The multicomponent analysis of estrogens in urine by ion exchange chromatography and GC-MS--II. Fractionation and quantitation of the main groups of estrogen conjugates

A method for the metabolic profiling of estrogen conjugates in urine is described. It mainly involves protection of carbonyl functions by ethoximation, solid extraction on Sep-Pak C18 cartridges, a number of ion exchange chromatographic steps and quantitation by capillary GC or GC-MS. The acetate form of DEAE-Sephadex is used to initially separate estrogen conjugates into four groups; unconjugated, monoglucuronides, monosulfates and double conjugates. Monoglucuronides are further subfractionated to A- and D-ring glucuronides by carbodiimide methylation of the carboxylic functions and chromatography on the free base form of DEAE-Sephadex. Double conjugates are subfractionated to disulfates and sulfoglucuronides by solvolysis and chromatography on the acetate form of DEAE-Sephadex. After the appropriate enzymatic hydrolysis or solvolysis procedures the liberated free estrogens are purified and fractionated by a series of anion exchange chromatographic steps. Finally, following trimethylsilyl ether derivatization estrogens are analysed by capillary GC or GC-MS. The method permits the quantitation of the main conjugates of all the important estrogen metabolites including catechol estrogens. The method is precise, the sensitivity depending on the quantitation mode employed GC or SIM GC-MS. The method was applied to seven late pregnancy urines the values of which are presented.     

Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

外切葡聚糖纤维二糖水解酶的分离纯化和部分性质研究

纤维素酶系包括内切葡聚糖酶,外切葡聚糖纤维二糖水解酶和葡萄糖昔酶三类酶．近年纤维素酶的结构与功能方面的研究有了很大进展[1～3]．目前,已有百余个不同菌种来源的纤维素酶的基因得到了克隆和表达,但主要为Trichodermareesei和Trichndermaviride产生的纤维素酶．TrichodemapseudokiningiiS-38是我们分离得到并易于培养的高产酶菌株[4]．我们采用固态发酵方法培养了该菌株,从发酵液中提纯了两个外切酶（cellobiohydrolase,EC3．21．91,CBH）组分,并测定了它们的部分酶学性质．1材料和方法1．1对硝基酚纤维二糖（P-nitrophenyl…     

The preparation of pregnancy urine for an estrogen profile.

A method is described for purifying the estrogen content of pregnancy urine with little loss of the labile estrogens. The procedure makes use of the initial 50-fold purification effected by their precipitation whith ammonium sulphate, with simultaneous elimination of most urinary corticosteroids and 50--60% of urinary ketosteroids. It also employs the antioxident ascorbic acid as an additive in most stages of the procedure. The mild organic-solvent-HIO partition system of Brown is used for separating the strongly polar, 2including all "labile" estrogens, and of the weakly polar estrogens, from neutral steroids. The remaining neutral steroid still interfering with the assays were removed by an ascorbic acid treated ion exchange resin (AG 1). The final residues were revealed by mass-spectroscopy to consist almost solely of estrogens. Gas-liquid chromatography in which just 2 chromatograms are required yields a total of 12 "estrogen" peaks (for 12 estrogens which are excreted in amounts greater than 0.1 mg/day) in normal pregnancy urine, including all the known labile estrogens. Identification as estrogen for all but a few minor peaks of the gas chromatogram was obtained by mass-spectroscopy. The practical significance of the method lies in the fact that some labile estrogens are much more important in the estrogen metabolism of pregnant and nonpregnant women than heretofore generally thought.     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

The multicomponent analysis of estrogens in urine by ion exchange chromatography and GC-MS--I. Quantitation of estrogens after initial hydrolysis of conjugates

A method for the multicomponent analysis of estrogens in urine after initial hydrolysis of the conjugates is described. Following protection of the carbonyl functions by ethoximation, estrogen conjugates were extracted on Sep-Pak C18 cartridges and purified on the acetate form of DEAE-Sephadex. The samples were subsequently hydrolysed by Helix pomatia juice and the hydrolysate was purified on the acetate form of QAE-Sephadex. Estrogens with vicinal cis-hydroxyls and diphenolic compounds were fractionated on the borate and bicarbonate form of QAE-Sephadex, respectively. Neutral steroids were removed by the free base form of DEAE-Sephadex after which estrogens were separated into two groups using Lipidex 5000 in a straight phase system. Following trimethylsilyl ether derivatization estrogens were analysed by selected ion monitoring (SIM). The method allows the quantitation of all the important estrogen metabolites including catechol estrogens. It is precise, accurate and sensitive permitting the quantitation of estrogens in urine of males and non-pregnant females.     

Apoplastic β-1, 3-Glucanases in Leaves of Tomato Systematically Infected by TMV

Apoplastic β-1, 3-glucanase was purified from leaves of tomato (Lycopersicon esculentum Mill. ) which were systematically infected by TMV (tobacco mosaic virus). The enzyme obtained through -20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, DEAE-Sephadex A-25 ion exchange chromatography and Sephadex G-75 gel filtration, showed homogeneity in PAGE, and SDS-PAGE which had two isoenzymes of 27 kD and 36 kD. The enzyme hydrolysed laminarin at an optimum pH of 4.8--5.2 and was stable between pH 4--8 and at an optimum temperature between 30--40℃, and stable at 40℃ after 1 hour of incubation, It had a Km of 9. 2 mg/mL. SDS-PAGE profiles of the proteins in the tomato leaf intercellular fluid had the bands of 22 kD, 27 kD and 36 kD that were β-1, 3-glucanases.     

Immunological and biochemical properties of metallothioneins of golden hamster,mouse and rat

Summary Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.     

Respiratory behaviour of sea-urchin spermatozoa. II. Sperm-activating substance obtained from jelly coat of sea-urchin eggs.

A sperm-activating substance (SAS) was obtained from the jelly coat of sea-urchin ova and its chemical properties were investigated in three sea-urchin species. The SAS was partially purified from the jelly coat of Pseudocentrotus eggs through several steps of purification by procedures consisting of charcoal adsorption, ion-exchange chromatography on DEAE-Sephadex A-25 column, and gel-filtration on Sephadex G-15 columns. The partially purified SAS was found to contain a ninhydrin-positive material and is inactivated by pronase digestion. The molecular weight of SAS was estimated as about 630 by gel-filtration through Sephadex G-25 and the isoelectric-point of SAS is located at about pH 5.3 by isoelectrofocusing method. The SAS is non-volatile, alcohol-soluble, and labile in a diluted alkaline or acid solution. The origin of SAS is discussed.     

Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

乳酸菌Enterococcuse faecalis TN-9低温蛋白酶的提纯及性质

对产自乳酸菌Enterococcuze fecalis TN-9的蛋白酶,进行了硫酸铵沉淀,DEAE—Sephadex A-25以及DEAE Cellulofine A-500离子交换层析的3步纯化和特性研究。纯化酶Native PAGE显示1条蛋白带。SDSPAGE和凝胶层析分子量分别为30ku及69ku。纯化酶最适作用温度为30℃,最适作用PH为7．5～8．0,在pH6．0～9．5和45℃以下条件下稳定,在0℃下显示了6．1％的相对活性,60℃以上热处理完全失去酶活。该酶被EDTA-2Na,Hg^2＋、Cu^2＋、Ni^2＋、Ag^2＋、Co^2＋及Pepstatin A不完全抑制。Zn^2＋对蛋白酶具有明显的激活作用。纯化酶作用于偶氮酪蛋白的Km和Vmax分别为0．098％和72mg／（h·mg）。该酶为N末端VGSEVTLKNS的明胶酶（Gelatinase）的一种,性质属于低温蛋白酶。     

Purification of a Latent Form of Polyphenoloxidase from La France Pear Fruit and Its Pressure-activation

A latent form, mostly soluble, of polyphenoloxidase of La France pear fruit (Pyrus communis) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography with DEAE-Sephadex A-25 and then DEAE-Toyopearl 650M, followed by gel permeation chromatography with Toyopearl HW-55s. The addition of 10% glycerol to the eluting buffer was needed for purification. The purified latent enzyme seemed to be a monomeric protein; the molecular weight was estimated to be 70,000 by gel permeation chromatography and 65,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was activated by pressurization at 400 MPa or higher or by treatment with SDS. The highest activity was obtained by pressurization at 600 MPa and 20°C for 6 h.     

The synthesis of [A 19-3-iodotyrosine] and [A 19-3,5-diiodotyrosine]insulin (porcine)

The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.     

Immunochemical characteristics of Streptococcus mutans serotype h carbohydrate antigen

Serotype h carbohydrate antigen was prepared from cell walls of Streptococcus mutans strain MFe28 of monkey origin. The h antigen was extracted from the cell walls with 5% trichloracetic acid at 4 C, and purified by DEAE-Sephadex A-25 ion exchange chromatography followed by Sephacryl S-300 gel filtration. The purified antigen was composed of galactose (75%), glucose (16%), and rhamnose (3%). Although the antiserum against whole cells of S. mutans MFe28 gave a strong cross reaction with serotype d S. mutans, serotype h-specific antiserum could be obtained by adequate adsorption. The precipitin reactions and hapten inhibition test using serotype h-specific antiserum showed that galactose, glucose, and their derivative sugars were markedly potent inhibitors. It was concluded that the serotype h antigen is immunologically distinguishable from the known serotypes of S. mutans, although it is closely related to serotype d antigen of S. mutans.     

A new inhibitor of elastase from the sea anemone (Anemonia sulcata)

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.     

Purification and some physicochemical properties of slow-reacting substance of anaphylaxis (SRS-A) from sensitized guinea pig lung.

The slow-reacting substance of anaphylaxis (SRS-A) generated by antigen challenge of sensitized guinea pig lung fragments was partially purified and the physicochemical properties of this activity were studied. The SRS-A recovered from antigen challenged lung preparations of 600 animals was used for the purification procedure. Treatment with organic solvents, extraction with 80% ethanol, Sephadex LH-20 chromatography with 80% ethanol, and DEAE-Sephadex A-25 chromatography in 60% methanol eluted with 0.0 to 0.1 M NaCl in 60% methanol was the purification sequence finally adopted. Overall recovery of SRS-A bioactivity was 60% with a specific activity of 2.52 units/ng of dry weight. This represented a 1.67 million-fold purification over the starting material. The DEAE-Sephadex A-25 step alone provided a 7600-fold purification. This highly purified SRS-A had an apparent molecular weight of 380 to 400 daltons. The bioactivity was acid labile and alkaline stable and was blocked by low concentrations of the SRS-A antagonist FPL 55712. The SRS-A was thermostable in aqueous media and displayed enhanced bioactivity after heating at 60 C for 60 min. These results indicate that we have developed a highly efficient new approach to the isolation of guinea pig SRS-A, which also may be useful in the study of SRS-A from other tissues or species. The physicochemical properties of guinea pig SRS-A appear to be very similar to those of SRS-A from other species.     

Physicochemical properties of 57 kDa thyroid hormone binding protein from rat liver nuclei

The nuclear thyroid hormone binding protein (NTHB) with the molecular weight of 57 kDa was obtained from rat liver nuclear extracts by using HPLC and DEAE-Sephadex A-25 ion exchange chromatography methods. Fluorescein isothiocyanate-labeled 3,5,3'-triiodo-L-thyronine (F-T3) was used as a fluorescent probe to identify the hormone binding protein. Purified NTHB has a single binding site for T3 with the apparent binding constant of (3.3 +/- 0.7) X 10(8) M-1. NTHB is an acidic protein with a pI of 5.0. The secondary structure of NTHB is characterized by about 42% helical and 18% beta-structure from CD measurements.     

海巴戟果水溶性多糖的分离纯化及清除自由基活性

采用热水提取乙醇沉淀获得海巴戟水溶性粗多糖,经DEAE-Sephadex A-50分离纯化得海巴戟果水溶性多糖（MOCI）。经纸层析、醋酸纤维薄膜电泳和SepharoseCl-4B柱层析纯度鉴定表明,MOCI为相对分子质量均一的纯多糖。抗衰老活性研究结果表明,MOCI能有效清除.OH和O2^-.自由基。     

Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro

1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro.