Characterization of bacterial L-(-)-tyrosine decarboxylase by isoelectric focusing and gel chromatography.

The purification of L-(-)-tyrosine apodecarboxylase (TAD) (E.C. 4.1.1.25), obtained from extracts of cells of Streptococcus faecalis, has been investigated by means of preparative isoelectric focusing, molecular sieve chromatography and hydrophobic interaction chromatography. Isoelectric focusing demonstrated two separate fractions possessing enzyme activity that had pI values of 4.5 and ca. 3.2. In the chromatographic methods, however, the activity was obtained in a single peak. It was found that hydrophobic interaction chromatography on phenyl-Sepharose was particularly suitable for purification purposes. The enzyme is very firmly bound to octyl-Sepharose CL-4B but retains most of its activity even in the bound state.     

Lipoprotein lipase activity of the fungusRhizopus microsporus

A method is described for the isolation and purification of lipoprotein lipase activity from the culture liquid of the fungusRhizopus microsporus. Some properties of the homogeneous enzyme have been studied: molecular weight 43,000, pI 3.7.     

Purification of phospholipase C by hydrophobic interaction affinity chromatography.

A simple procedure is described for the purification of phosphatidylcholine-hydrolyzing phospholipase C(PLC). Lecithin, the substrate for PLC, was ligated hydrophobically to octyl-Sepharose in 2 M (NH4)2SO4. The washed lecithin-conjugated resin was then used to purify PLC from crude preparations by affinity chromatography. PLC binds to the lecithin moiety in the presence of Zn2+ and is eluted with an acidic buffer containing EDTA. PLC activity was recovered in the eluate. Both sodium dodecyl sulphate polyacrylamide gel electrophoresis and pI electrofocusing showed that the eluate contained a single monomeric protein with an apparent molecular mass of 66 kDa and a pI of 5.5.     

新型亲和去垢小柱净化-液相色谱-串联质谱法分析水稻叶片蛋白质组

采用亲和去垢小柱净化,建立了水稻叶片蛋白质组的纳升液相色谱-串联质谱分析方法。水稻叶片蛋白质分别采用酚提取法结合十二烷基硫酸钠(sodium dodecyl sulfate,SDS)裂解,裂解液经亲和去垢小柱净化,酶解肽段用纳升液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱(nanoLC-LTQ/Orbitrap MS)分析,相关数据库检索鉴定。比较了超滤辅助样品制备法(FASP法)、亲和去垢小柱法和丙酮沉淀法对SDS去除效率及对蛋白质鉴定结果的影响。结果表明:3种方法均有较好的SDS去除效果(去除效率均大于95%);尽管3种方法鉴定的蛋白质种类具有一定的互补性,但以亲和去垢小柱法鉴定的蛋白质数目最多,为563种,远多于FASP法和丙酮沉淀法的196和306种;此外,亲和去垢小柱法适合于各种相对分子质量和不同pI值蛋白质的净化,而FASP法和丙酮沉淀法中不同相对分子质量和pI值蛋白质均有类似程度的损失。采用本文建立的方法,一次进样分析可鉴定出水稻叶片蛋白质多达588种;肽段匹配数≥2的296个蛋白质的生物学功能主要分为结合活性、酶活性、转移运输活性和结构组成等。该蛋白质分析方法可为开展水稻蛋白质组学研究提供技术参考。     

Chick 28,000 Mr vitamin D-dependent calcium-binding protein in intestine, kidney and cerebellum. Purification using chromatofocusing

A purification method has been developed for chick 28,000 Mr vitamin D-dependent calcium-binding protein, involving Blue Sepharose CL-6B column chromatography, heat treatment and chromatofocusing with a microparticulate anion exchanger (Mono P). It allowed the rapid and reproducible purification of milligram amounts of homogeneous calcium-binding protein with good yields from chick intestine, kidney and cerebellum. The calcium-binding proteins thus obtained have the same molecular weight of 28,000, heat stability, calcium binding capability and apparent isoelectric point of 4.0. These physico-chemical properties are in good agreement with those of proteins isolated by a previous procedure, which gave a low and variable yield of calcium-binding protein.     

Lipoprotein lipase activity of the fungusRhizopus microsporus

A method is described for the isolation and purification of lipoprotein lipase activity from the culture liquid of the fungusRhizopus microsporus. Some properties of the homogeneous enzyme have been studied: molecular weight 43,000, pI 3.7.Institute of Microbiology, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 373–376, May–June, 1981.     

Purification and characterization of cAMP-factor from Streptococcus agalactiae by hydrophobic interaction chromatography and chromatofocusing

CAMP-factor from Streptococcus agalactiae (group B streptococcus) was purified 60-fold from the culture supernatant to electrophoretic homogeneity in 57% yield. The purification procedure involved ammonium sulphate precipitation, ultrafiltration, hydrophobic interaction chromatography on Octyl-Sepharose and chromatofocusing on polybuffer exchanger PBE 94. The purified CAMP-factor consists of a single polypeptide chain with an apparent molecular weight of 25 kD and an isoelectric point of 8.9. The properties of the CAMP-factor demonstrated by charge-shift electrophoresis were consistent with those of an amphiphilic polypeptide.     

Purification and characterization of hamster hepatic microsomal N,O-acetyltransferase.

A microsomal N,O-acetyltransferase which activates carcinogenic arylacetohydroxamic acids was purified 75-fold from hamster liver sequentially by anion exchange column chromatography, chromatofocusing, gel filtration, and hydroxyapatite column chromatography. The purified enzyme, AT-2, was a glycoprotein with a molecular weight of 60000 and a pI value of 5.4. The N-terminal amino acid sequence of AT-2 was: 60000 and a pI value of 5.4. The N-terminal amino acid sequence of AT-2 was: Asp-Ser-Pro-Ser-Pro-Ile-Arg-Asn-Thr-His-Thr-Gly-Gln-Val-Arg-Gly-Leu-Val- His- Lys-. This sequence was highly homologous to that of the form 2 carboxylesterase of rabbit liver, but not to that of major hepatic microsomal carboxylesterases of hamster and other species. AT-2 catalyzed the hydrolysis of 4-nitrophenyl acetate and the N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene. Both enzyme activities were strongly inhibited by paraoxon, but not by iodoacetamide. These results demonstrate that this N,O-acetyltransferase is a member of carboxylesterase (EC 3.1.1.1).     

Purification and characterization of transglutaminases from the genital tract of the male rat.

In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.     

Small-column chromatofocusing of cerebrospinal fluid and serum immunoglobulin G

Chromatofocusing performed by the Pharmacia fast protein liquid chromatographic system equipped with a specially designed small column was applied for examinations of submilligram quantities of cerebrospinal fluid and serum immunoglobulin G. The separations were based on a pH gradient between 9.5 and 6.0. Mono- or oligoclonal immunoglobulin G components having pI values within the optimal working range of the gradient were easily identified and the findings differed clearly from those of normal immunoglobulin G. The capacity to detect abnormal immunoglobulin G components compared well with previous experiences from the commercially available Mono P column. The small column offers advantages by having a shorter separation time, a decreased dilution of sample in the eluate and a lower consumption of start and eluent buffers.     

Ovine luteinizing hormone. VI. Analysis of the misclassification errors in the separation of intrapituitary isohormones by chromatofocusing.

Luteinizing hormone (LH) in extracts of the ovine (o) anterior pituitary gland elutes as eight or more distinct peaks when analyzed by chromatofocusing on pH 10.5-7 gradients [Keel et al., Biol. Reprod., 36 (1987) 1102]. In order to examine the efficacy of this approach to identify the distinct charge isomers of oLH, a pool of pituitary extracts was de-salted by flow dialysis and chromatofocused on a pH 10.5-7 gradient. The immunoreactive oLH eluted in nine distinct peaks which were coded with letters beginning with the most basic form. The fractions corresponding to each peak were pooled, dialyzed and lyophilized. Each peak was then re-chromatofocused on a pH 10.5-7 gradient except for the immunoreactive oLH eluting in peak A' because of the small amount present in this peak. Each peak, except for F and H, also consisted of a small percentage of immunoreactive oLH associated with adjacent peaks. This was plausible because chromatofocusing does not generally yield baseline resolution of peaks. Peak H eluted in a broad manner and was contaminated with significant amounts of isohormones F, G and Z. In contrast, peaks B, E, F, G and Z almost completely eluted in the anticipated regions. Thus, it appears that analysis of oLH charge isomers by chromatofocusing yields minimal misclassification errors and that the misclassification errors observed are associated with molecular forms which comprise a relatively small percentage of the oLH in pituitary extracts.     

Properties of androsterone-sulfating sulfotransferase in female rat liver

Some properties of androsterone (AD)-sulfating sulfotransferase (ST) present in female rat livers were characterized. Based on the substrate specificities of the enzyme preparation obtained by anion exchange chromatography and 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity chromatography, AD-ST was supposed to be among isoenzymes of hydroxysteroid STs. The identity of the AD-ST with the isoenzymes of hydroxysteroid ST, however, remains unclear at present. The enzyme preparation revealed a wide range of native molecular weight with a major Mr of some 600000. The AD-ST did not appear to have a homogeneous isoelectric point, because the enzymatic activity was spread over a wide range of the pH gradient, centering around pH 6.6 on chromatofocusing. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the AD-ST showed a subunit with Mr of 30000, which was similar to the hydroxysteroid STs purified previously. Under denaturing conditions the subunit was demonstrated to be composed of three protein species containing distinct pI values (pI 6.1, 6.7 and 7.2). The AD-ST was thus supposed to be an oligomer with high molecular weight, in which the subunits of different pI values are assembled in various association numbers.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

Purification and Properties of the diamine oxidase of pea seedlings

Enzymes have been used extensively in many industries for the last 20 yrs. The purpose of this study was the isolation, purification, and specification of diamine oxidase (DAO) of pea seedlings. The relationship between enzyme activity and growth conditions has been investigated. DAO that was extracted from pea seedlings was purified by centrifugation, thermal denaturation, fractionation with ammonium sulfate, precipitation of inert components, column electrophoresis, and DEAE-cellulose column chromatography. It was found that the final enzyme preparation is 400-fold purer than the original extract at the end of the purification steps. The molecular weight, isoelectric point, and copper content of the purified enzyme also were determined.     

Chitinase and beta-N-acetylglycosaminidase in the digestive juice of Helix pomatia.

A beta-N-acetylglucosaminidase from Helix pomatia digestive juice was separated and partly purified by gel chromatography. The optimal pH for the degradation of p-nitrophenyl-N-acetyl-beta-D-glucosaminide was 3.4. The molecular weight was around 160 000 and the pI = 4.95. In the same gel chromatography run two chitinase active peaks were also obtained. These chitinase active peaks were also obtained. These chitinases, with molecular weights around 26 000 and 13 000, had somewhat different pH activity curves with optima at 4.2 and 4.3. By isoelectric focusing the first peak with molecular weight around 26 000 was divided in two chitinase active regions with pI at 5.7 and 3.5. The second peak with molecular weight around 13 000 had a pI at 7.3.     

Separation and purification of uridine diphosphate-glucuronosyltransferases by chromatofocusing on a high-performance liquid chromatograph

A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity

The development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1-0.5; rocket immunoelectrophoresis, 0.5-0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico-chemical differences between different molecular forms of the lipase, e.g. forms with different pI.     

Purification and properties of multiple forms of dihydrodiol dehydrogenase from monkey liver

Two major and two minor forms of dihydrodiol dehydrogenase with similar molecular weights of around 36000 were purified from monkey liver cytosol. All the forms oxidized trans-dihydrodiols of benzene and naphthalene and reduced aromatic aldehydes, but showed differences in charge, specificity for other substrates and inhibitor sensitivity. One major (pI 8.7) and one minor (pI 7.9) form of the enzyme exhibited high activity for alicyclic alcohols and sensitivity to o-phenanthroline. The other major form (pI 6.2) oxidized 3 alpha-hydroxysteroids and was inhibited by dexamethasone and indomethacin, whereas the other minor form (pI 5.8) showed high reductase activity for aldehydes including D-glucuronate and sensitivity to barbital and sorbinil, and cross-reacted with human aldehyde reductase. The results indicate that the multiple forms of monkey liver dihydrodiol dehydrogenase are indanol dehydrogenases, 3 alpha-hydroxysteroid dehydrogenase and aldehyde reductase.