蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

Identification and partial purification of an ATP-stimulated alkaline protease in rat liver.

Extracts from rat liver contain a sulfhydryl-dependent endoprotease which degrades [methyl-14C]globin or 125I-hemoglobin to acid-soluble peptides. This enzyme was isolated from the 100,000 x g supernatant of the homogenate. It showed a pH optimum between 7.5 and 9.5 and very little activity below pH 7.0. The enzyme has an apparent molecular weight of 550,000 as determined on Sepharose 6B column chromatography and sucrose density gradient centrifugation. ATP, at physiological concentrations, as well as pyrophosphate, stimulated the protease activity in these partially purified preparations up to 3-fold. Nonionic detergents such as Triton X-100 increased proteolytic activity and the stimulation by ATP. Other nucleotide triphosphates and ADP also increased proteolysis but less effectively than ATP. Sodium phosphate, creatine phosphate, and EDTA had no stimulatory effect.     

Plant nucleases. I. Separation and purification of two ribonucleases and one nuclease from corn

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.     

Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization.

Lipoamide dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.     

Plant Nucleases. II. Properties of Corn Ribonucleases I and II and Corn Nuclease I

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.

Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.

Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.

The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.

     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

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.     

Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris,a purple non-sulfur bacterium producing hopanoids and tetrahymanol

The squalene-hopene cyclase of the hopanoid- and tetrahymanol-producing Rhodopseudomonas palustris was released from the isolated membranes by CHAPS and purified to homogeneity by succesive chromatography on DEAE Sephacel, Octyl Sepharose, and Blue Sepharose. The enzyme has a molecular weight of 70 kDa as determined by SDS-PAGE and an isoelectric point at about pH 5.O. The enzyme activity has a maximum at 30°C and at pH 6.5. No production of tetrahymanol could be demonstrated by using either crude or purified cyclase preparations.     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

The isolation of pig liver monoamine oxidase

Monoamine oxidase from pig liver has been isolated and purified approximately three hundred-fold. This enzyme has a molecular weight of 1,200,000, is highly polymeric, and contains subunits of molecular weight 146,000, as determined by Sephadex chromatography. The apparent K_m at 25°C is 1.28 × 10^?6 M at pH 9.0 (0.05 M glycine) and 1.74 × 10^?5 M at pH 7.2 (0.2 M phosphate) using benzylamine as a substrate. This enzyme contains approximately 8 copper(II) ions per 1,200,000 molecular weight.     

Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.

The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum

The F₄₂₀-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent K_m of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH₂ nor FADH₂ could serve as electron donors; NAD was not utilized as electron acceptor.     

Purification and partial characterization of glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions has been purified to homogeneity. The purification procedure involves affinity chromatography on ADP-agarose type 2 as the major purification step. The recovery in the purification is 70%. The specific activity of the purified enzyme is about 10-times higher in the gamma-glutamyl transferase assay than in the coupled biosynthetic assay. The molecular weight was determined to 530,000 by native gradient polyacrylamide gel electrophoresis and to 500,000 by gel filtration. The subunits have an apparent molecular weight of 52,000. Glutamine synthetase isolated from Rsp. rubrum which had been exposed to ammonium ions ('switch-off') before harvest had about 20% of the transferase activity compared with the enzyme purified from nitrogen-starved cells. The low-activity form showed two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.     

Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

NgoII, a restriction endonuclease from Neisseria gonorrhoeae.

EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.     

Isolation,Purification and Some Properties of Invertase from Leaves of Mandarin Orange

Highly active acid invertase was found in the young leaf extract of mandarin orange Citrus reticulata Blanco). The invertase was isolated and purified from the young leaf extract of mandarin orange through the procedures of ammonium sulphate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. 6.4% of the invertase activity was recovered. Invertase was 179.2-folds purified. The purified invertase preparation was homogeneous as shown in polyacrylamide gel electrophoresis and Sephacryl S-200 molecular sieve chromatography. The molecular weight of the native invertase determined by gel filtration was 80 kD. The invertase consists of two identical subunits with apparent equal subunit weight of 40 kD as determined on SDS-PAGE. The invertase followed typical Michaelis-Menten Kinetics with apparent Km Of 1. 6 × 10-2 mol/L for sucrose. Vmax of the invertase was 100 mg reducing sugar · mg-1 protein · h-1 The optimum pH was 5.0 (stable from 4.5—5.5). The optimum temperature was 55℃.