5 to 7.6. The number of these isoforms was a function of growth pH. Strain pho-2A did not produce active Pi-repressible alkaline phosphatase (the pho-2 gene codes for its amino acid sequence), which gives an indication that these isoforms are encoded by the same structural gene.     

长江中下游部分湖泊沉积物碱性磷酸酶分布及其作用研究

沉积物磷负荷在湖泊富营养化的发生与恢复过程中具有关键作用,其释放受物理、化学与生物机制调节,而碱性磷酸酶催化有机磷的矿化,故当为促进沉积物磷循环的重要因素。本文讨论了长江中下游部分湖泊沉积物碱性磷酸酶分布及其在磷释放过程中的作用。五里湖疏浚与未疏浚区以及太湖、巢湖、龙感湖、东湖、月湖、龙阳湖、莲花湖等不同湖泊的不同区域表层沉积物碱性磷酸酶活性(APA)明显不同,这种空间异质性与湖泊富营养化程度相联系。此外,APA随沉积物的深度递减,或在中间与较深层次出现峰值,且具明显的季节性。上述事实以及APA对抑制剂的不同响应方式暗示酶存在形态的多样性(同工酶)。苯丙氨酸(Phe)明显提高月湖与五里湖沉积物APA,沉积物与Phe相互作用并静置一天之后,生物可利用性磷(SRP)的释放量明显增加。再者,Phe可抑制月湖沉积物APA,沉积物与Phe相互作用并静置一天之后,SRP释放量无明显变化,溶解有机磷(DOP)的释放量则明显增加。因此,释放的SRP部分来自某些活跃的有机磷的酶促水解,沉积物碱性磷酸酶在内源磷的释放以及富营养化过程中具有重要作用。     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

泡囊丛枝（VA）菌根对玉米际磷酸酶活性的影响

以玉米为材料,利用三室隔网培养方法,研究了缺P土壤上施用植酸和卵磷脂时接种几种菌根真菌（Glomus mosseae,Glmous versiformea,Gigaspora margarita)对根际土壤酸性磷酸酶和碱性磷酸酶活性的影响,玉米生长70d后,收获测定距根表不同距离土壤中的磷酸酶活性,结果表明,接种菌根真菌增加了根际土壤酸性和碱性磷酸酶活性,Gigaspora margarita菌根菌的作用大于其它2个菌极菌,不同P源对磷酸酶活性有明显影响。     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０~（－４）ｓ~（－１）、６．３８×１０~（－４）ｓ~（－１）、２．１７×１０~（－３）ｓ~（－１）、２．３３×１０~（－３）ｓ~９－１）、５．１７×１０~（－３）ｓ~（－１）;而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０~（－４）ｓ~（－１）、３．５７×１０~（－４）ｓ~（－１）、５．８６×１０~（－４）ｓ~（－１）、１．１４×１０~（－３）ｓ~（－１）、３．４５×１０~（－３）ｓ~（－１）;表现为失活与构象伸展变化基本平行。     

Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: Purification,characterization and potential application as a laundry detergent additive

Two detergent stable alkaline serine-proteases (BM1 and BM2) from Bacillus mojavensis A21 were purified. The molecular weights of BM1 and BM2 enzymes determined by SDS–PAGE were approximately 29,000 Da and 15,500 Da, respectively. The optimum pH values of BM1 and BM2 proteases were shown to be 8.0–10.0 and 10.0, respectively. Both enzymes exhibited maximal activity at 60 °C, using casein as a substrate.     

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon nanotubes (SWNTs) modified glass carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK_a of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.     

A synthetic analog of prostaglandin E(1) prevents the production of reactive oxygen species in the intestinal mucosa of methotrexate-treated rats

Administration of methotrexate to rats results in severe enterocolitis and death. Previous our studies showed that a synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] ameliorated the anticancer agent-induced enterocolitis of rats. In the current study, we have focused on the biochemical effect of OP-1206 on the methotrexate-induced intestinal inflammation implicating reactive oxygen species (ROS). Methotrexate (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days. On the 6th day, the chemiluminescence from the jejunum was measured to evaluate the generation of ROS. Spontaneous chemiluminescence from the jejunum of the methotrexate-treated rats increased significantly, compared with the control. Luminol-enhanced chemiluminescence from inflamed mucosal scrapings from the jejunum of the methotrexate-treated rats indicated more remarkable enhancement than the control rats. The treatment of OP-1206 with methotrexate showed significantly lower chemiluminescence of both the jejunum and mucosal scrapings than those of the methotrexate-treated rats. The alkaline phosphatase (ALP) activity, as a marker of small intestinal differentiation, in the intestinal mucosa of the methotrexate-treated rats decreased remarkably, but that of the methotrexate and OP-1206-treated rats was significantly higher than that of the methotrexate-treated rats. Thus, OP-1206 may possibly help the anticancer chemotherapy by protecting the small intestine from the methotrexate-induced damage.     

Application of protease from Nocardiopsis sp. as a laundry detergent additive

The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo^®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K⁺, Na⁺, and Mg⁺⁺ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg).     

Enhancement of bone repair with a helper-dependent adenoviral transfer of bone morphogenetic protein-2

Regional gene therapy, which involves the delivery of growth factors to a specific anatomic site, has the potential to enhance bone formation in clinical application. Helper-dependent adenoviral vectors, which have deleted all of the viral coding regions, have been shown to be safe and highly efficient with long-lasting transgene expression. In this study, we constructed a helper-dependent adenoviral vector producing bone morphogenetic protein-2 (AdHDBMP-2). The AdHDBMP-2 increased the alkaline phosphatase activity of W-20-17 cells in vitro. In addition, when AdHDBMP-2 infected rat bone marrow cells were implanted into the hindlimbs of SCID mice, orthotopic bone formation was shown at 2 weeks. To our knowledge, this is the first study to demonstrate bone formation with the helper-dependent adenoviral vector with the BMP-2 expression cassette. This type of gene therapy vector could prove to be highly useful for bone augmentation in patients with bone loss associated with trauma, revision total joint arthroplasty, or cancer.