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1.
菜心和水稻绿叶中不同等电点的乙醇酸氧化酶   总被引:3,自引:0,他引:3  
The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%~20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.  相似文献   

2.
The relationships among concentrations of copper and zinc, the oxidase activity of ceruloplasmin (Cp) in serum, and Cu,Zn-SOD (superoxide dismutase) activity in erythrocytes were investigated in men with atherosclerosis obliterans (AO) and a control group. The oxidase activity of Cp was measured with o-dianisidine dihydrochloride as a substrate, and Cu,Zn-SOD activity in erythrocytes by using the RANSOD kit. The lipid profile and uric acid concentration were determined in AO and control groups. The results showed higher copper and zinc concentrations in serum in the AO group (20.0±3.5 and 18.0±3.2 μmol/L, respectively) in comparison with the control group (15.6±2.3 and 14.7±1.9 μmol/L). The Cp activity in serum was higher in the AO group (174.2±61.8 U/L) than in the control group (93.7±33.9 U/L), and a significant difference was found in the activity of Cu,Zn-SOD in erythrocytes (2389±1396 and 1245±365 U/g Hb, respectively) between both groups. The activity of Cu,Zn-SOD was positively correlated with copper in the control group (r=0.73), but not in AO, and negatively with uric acid concentration (r=−0.63) in the AO group. The oxidase activity of Cp was correlated with copper, but not zinc, in AO and control groups (r≥0.65). Negative correlation coefficients were calculated for uric acid and copper and zinc concentrations in the AO group (−r≥0.61). Increased copper concentrations and oxidase activity of Cp in serum in AO and the activity of Cu,Zn-SOD in erythrocytes could result from atherosclerotic disease, accompanied by chronic ischemia of a lower limb. These results suggest also that relationship between copper concentration and Cu,Zn-SOD activity in erythrocytes found in the serum of healthy subjects may be disturbed in pathologic conditions.  相似文献   

3.
Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.  相似文献   

4.
Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.  相似文献   

5.
The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.  相似文献   

6.
The net photosynthetic efficiency in C3 plants (such asrice, wheat and other major crops) can be decreased by30% due to the metabolism of photorespiration [1], inwhich glycolate oxidase (GO) serves as a key enzyme. Itis known that GO, with flavin mononucleotide (FMN) asa cofactor, belongs to flavin oxidase [2]. But it differs fromother flavoproteins in that FMN is loosely bound to itsapoprotein and there exists a dissociation balance betweenthem, which indicates that FMN probably regulate…  相似文献   

7.
Summary. The levels of polyamines (putrescine, spermidine and spermine) and polyamine oxidase in plasma of patients with chronic renal failure were determined. The level of putrescine was increased but the level of spermine was decreased in the plasma of these patients. The patients also had increased plasma polyamine oxidase activity leading to increased degradation of spermine. As acrolein was a major toxic compound produced from spermine by polyamine oxidase, the levels of free and protein-conjugated acrolein in plasma were also measured. Acrolein levels were enhanced in plasma of patients with chronic renal failure. The accumulated acrolein found as protein conjugates was equivalent to 170 μM, which was about 5-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by spermine oxidase in plasma. An increase in putrescine, spermine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. After patients with chronic renal failure had undergone hemodialysis, their levels of plasma polyamines, spermine oxidase and acrolein returned towards normal. It is likely that acrolein produced from spermine accumulates in the blood due to decreased excretion into urine and may function as a uremic “toxin”.  相似文献   

8.
Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.  相似文献   

9.
We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 μg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 7, pp. 948–954.  相似文献   

10.
EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpression engineering bacteria in which the specific expression products increased to about 20% of total cellular protein.By using chromatography on DEAE cellulose column,phosphocellulose column and FPLC of Resource Q,the enzyme was purified from soluble protein fraction.The inclusion bodies were solved and renatured,and the enzyme was purified from this part of protein with higher specific activity by using FPLC of Resource Q.Detection showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).  相似文献   

11.
菠菜乙醇酸氧化酶同工酶GO Ⅰ的纯化和特性   总被引:1,自引:0,他引:1  
从菠菜绿叶中获得SDS-PAGE为40000±2000M  相似文献   

12.
菠菜中的乙醇酸氧化酶是一个同工酶   总被引:8,自引:4,他引:4  
乙醇酸氧化酶(EC.1.1.3.15.GO)是光呼吸途径的关键酶,降低其活性可提高C3植物如水稻的产量,在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下,对GO的研究具有重要的理论意义和实际应用价值.在光呼吸途径被提出后的几十年间,人们对如...  相似文献   

13.
乙醇酸氧化酶 (EC 1 1 3 15 ,GO)被认为只含4 0kD一种碱性亚基 ,是因为从多种植物中获得了具GO活性的蛋白 ,SDS PAGE后呈约 4 0kD单带[1] .菠菜GOcDNA编码约 370个氨基酸 ,即 4 0kD多肽 ,其碱 酸性氨基酸的比例高达 0 96 ,富含碱性氨基酸[2 ] .已克隆的GOcDNA在E .coli中表达  相似文献   

14.
菠菜乙醇酸氧化酶同工酶的亚基组成   总被引:3,自引:1,他引:2  
我们首次报告 ,菠菜有GOⅠ (pI≈ 7.4 )、GOⅡ(pI≈ 9.4 )和GOⅢ (pI≈ 8.3 ) 3种乙醇酸氧化酶(GO)同工酶 ;GOⅠ只含 4 0± 2kD一种亚基 ,而GOⅡ和GOⅢ的亚基组成未被研究 ;3种同工酶之间均有免疫同源性[1-4 ] .水稻也存在 3种GO同工酶 ,其中GOⅡ (pI >8.3 )能被乙醇酸所诱导 用柱层析法纯化可获得经SDS PAGE后为 4 3± 2kD单带的水稻GOⅠ[5~ 7] .以上初步解释了前人报告GO电荷不均一的原因[5] .最近从菠菜分离得到含GOⅡ的蛋白和含GOⅢ的蛋白 ,其SDS PAGE分别为 67± 2kD和 4 0±…  相似文献   

15.
乙醇酸氧化酶(GO)只含40 kD亚基却具有多个等电点(pI).我们曾报告GO含40 kD酸性亚基和68 kD碱性亚基,组成3种GO同工酶[1~6].后来证实,68 kD亚基由14个2 kD富苯丙氨酸的碱性短肽(BOP)和40 kD亚基组成[7].现在进一步证实,不同数量的BOP和乙醇酸氧化酶结合,组成多种GO-BOP复合体GC.这些复合体具有不同的pI、乙醇酸氧化酶活性和吸收曲线,但SDS-PAGE和免疫反应相同.在低电压和短时间的醋酸薄膜电泳中,GO和BOP不会解离,GC呈现强碱性(pI>10.0);在高电压的毛细管等电聚焦电泳(cIEF)中,GO和BOP发生不同程度解离,导致GC不聚焦,或聚焦为不同带,pI为7.4/6.8/6.2/5.3.BOP存在于各种动植物和微生物以及血纤维蛋白原中.  相似文献   

16.
通过缩短DEAE-Cellulose柱长度,加快流速并采用pH8.8的80mmol.L-1Tris-HCl为洗脱液,可在9小时内快速地从菠菜、菜心和豆角绿叶中纯化得到乙醇酸氧化酶。该酶具高活性(54.6~197.0U.mg-1)及高等电点(pI>10.0)。产率为4.1%~71.5%,纯化倍数为21.6~122.68。经SDS-PAGE检测均有40kD带,表明3种植物乙醇酸氧化酶的亚基大小无区别。  相似文献   

17.
通过缩短DEAE-Cellulose柱长度, 加快流速并采用pH8.8的80 mmol.L-1 Tris-HCl为洗脱液, 可在9小时内快速地从菠菜、菜心和豆角绿叶中纯化得到乙醇酸氧化酶。该酶具高活性(54.6~197.0 U.mg-1)及高等电点(pI >10.0)。产率为4.1%~71.5%, 纯化倍数为21.6~122.68。经SDS-PAGE检测均有40 kD带,表明3种植物乙醇酸氧化酶的亚基大小无区别。  相似文献   

18.
An extracellular alpha-amylase (1,4-alpha D-glucan glucan hydrolase; EC 3.2.1.1) was isolated from the cell free broth of Streptomyces megasporus SD12 grown in glucose, soluble starch and raw starch. The enzyme was purified 55-fold with a specific activity of 847.33 U mg-1 of protein and with a yield of 36% activity. The apparent molecular mass of the enzyme was 97 kDa, as estimated by SDS-PAGE. The pI of the enzyme was 5.4 and it was stable at a pH range of 5.5 to 8.5 with an optimum pH 6. The enzyme was stable upto 85 degrees C with a half life of 60 min. With soluble starch as substrate the enzyme exhibited a K(m) and kcat value of 4.4 mg ml-1 and 2335 U min-1 mg-1 of protein respectively. The major end products of starch hydrolysis were maltotriose and maltose depending on the incubation period. The production of the enzyme with agricultural wastes as substrates was 643 to 804 U min-1 mg-1 of protein in submerged fermentation whereas solid state fermentation could produce only 206 U min-1 mg-1 of protein.  相似文献   

19.
以黑豆幼苗为材料,研究了水分胁迫下其非酶促保护物质和保护酶系对活性氧的清除作用。结果显示:黑豆皮红色素(17.36%)对DPPH.的清除率高达93.6%,可溶性糖(7.227%)对O2-与.OH的清除率分别为47.71%与68.16%,谷胱甘肽(0.8033mg/g)对O2-与.OH有较明显的清除作用,三种非酶促保护物质随着浓度的升高其清除能力呈逐渐增强的趋势;在水分胁迫下,黑豆幼苗超氧化物歧化酶(33~14 U.mg-1.min-1.2~6d-1),过氧化氢酶(2.87~2.01 mg.mg-1.min-1.3~6d-1)与过氧化物酶(31~14 mg.mg-1.min-1.3~6d-1)三种保护酶活性呈现先升高后降低的变化趋势。研究表明:黑豆体内清除活性氧防御系统作用十分明显,且具有较强的抗氧化、抗衰老、抗逆能力。  相似文献   

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