Purification and kinetic analysis of acetylcholinesterase from western corn rootworm,Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Acetylcholinesterase (AchE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 μmol/min/mg and a yield of 23%. The V_max values for hydrolyzing acetylthiocholine (ATC), acetyl-(β-methyl)thiocholine (AβMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140.5, 150.2, and 18.8 μmol/min/mg, respectively, and K_m values were 19.7, 18.5, 14.1, and 11.0 μM, respectively. The first three substrates showed significant inhibition to the AchE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25–2 mM but activation at >4 mM. AchE activity was almost completely inhibited by 1 μM eserine and BW284C15, respectively, but only 12% of AchE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AchE from WCR was a typical insect AchE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AchE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AchE agreed quite well with the previously published in vivo bioassay data. Arch. Insect Biochem. Physiol. 39:118–125, 1998. © 1998 Wiley-Liss, Inc.     

A comparison of the cholinesterases of an oyster (Crassostrea virginica) and a clam (Macrocallista nimbosa).

Cholinesterase activities in the hearts and ganglia of an oyster (Crassostrea virginica) and a venerid clam (Macrocallista nimbosa) were measured and compared. Tissue extracts were partially purified by ammonium sulfate fractionation followed by gel column chromatography. Enzymatic activity was assayed spectrophotometrically; substrates were acetyl-, butyryl-, and propionylthiocholine (ATC, BTC, PTC). Kinetic constants characterizing each enzyme were derived. At all substrate concentrations, the hydrolysis rates of both clam enzymes were in the order: BTC greater than PTC greater than ATC. With oyster enzymes the ranking was ATC greater than or equal to PTC greater BTC. The specific activities of oyster heart and ganglion enzymes were similar. In contrast, clam ganglion extracts were 75-100 times more active than clam heart extracts and, with any substrate, had greater activity than either oyster enzyme. All enzyme preparations proved to be homogeneous on the bases of constant substrate activity ratios in successive column fractions, and of intermediate velocities with mixed substrates. Six cholinesterase inhibitors were tested. The specific acetylcholinesterase antagonist, B.W. 62C47, WAS MUCH MORE EFFECTIVE AGAINST OYSTER ENZYMES, WHILE THE SPECIFIC ANTIBUTYRYLCHOLINESTERASE, ISO-OMPA, almost totally inhibited calm enzyme activity, but had little effect on oyster. Eserine was the most effective inhibitor of both enzymes. In conclusion, the enzymes in oyster tissues are acetylcholinesterases, while clam enzymes are butyrylcholinesterases. Nevertheless, clam ganglion esterase is sifficiently active to hydrolyze the physiological substrate, acetylcholine. These results explain the long-observed differences in isolated heart pharmacology between ostreid and venerid bivalves.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

中华稻蝗谷胱甘肽S-转移酶的分离纯化

采用硫酸铵沉淀法和GSH-agarose亲和层析法,对中华稻蝗Oxya chinensis(Thunberg)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化.结果表明:经硫酸铵沉淀,饱和度在60%-80%下沉淀中GSTs比活力较高,饱和度90%时比活力达到最高...     

Anticoagulant activity of fucosylated chondroitin sulfate isolated from Cucumaria syracusana

Fucosylated chondroitin sulfate (FCScs) isolated from sea cucumber Cucumaria syracusana was characterized by Fourier Transform InfraRed spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy and high performance size exclusion chromatograph, a multi-angle laser light scattering detector, a viscometer and a differential refractive index (dRI) detector (HPSEC-MALLS-dRI). The anticoagulant activities of FCScs were studied by the classical clotting time assays and the purified systems containing thrombin and antithrombin or heparin cofactor II. The effect on thrombin generation was investigated using calibrated automated thrombography (CAT). The results obtained showed that the FCS with high sulfate content 31 % and relatively low average molecular weight of 36.3 kDa was isolated from C. syracusana in amount of ∼ 35.6 mg/g dry body wall. Structural analysis of this polysaccharide revealed the presence backbone structure of chondroitin sulfate chain branched by two types of fucose 2,4-O-di and 3,4-O-disulfated residues in respective ratios of 57.5 and 42.5 %. The FCScs exhibited a high anticoagulant activity mediated essentially by heparin cofactor II (HCII) and to lesser extent by antithrombin (AT) with IC₅₀ values of 0.05 μg/mL and 0.09 μg/mL, respectively. Furthermore, the results of CAT assay showed that the velocity index decreases 3-times at 50 μg/mL in comparison with normal plasma. The overall results showed high anticoagulant activity attributed to the high sulfate content and abundance of disulfated fucose branches of FCScs which made it a promising candidate of anticoagulation drug.     

An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species.     

Cholinesterase activity during embryonic development in the blood-feeding bug Triatoma patagonica

Triatoma patagonica Del Ponte (Hemiptera: Reduviidae), a vector of Chagas' disease, is widely distributed in Argentina and is found in sylvatic and peridomiciliary ecotopes, as well as occasionally in human dwellings after the chemical control of Triatoma infestans. Anti-cholinesteratic products can be applied in peridomiciliary areas and thus knowledge of cholinesterase activity during embryonic development in this species might contribute further information relevant to effective chemical control. Cholinesterase activity was characterized by reactions to eserine 10(-5) m, to increasing concentrations of substrate and to varying centrifugal speeds. Acetylcholinesterase activity was detected on day 4 and was significant from day 5. A reduction in cholinesterase activity towards acetylthiocholine (ATC) was observed on days 9 and 10 of development. Cholinesterase activity towards ATC and butyrylthiocholine (BTC) in homogenates of eggs was inhibited by eserine 10(-5) m. The shape of the curve indicating levels of inhibition at different concentrations of ATC was typical of acetylcholinesterase. Activity towards BTC did not appear to be inhibited by excess substrate, which parallels the behaviour of butyrylcholinesterases. Cholinesterase activity towards ATC was reduced in supernatant centrifuged at 15 000 g compared with supernatant centrifuged at 1100 g. The cholinesterase system that hydrolyzes mainly ATC seems to belong to the nervous system, as indicated by its behaviour towards the substrates assayed, its greater insolubility and the fact that it evolves parallel to the development of the nervous system. Knowledge of biochemical changes associated with the development and maturation of the nervous system during embryonic development would contribute to the better understanding of anti-cholinesteratic compounds with ovicidal action that might be used in control campaigns against vectors of Chagas' disease.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.     

Bovine endothelial cell plasminogen activator inhibitor. Purification and heat activation

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant. The yield was 2.2 mg from 1.61 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 kDa. The amino-terminal 40-amino-acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI-1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI-1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 X 10(5) U/mg) and 4 M guanidine-HCl (1.5 X 10(5) U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100 degrees C for 2.5 min, generating the specific activity of 1.0 X 10(6) U/mg. The heat-activated PAI lost its activity during incubation at 56 degrees C for 30 min, but repeated heat at 100 degrees C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37 degrees C, 56 degrees C and 80 degrees C, however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat-induced, specific conformational change, but tends to be masked again during renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form.     

用国产微孔玻璃珠初步纯化重组人尿激酶原

用国产微孔玻璃珠从CD-1工程细胞株培养上清中纯化尿激酶原,探索了微孔玻璃珠结合尿激酶原的条件和洗脱方法,比较了硫酸铵线性梯度洗脱或25％乙二醇脱尿激酶原活性蛋白的结果,最后确定洗脱条件为0.25mol/L Tris、1～2mol/L(NH_4)_2SO_4、pH9.3。经此一步纯化,尿激酶原比活性达到15329IU/mg,回收率为78％,还原条件下SDS-PAGE分析分子量为52×10~3的单链尿激酶原占74％。     

枯草芽孢杆菌SA-22 β-甘露聚糖酶的纯化及其特性

利用硫酸铵沉淀、羟基磷灰石柱层析、Sephadex G-75凝胶过滤和DEAE-52离子交换柱层析的方法,将枯草芽孢杆菌SA-22 β-甘露聚糖酶纯化了30.75倍,同时,该酶比活达到3478056 u/mg,收率达到23.43%。利用SDS-PAGE凝胶电泳和Sephadex G-75凝胶过滤的方法测得枯草芽孢杆菌SA-22 β-甘露聚糖酶的分子量分别为38 kD和34 kD。实验发现该酶的最适pH为6.5,在pH 5～10的范围内稳定;该酶最适温度为70℃,在50℃保温4h后其活力不变,在60℃保温4 h后剩余酶活为74.2%,70℃的酶活半衰期为3h。实验还发现Hg²⁺对酶活力有明显抑制作用。该酶对槐豆胶和魔芋胶的K_m和V_max值分别为11.30mg/mL, 4.76mg/mL和188.68(μmol·mL^-1·min^-1), 114.94(μmol·mL^-1·min^-1)。     

Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.     

石榴皮多酚提取物及纯化物对α-葡萄糖苷酶的抑制作用研究

采用紫外分光光度法研究了石榴皮多酚提取物及其2种纯化物(P-1和P-2)对α-葡萄糖苷酶体外抑制作用以及纯化物对该酶的抑制作用类型.结果显示,石榴皮多酚提取物和纯化物对α-葡萄糖苷酶活性均表现出较强的抑制作用,且其作用大小与浓度呈明显的剂量-效应关系;3种多酚样品中,纯化物P-2的抑酶活性最强,纯化物P-1次之,提取物最弱,它们对α-葡萄糖苷酶的半数抑制浓度(IC50,mg/mL)分别为0.045、0.185和0.278.石榴皮多酚纯化物P-2对α-葡萄糖苷酶抑制作用类型为反竞争性抑制;浓度为0.01 mg/mL时该纯化物对α-葡萄糖苷酶的抑制常数Ki为1.22 μg/mL.     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

Separation of Neurospora Crassa Myo-Inositol-1-Phosphate Synthase from Glucose-6-Phosphate Dehydrogenase by Affinity Chromatography

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD⁺) and the coenzyme analogues (5′AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 60 μq of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.     

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.