菠菜叶片蔗糖磷酸合成酶的纯化

经硫酸铵分部沉淀,DEAE-纤维素(DE 52),Sepharose 6B和 AH—4B连续三次柱层析,得到纯化88倍电泳均一的菠菜叶片蔗糖磷酸合成酶。电泳分析该酶分子量为490 kD,是由八个分子量为60 kD的相同亚基组成的寡聚体,等电点为PI=4.l,其最适pH值为6.9。     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值,在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）;在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ）,４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４）,表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。     

Purification and Characterization of Glutamine Synthetase from the Basidiomycete Pleurotus ostreatus

The purification and some properties of glutamine synthetase (GS) from the mycelium of the basidiomycete Pleurotus ostreatus are described. The enzyme was purified to apparent homogeneity with ion exchange chromatography and a Dyematrex Green A column as the major purification steps. The GS has a molecular weight of 470 kDa and is composed of eight subunits with a molecular weight of 58 kDa. A tetrameric form of the enzyme may also be active. The apparent K _m values for the biosynthetic reaction varied in different mycelial extracts from 2.5 to 3.5 mM and from 0.02 to 0.06 for glutamate and ammonium respectively. In the transferase reaction, K _m values of 48 mM and 6.2 mM were found for L-glutamine and hydroxylamine, respectively. From the divalent cations tested, Mn²⁺ showed the strongest stimulatory effect both on the transferase and the biosynthetic reaction. ADP was the only nucleotide having an activating effect on the transferase reaction. The biosynthetic reaction was strongly inhibited by AMP and the transferase reaction by carbamoylphosphate. L-Alanine and glycine inhibited both reactions. Received: 21 February 1996/Accepted: 12 March 1996     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Sucrose Phosphate Synthetase from Various Plant Origins

Some properties of sucrose-P synthetases obtained from various plant tissues, including sweet potato roots, potato tubers and leaves of barley, rape and ladino clover were studied. The specific enzyme activity of the sucrose-P synthetase from sweet potato roots was much lower than that of the sucrose synthetase of the other tissues. The enzyme activity decreased gradually as the roots developed. The optimum pH did not differ between enzyme preparations from sweet potato roots and barley leaves. Manganese chloride exhibited a marked stimulative effect on the sucrose-P synthetase from sweet potato roots and potato tubers, whereas it was inhibited the barley leaf enzyme.

Kinetic studies of sucrose-P synthetase showed that the behavior of the enzyme to the substrates did not differ in the enzyme sources examined. The substrate saturation curve of the enzyme with respect to fructose-6-P was sigmodal in shape, giving a straight line with a slope of 1.35~1.5 (n value) in a plot of the data using the empirical Hill equation. On the other hand, enzymes from all the various tissues exhibited a hyperbolic substrate saturation curve for UDP-glucose, obeying the ordinary Michaelis-Menten type reaction. Manganese chloride had no effect on the Km for UDP-glucose, the S_0.5 for fructose-6-P and the n value of the enzyme from potato tuber tissues.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Purification and Characterization of Glutamine Synthetase and NADP-Glutamate Dehydrogenase from the Ectomycorrhizal Fungus Laccaria laccata

Glutamine synthetase (GS) and NADP-dependent glutamate dehydrogenase (NADP-GDH) play a key role in nitrogen assimilation in the ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke) strain S 238. The two enzymes were purified to apparent electrophoretic homogeneity by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl and affinity chromatography, and DEAE-5PW fast protein liquid chromatography. This purification scheme resulted in a 23 and 62% recovery of the initial activity for GS and NADP-GDH, respectively. Purified GS had a specific activity of 713 nanomoles per second per milligram protein and a pH optimum of 7.2. Michaelis constants (millimolar) for the substrates were NH₄⁺ (0.024), glutamate (3.2), glutamine (30), ATP (0.18), and ADP (0.002). The molecular weight (M_r) of native GS was approximately 380,000; it was composed of eight identical subunits of M_r 42,000. Purified NADP-GDH had a specific activity of 4130 nanomoles per second per milligram protein and a pH optimum of 7.2 (amination reaction). Michaelis constants (millimolar) for the substrates were NH₄⁺ (5), 2-oxoglutarate (1), glutamate (26), NADPH (0.01), and NADP (0.03). Native NADP-GDH was a hexamer with a M_r of about 298,000 composed of identical subunits with M_r 47,000. Polyclonal antibodies were produced against purified GS and NADP-GDH. Immunoprecipitation tests and immunoblot analysis showed the high reactivity and specificity of the immune sera against the purified enzymes.     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Sucrose Synthetase of Sweet Potato Roots

The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the enzyme was determined to be about 540,000 by the Sephadex gel filtration chromatography.     

Purification and Properties of Formyltetrahydrofolate Synthetase from Spinach

Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH₄ and formate were required specifically as the substrates, and Mg⁺⁺ and ATP were essential components. The Michaelis constants for dl-FAH₄, formate, ATP and magnesium chloride were 1.7×10^?3 m, 1.7×10^?2 m, 4.1×10^?4 m and 3.3×10^?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N¹⁰-formyl-FAH₄ spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed.     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

Isolation and Fractionation of Water-soluble Polysaccharides from Bamboo Shoot

It was established that the water-soluble polysaccharides of bamboo shoot other than starch consisted of a water-soluble xylan, an arabinogalactan and an α-glucan by isolation and fractionation of the water-soluble polysaccharides extracted with dimethyl sulfoxide from a preparative meal of bamboo shoot. On the basis of the examination of several properties of these polysaccharides, the presence of a galactan first isolated from bamboo shoot was discussed from points of plant biogenesis.     

假单胞菌L-半胱氨酸合成酶的纯化和性质研究

假单胞菌TS-1138的细胞浆液通过硫铵沉淀、SephadexG-75凝胶过滤、DEAE-Cellulose52离子交换、SephadexG 100凝胶过滤等分离纯化手段分别将从L-ATC合成L 半胱氨酸的两个酶——L-ATC水解酶和L-SCC水解酶纯化了 83.9和 90.3倍。SDS-PAGE鉴定均为单一条带 ,两种酶的相对分子质量分别为 37.5和 42.8kD ;酶反应的最适温度均为 35℃ ,最适pH分别为 7.0和 8.0 ;酶的米氏常数分别为 0.67mmol L和 0.15mmol L ,     

Purification and Partial Characterization of the B Subunit of Serratia marcescens Tryptophan Synthetase

A trpE mutant of Serratia marcescens (E-7) was isolated, and the multimeric enzyme tryptophan synthetase (EC 4.2.1.20) was purified to homogeneity from derepressed cells. The A and B subunits were resolved, and the B subunit was partially characterized and compared with the Escherichia coli B subunit as part of a comparative evolution study of the trpB cistron of the trp operon in the Enterobacteriaceae. The S. marcescens B subunit is a dimer (beta(2)), and its molecular weight was estimated to be 89,000. The separate subunits (beta monomers) had molecular weights of approximately 43,000. The B subunit required pyridoxal phosphate for catalytic activity and had an apparent K(m) of 9 x 10(-6) M. The N terminus of the B subunit was unavailable for reaction with terminal amine reagents (blocked), whereas carboxypeptidase digestion released a C-terminal isoleucine. Using S. marcescens B antiserum in agar immunodiffusion gave an almost complete reaction of identity between the B subunits of S. marcescens and E. coli. The antiserum was used in microcomplement fixation, allowing for a comparison of the overall antigenic surface structure of the two B subunits. The index of dissimilarity for the heterologous E. coli enzyme compared with the homologous S. marcescens enzyme was 2.4, indicating extensive similarity of the two proteins at their surfaces. Comparative antiserum neutralization of B-subunit enzyme activity showed the E. coli enzyme to cross-react 85% as well as the S. marcescens enzyme. With regard to the biochemical and immunochemical parameters used in this study, the S. marcescens and E. coli B subunits were either identical or very similar. These findings support the idea that the trpB cistron of the trp operon is a relatively conserved gene in the Enterobacteriaceae.     

Biosynthesis of the Diterpene Phytoalexin Casbene: Partial Purification and Characterization of Casbene Synthetase from Ricinis communis

Casbene synthetase from 67-hour seedlings of Ricinis communis L. which had been treated with Rhizopus stolonifer spores was purified 700-fold by a combination of ammonium sulfate fractionation, QAE A-50 Sephadex chromatography, and G-100 Sephadex chromatography. Polyacrylamide disc gel electrophoresis revealed that the purified fraction was heterogeneous. No casbene synthetase was detected in extracts of seedlings which had not been exposed to the fungal spores; maximum activity was obtained from seedlings 14 hours after exposure to spores.     

Sucrose Synthetase of Rice Grains and Potato Tubers

ABSTRACT

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.     

菠菜叶片蔗糖磷酸合成酶的调节

电泳均一的菠菜叶片蔗糖磷酸合成酶的活力受G6P,Mg~(2 ),Mn~(2 )的调节;G6P对此酶的促进作用在F6P浓度较低时表现得比较明显;此酶对Mn~(2 )较对Mg~(2 )敏感,Mg~(2 ),Mn~(2 )对此酶的促进作用可被EDTA解除。底物F6P的饱和曲线为S型,底物UDPG的饱和曲线为双曲线型。NADP是此酶的负效应剂,NADP对F6P表现为混合型抑制,使V_m(F6P)降低和K_m(F6P)增大,3mmol/L NADP使F6P的K_m值从2.5mmol/L上升至3.8mmol/L,但不影响希尔系数,n=1.3。NADP对UDPG表现为K_m不变的非竞争性抑制,K_m(UDPG)=3.8mmol/L。     

The Structure of an Arabinogalactan Isolated from Bamboo Shoot

It was found that azide bound to alcohol oxidase non-covalently and caused a color change from yellow to red. Alcohol oxidase was purified with a high yield from methanol-limited chemostat-grown cells of Candida boidinii S2 as an enzyme-azide complex by a simple procedure. That is, a cell-free extract was prepared from cells treated with a cationic detergent, Cation M2, and alcohol oxidase amounted to more than 80% of the soluble protein. Inactivation of the enzyme by H₂O₂ and aldehydes was decreased in the complex. The results were discussed as to the production of various aldehydes with the yeast cells.     

Overexpression and Purification of Asparagine Synthetase from Escherichia coli

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUCI8, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15%, of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.