Isolation and characterization of glutathione-S-transferase isozyme Q3 from the northern quahog,Mercinaria mercinaria

Three glutathione-S-transferase (GST) isozymes (Q1, Q2, and Q3) from the northern quahog (Mercinaria mercinaria) were purified and separated with a combination of affinity and ion exchange chromatography. SDS-PAGE analysis of the separated quahog GSTs indicated there are four distinct subunits of the enzyme with molecular masses ranging between 23 and 27 kDa. The electrophoretic analysis in combination with GST information from literature indicates that among the quahog GST isozymes, there is a single homodimer and two heterodimers. Enzymatic kinetic analysis of the homodimeric quahog GST (Q3) using 1-chloro-2,4-dinitrobenzene and glutathione as reactants resulted in V _max and K _m values of 33.2 mol min^–1 mg^–1 and 0.40 mM, respectively. A pH profile analysis of the Q3 GST indicates that the optimum catalytic pH is 7.6. The Q3 isozyme composes about 28% of the ion exchange purified GSTs but accounts for only 9% of the total GST enzymatic activity (25 mol min^–1 mg^–1). An analysis investigating the dependence of the Q3 GST activity on temperature resulted in a retention of enzymatic activity (50–30% at temperature extremes from –13°C to 100°C), suggesting a unconventional role for the Q3 GST in quahog metabolism.     

Copepod parasites of a commercial clam (Meretrix meretrix) from Phuket,Thailand

Three species of copepods are reported from hard clams, Meretrix meretrix (L.), obtained from the market in Phuket, Thailand. They are: Conchyliurus bombasticus Reddiah (Clausidiidae), Ostrincola portonoviensis Reddiah (Myicolidae), and Lichomolgus similis Ho & Kim (Lichomolgidae). The first two species are redescribed based on the newly collected material. Conchyliurus fragilis Reddiah is proposed to be relegated to a synonym of C. bombasticus. L. similis is recorded for the first time from the Indian Ocean.     

Purification and properties of a glutathione-S-transferase from corn which conjugates s-triazine herbicides

A glutathione-S-transferase involved in atrazine conjugation was purified 43-fold from corn with a total yield of 36%. The purified enzyme has a MW of 45 000 as determined by gel filtration. The estimated activation energy of the enzyme is 6.4 kcal/mol and the optimum pH for activity between 8 and 8.5. Substrate specificity studies with s-triazines indicated that atrazine was the best substrate followed by simazine and propazine. The Cl group at the 2-position was essential for enzyme activity, and replacement by a SCH₃ group resulted in a total loss of activity. The absence of an alkyl group resulted in a reduction of conjugation and 2-chloro-4,6-bis-amino-s-triazine was the poorest substrate. With insecticidal substrates (organophosphates), conjugating activity was observed only with diazinon and little or no activity was observed with ethyl parathion, malathion and etrimfos. No activity was found using methyl iodide as a substrate. The purified enzyme has properties similar to those of an aryl-S-transferase. Quinones were inhibitors of this enzyme.     

猪肝微粒体NADH－细胞色素b_5还原酶的纯化及特性分析

采用硫酸铵分级分离,ＳｅｐｈａｄｅｘＧ－１００凝胶过滤,ＤＥＡＥ－纤维素离子交换层析以及５＇－ＡＭＰ～Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,从猪肝微粒体中纯化得到可溶性的ＮＡＤＨ－细胞色素ｂ５还原酶,提纯倍数为７５０～８００。总回收率为４０％左右。纯化的酶是典型的黄素蛋白吸收光谱,Ａ２７３／Ａ４６０比值为５．８。在ＳＤＳ－聚丙烯酸胺凝胶电泳板上呈单一的蛋白质区带,分子量为３２ｋｄ。ＮＡＤＨ和２,６－二氯酚靛酚的Ｋｍ值分别为２４和４５μｍｏｌ／Ｌ,以２,６－二氯酚靛酚为底物时,该还原酶的催化作用可能为乒乓机制。该酶的纯化为分子水平研究其反应机制及制备相应的抗体以建立免疫学检测方法创造了条件。     

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

抗脂多糖单克隆抗体特性及纯化的研究

用Ｅ．ｃｏｌｉ０１１１：Ｂ４死菌体及其脂多糖（ＬＰＳ）免疫ＢＡＬＢ／Ｃ小鼠,取其脾细胞与ＳＰ２／０细胞融合获得６株稳定分泌抗ＬＰＳ特异性单克隆抗体细胞株,其中一株为ＩｇＧ２ａ类,５株为ＩｇＭ类,轻链均为Ｋ型;染色体数目为９０－９８条;５株ＩｇＭ类单克隆抗体识别５种不同的抗原表位;相对亲和力在１０~８～１０~（１０）之间;１Ｂ１２单抗经ＳｅｐｈａｄｅｘＧ－１５０纯化后,经还原性ＳＤＳ－ＰＡＧＥ显示只有７０ＫＤ的重键和２５ＫＤ的轻链。     

胡萝卜愈伤组织伸展蛋白纯化及某些生化特性的研究

本文报道了胡萝卜愈伤组织伸展蛋白的柱色谱纯化,电泳性质,氨基酸组成及其电镜观察结果。用CM-cellulose柱色谱纯化胡萝卜愈伤组织伸展蛋白时,仅发现有一个组分;它的电泳性质与胡萝卜根产生的第一种类型伸展蛋白相同;它的羟脯氨酸/絲氨酸克分子数比例大约为4/1(羟脯氨酸,42.1mol％;丝氨酸,12.8mol％);此外,在电子显微镜下观察,伸展蛋白具有典型棒状分子结构。     

旋扭山绿豆（Desmodiumintortum）凝集素的纯化与特性

旋扭山绿豆（Ｄｅｓｍｏｄｉｕｍｉｎｔｏｒｔｕｍ）凝集素的纯化与特性彭建宗,程双奇,陈兆平,莫熙穆（华南师范大学生物系,广州５１０６３１）关键词凝集素;旋扭山绿豆;亲和层析;糖蛋白豆科植物和根瘤菌之间的共生结瘤固氮关系早有报道。例如大豆只被Ｂ．仰。６。...     

Purification and Characterization of a Novel Chemorepellent Receptor from Tetrahymena thermophila

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote. Received: 28 July 1997/Revised: 14 November 1997     

Purification and Characterization of Carboxypeptidases from the Sarcocarp of Watermelon

Two kinds of carboxypeptidases (F–I, F–II) were purified from the sarcocarp of watermelon (Citrullus vulgaris, var. Shimao). F–I was not purified to homogeneity. F–II was homogeneous on ultracentrifugal analysis, but a trace of impurity was detected at high concentrations by disc electrophoresis.

F–I was optimally active and stable at pH 5.0~5.5 and was strongly inhibited by DFP and HgCl₂, but not by EDTA. The molecular weight and isoelectric point were 89,000 and 4.4, respectively.

F–II was optimally active at pH 5.0 ~ 5.5 and was most stable at pH 5.5 ~ 7.0. It was completely inhibited by DFP and HgCl₂, but not by EDTA and 1, 10-phenanthroline, and it hydrolyzed an oligopeptide containing proline, glutamic acid, lysine and several neutral amino acids, sequentially from the C-terminal. The molecular weight and isolelectric point were 110,000 (5.1 S) and 5.0, respectively.

The similarity of enzymatic properties of both the present enzymes to those of other plant carboxypeptidases and pig kidney cathepsin A are discussed.     

Purification and Characterization of N-Acetylglucosamine-6-phosphate Deacetylase from a Psychrotrophic Marine Bacterium, Alteromonas Species

A psychrotrophic bacterium, strain Mct-9, which produced an N-acetylglucosamine-6-phosphate deacetylase, was isolated from a deep-seawater sample in the Mariana Trough. The Mct-9 strain was identified as Alteromonas sp. The native enzyme had a molecular mass of 164,000 Da, and was predicted to be composed of four identical subunits with molecular masses of 41,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine (GlcNAc), GlcNAc-6-phosphate, and GlcNAc-6-sulfate. Considering the low K _m and high k _cat /K _m for GlcNAc-6-phosphate, it probably acts as a GlcNAc-6-phosphate deacetylase in vivo. The enzyme was functional in the temperature range of 5° to 70°C and displayed optimal activity at 55°C. The optimal temperature was higher than that of the deacetylase from the mesophilic bacterium Vibrio cholerae non-O1. The characteristics of the GlcNAc-6-phosphate deacetylase from Alteromonas sp. are unique among psychrotrophs and psychrophiles, whose intracellular enzymes are mostly thermolabile. Received May 6, 1999; accepted August 16, 1999.     

Purification and Characterization of the polygalacturo nase from Tomato Fruits

The polygalacturonase (PG) isolated fron the pericarp of fully ripe tomato fruits was purified by (NH4)2SO4 precipitation, carboxyme-thylsepharose ion-exchang column and sephadex G-75 gel filtration. Specific activity of purified PG was 1.5 μmol galacturonic acid mg-1 protein min-1, which was 30 times as high as that of the crude extract with 1.7mol NaC1. When the elution separated by second sephadex G-75 column was analyzed by SDS-PAGE, only a single protein band was detected. It was shown by heat and pH experiments of the purified enzyme that the enzyme activity retained 50%, after treatment with heat at 50℃ for 10 min, and that the optimal pH was 4.6.     

Purification and Characterization of Fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K _eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K _eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Purification and Characterization of Clathrin-Coated Vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas rein-hardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter±SD of 95±17 nm, n=300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

黑木耳漆酶纯化及部分漆酶特性的研究

黑木耳漆酶研究可为漆酶的进一步分离纯化、基因克隆表达和大规模生产应用奠定基础。对黑木耳"黑29"菌株漆酶粗酶液进行硫酸铵分级沉淀后,通过Native SDS-PAGE电泳检测,存在3种漆酶LacA、LacB、LacC,分子量分别为60,34,19 kD。经硫酸铵分级沉淀和DEAE-Sephacel柱层析技术分离得一纯化成分LacC,纯化倍数7.60,酶活性回收4.28%。对LacC的pH、温度、金属离子和Km值等部分酶学性质进行研究发现,该酶氧化ABTS的Km值为1.18×10-6mol/L,催化氧化底物ABTS的最适pH为3.8,在pH 3.0~4.6表现出较强的稳定性;最适反应温度为55℃,低于50℃时有较好的热稳定性;金属离子Ag+对漆酶有激活作用,而Fe3+、Mn2+、Co2+则有抑制作用。     

特异腐质霉纤维素酶的分离纯化与性质

特异腐质霉Humicola insolens(YH-8)能高效合成高稳定性的纤维素酶.由该菌株所产的纤维素酶粗液经过1.5倍酒精沉淀、DEAE-SephadexA-50离子交换层析,SephadexG-100凝胶过滤,得到电泳纯的纤维素酶组分一个.对这个组分的催化性质(以羧甲基纤维素为底物)进行了研究,该酶的最适催化温度60℃,pH值为5.5,在50℃以下酶的稳定性较好,70℃条件下底物对酶有较强的保护作用,该酶的pH稳定性范围为4～8,zn2 ,ca2 ,Mg2 ,K ,Li 对酶活有促进作用,Mn2 、Co2 、Fe2 、Fe2 对酶活起抑制作用,酶对CMC-Na和水杨素有分解作用,而不分解脱脂棉和滤纸.     

Purification and Characterization of Kininogens from Sheep Plasma

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep ( Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. K_i values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. K_i values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白,并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ,１７．５ｋＤ）组成的大分子,其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性,Ｃａ~（２＋）离子也能提高其活性,Ｍｇ~（２＋）离子无明显影响。钒酸盐,碘乙酸,对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。     

Purification and Characterization of Myosin from Calf Brain

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.