An evaluation of the use of Sep-Pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.     

Quantitative recovery, selective removal and one-step purification of human parotid and leukemic lysozymes by immunoadsorption

Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti-(human leukemic lysozyme) IgG to epoxy-activated Sepharose 6B. Lyophilized parotid saliva (21) was resuspended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non-adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino-terminal analyses, and immunoelectrophoretic analysis. The one-step purification procedure yielded a 1370-fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques.     

Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography.

A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [14C]HMG CoA was used as the substrate and the product formed, i.e., [14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05 M. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.     

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.     

Separation of oligomannose-type sugar chains having one to five mannose residues by high-performance liquid chromatography as their pyridylamino derivatives

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.     

Cytokinin and inhibitor activities in the avocado fruit mesocarp

No cytokinin activity was found in methanolic mesocarp extracts after purification with petroleum ether and ethyl acetate. Cytokinin activity did appear, however, in aqueous fraction after acid hydrolysis or passage through Dowex 50 (H⁺) ion exchange columns. The level of this activity, the fruit growth rate, and the cell division rate were found to be positively correlated (with each other).     

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.     

Two-dimensional high-performance liquid chromatographic determination of 5-hydroxyindoleacetic acid and homovanillic acid in urine

The biomedically and neurochemically important compounds 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) have been simultaneously determined in human urine after reverse-phase two-dimensional high-performance liquid chromatography. A 10-fold-diluted urine sample (20 microliters) is first separated on a C18 column (30 X 0.39 cm) using an 85% pH 6.0 phosphate buffer/15% methanol solvent system. The elution volume containing both 5-HIAA and HVA (Rt approximately 3 min) is collected. Recoveries (mean +/- SD) for this purification step, which is monitored using fluorometric detection, were usually above 90%. After acidification of the approximately 2 ml collected fraction, 100 microliters is reinjected on a C18 column and separated (Rt: 5-HIAA, 4 min; HVA 5.5 min) using an 80% pH 3.5 phosphate buffer/20% methanol mobile phase. The compounds are determined by flow-through amperometry with absolute detection limits of approximately 25 pg. Both 5-HIAA and HVA are well resolved from other electroactive species present and are easily determined at normal and greatly reduced concentrations in human urine.     

哺乳类(兔)红细胞分化因子的纯化及特性分析

本文报道从哺乳动物(兔)红细胞分离、提纯红细胞分化因子(ErythroidDifferentiation Factor,简称EDF及对其性质的分析。按Bishop方法给新西兰兔注射盐酸苯肼(2.5％)0.3mL/kg体重/日连续6days致贫血,收集血液用生理盐水洗去白细胞后,得到的网织红细胞用冻融法使细胞裂解,用乙醇、氯仿选择性变性除去血红蛋白,凝胶过滤,连续经DEAE-纤维素及CM-纤维素柱层析,取得较原裂解液纯化了31 000倍的活性物质EDF,产率为9.1％,分子量15kD,在SDS-PAGE电泳图中为单一蛋白带。EDF对热相对稳定,100℃处理10min仍保持一定活性,对蛋白酶敏咸而对DNA酶I及RNA酶不敏感。EDF对体外培养细胞的生长抑制作用已在体外实验体系得到验证。     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB2 by HPLC on silica columns is reported.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB₂ by HPLC on silica columns is reported.     

Inhibition of chick embryo DNA ligase by dATP: its use in enzyme purification.

Highly purified chick embryo DNA ligase (EC.6.5.1.1) obtained in our laboratory using classical methods, mainly column chromatographies shows a bimodal pH activity and an high affinity inhibition by dATP. A single step passage of crude extract containing DNA ligase through an anion exchange resin (Dowex AG₁X₂) saturated with dATP allows an important purification of the enzyme retained on the column at pH 7.5 and eluted at pH 8.6. Specific activity of the purified enzyme preparation is more than 600 fold higher than that of the crude extract. Analysis of the eluant by polyacrylamide gel electrophoresis shows a main protein containing the enzyme activity.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

粘虫咽侧体静止激素的初步分离纯化

用放射化学的方法,检测粘虫Mythimna separata幼虫脑提取物中咽侧体静止激素(Allatostatin,AS)样的活性物质。发现粘虫幼虫脑中含有AS样的活性物质,可抑制离体咽侧体(Corporaallata,CA)的保幼激素(Juvenile hormone,JH)的生物合成。用1个脑当量的幼虫脑提取物,对CA的JH合成的抑制率达51%。幼虫脑提取物经胰蛋白酶水解后,AS活性显著降低,表明幼虫脑中的活性物质是肽类或蛋白质类物质。幼虫脑提取物用Sep-Pak柱初步纯化,有活性的组分经高压液相色谱(HPLC)分离,洗脱组分的AS活性测定表明,有AS活性的组分主要集中在组分1～20和组分30～60,其中对离体CA的JH合成的抑制大于50%的组分有3、5、11、40、54和60。     

Two-step high-performance liquid chromatography method for the determination of myo-inositol and sorbitol

A simple two-step HPLC method for the separation and quantitation of myo-inositol and sorbitol in extracts of glomeruli from rat kidneys is described. The limit of detection is 2 ng. The procedure involves fractionation of the sugar alcohols on a Waters Sugar Pak column, preparation of the p-nitrobenzoate derivatives, and further purification with quantitation by absorbance at 254 nm using a Waters mu Porasil column. The applicability of the procedure to determination of sorbitol and myo-inositol in biological samples was demonstrated by the finding of marked alterations in sorbitol and myo-inositol content of glomeruli isolated from diabetic compared to that from normal rat kidneys.     

Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres

The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

The gill sialic acid content as an index of environmental stress in rainbow trout, Salmo gairdneri, Richardson

The gill sialic acid content in Rainbow Trout exposed to several aqueous concentrations of ammonia NH₃ and/or to a high environmental pH was determined by two different methods. The results showed Warren's method was adequate for the biological material, even without purification of the extracts by Dowex 2.8 resin. A significant increase of the sialic acid concentration occurred in fish exposed to ammonia.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure

We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.