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1.
鲫鱼酸性磷酸酶酶学特性及不同效应物对酶活力的影响 总被引:3,自引:0,他引:3
经NaAc-HAc缓冲液(pH5.0)抽提,正丁醇处理,硫酸铵分级沉淀,DEAE-32离子交换层析,SephadexG-150凝胶过滤纯化,从鲫鱼内脏中分离纯化出电泳纯的酸性磷酸酶。该酶提纯倍数为30.82,比活力195.06U/mg。研究表明,该酶催化对硝基苯磷酸二钠水解反应,最适pH4.8,pH小于4和大于7时不稳定;最适温度45℃,温度高于50℃不稳定;米氏常数为0.23mmol/L,利用SDS-PAGE测定酶亚基分子量为33.3kD。化学修饰剂SUAN、PMSF、DTT、NBS对该酶活力影响不大,BrAc和IAc有明显抑制作用。金属离子对该酶催化活力有不同影响,Na+、K+、Ni2+、Co2+影响不显著,Mg2+、Ca2+、Ba2+、Mn2+有激活作用,Ag+、Cu2+、Pb2+、Cd2+有抑制作用,其中Mg2+、Ca2+、Pb2+、Cd2+对鲫鱼酸性磷酸酶荧光光谱的影响表明金属离子对酶活力的影响与酶构象改变有关。
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2.
Prawn (Penaeus penicillatus) acid phosphatase (EC 3.1.3.2) catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in sea water on the enzyme activity results in the loss of the biological function of the enzyme, which leads to disruption of phosphate metabolism in cells. This paper analyzes the effects of methanol on the activity and conformation of prawn acid phosphatase. The results show that low concentrations of methanol can lead to reversible inactivation. Inhibition of the enzyme by methanol is classified as non-competitive inhibition, and the inhibition constant (Ki) is 8.5%. Conformational changes of the enzyme molecule in methanol solutions of different concentrations were measured using fluorescence emission, differential UV-absorption, and circular dichroism spectra. Increased methanol concentrations caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with methanol had two negative peaks, at 222 and 270 nm, and a positive peak at 236 nm. The changes in the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed some. These results suggest that methanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity. 相似文献
3.
M A Nesmeianova O B Maraeva M N Kolot I S Kulaev 《Biokhimii?a (Moscow, Russia)》1978,43(10):1783-1789
The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation. 相似文献
4.
Gonnety JT Niamké S Meuwiah Faulet B N'guessan Kouadio EJ Kouamé LP 《The Italian journal of biochemistry》2007,56(2):149-157
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. 相似文献
5.
An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo. 相似文献
6.
1. Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431-441). The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100. 2. The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0. A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent. 3. The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature. Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility. 4. A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible. 相似文献
7.
Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy. 相似文献
8.
缺磷条件下的小麦根系酸性磷酸酶活性研究 总被引:34,自引:2,他引:34
1 引 言植物根可向根际分泌许多有机化合物 ,其中有许多物质都能促进植物对矿质养分的吸收 .作为必需大量营养元素的P ,在土壤中以无机磷酸盐阴离子的形式被吸收 ,而有机磷酸酯必须被水解成无机P后才能进入植物根 ,在这一过程中有一非常重要的步骤 ,就是由微生物、菌根外真菌和植物根分泌酸性磷酸酶 .土壤中的有机P一般占全P的 30 %~ 5 0 % ,有的可达95 % .因此 ,如何发挥植物自身利用土壤有机P的潜力已成为目前植物营养学研究的热点之一 .Goldstein等[3 ] 研究P胁迫条件下悬浮培养细胞时发现 ,抑制植物生长和诱导酸性… 相似文献
9.
Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication. 相似文献
10.
Partanen S 《The Histochemical journal》2001,33(7):387-395
The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases. 相似文献
11.
Lysosomal acid phosphatase activity in human and mouse cells was separated into multiple zones by starch gel electrophoresis. One of the two major zones in the mouse was apparently extinguished when genetic information from man and the mouse was combined in proliferating man-mouse somatic cell hybrids. The evidence suggested that the absence of the mouse lysosomal acid phosphatase (mAP-1) was influenced by the human genome. The gene coding for human acid phosphatase (hAP-1) was shown to be unlinked to the presumed human component which extinguished the mouse acid phosphatase (mAP-1). The mechanism of “extinction” is postulated to be a modification in the processing of the mouse lysosomal enzyme. A dimeric structure was suggested for acid phosphatase-1 of man, mouse, and rat since a single hybrid enzyme was expressed in man-mouse and mouse-rat somatic cell hybrids. 相似文献
12.
利用电镜酶细胞化学方法,观察盘基网柄菌细胞分化和凋亡过程中酸性磷酸酶的变化。在细胞丘阶段,酶反应颗粒出现在线粒体内自噬空泡内,随着内自噬空泡的逐渐增大,线粒体内的酶反应颗粒逐渐增多,线粒体内嵴结构不断破坏,直至遍布整个空泡化的线粒体内;当细胞发育至前孢子细胞时,由于嵴结构被完全破坏,酶反应颗粒主要集中在前孢子细胞空泡的单层膜上,空泡化的线粒体内酶反应颗粒逐渐消失。在凋亡的柄细胞中,自噬泡内酶反应强烈,凋亡中期的前柄细胞的细胞核中出现酶反应颗粒,均匀分布在细胞核中,直至细胞核与自噬泡融合。在孢子细胞外被与质膜间也观察到非溶酶体酸性磷酸酶。所得结果证实:线粒体内自噬小泡具有消化功能;自噬泡内酶活性与细胞器消亡有关;细胞核中的酸性磷酸酶可能作为一种非溶酶体酸性磷酸酶参与细胞核中核蛋白的脱磷酸化过程,与发育相关基因表达有关 相似文献
13.
Agarose gel isoelectrofocusing is used to separate the isozymes of human prostatic acid phosphatase with retention of enzyme activity. The native blotting of the isozymes onto a nitrocellulose membrane increases the sensitivity of the enzyme stain and is suitable for analysis of isozymes in prostate tissue, which contains little nonprostatic acid phosphatase. The specificity of the transfer is increased by treating the membrane with antibody to human prostatic acid phosphatase prior to the transfer. The specificity of the antibody is conferred to the membrane resulting in a transfer specific for prostatic acid phosphatase. The immunotransfer procedure is applicable to serum which contains appreciable amounts of nonprostatic acid phosphatase. 相似文献
14.
G Rune 《Histochemistry》1984,80(3):299-306
The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemistry for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm. In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis. 相似文献
15.
T Braten 《Journal of cell science》1975,17(3):647-653
Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization. 相似文献
16.
Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma 总被引:2,自引:0,他引:2
The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis. 相似文献
17.
Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues. 相似文献
18.
19.
George A. Marzluf 《Journal of bacteriology》1973,115(1):146-152
20.
Abdul Waheed Robert L. Van Etten V. Gieselmann K. von Figura 《Biochemical genetics》1985,23(3-4):309-319
The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft. 相似文献