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PubMed1991

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.

Animals↗

PubMed2000

Indirect determination of captopril by AAS.

An indirect method is described for the determination of captopril (KPL) in pharmaceutical preparations by atomic absorption spectrometry (AAS). The procedure is based on the complexation of KPL with an excess of Pd(II) ion. The unreacted Pd(II) was resoluted on a cationic ion-exchanger resin, while Pd(II)-KPL sequestrate was not retained. The effluent Pd(II) sequestrade was measured by AAS. The absorbance is found to increase linearly with increasing KPL concentration, because the amount of Pd(II) is related to the concentration of KPL, which is corroborate by the calculated correlation coefficient value of 0.9939. The system obeys Beer's law for 1-40 microg ml(-1), S.D. was found to be 0.039 (n = 5). The Pd(II)-KPL complex was obtained in the solid phase. Characterization of the complex was performed by elemental analysis, TG, conductance measurements and IR, 1H-NMR spectroscopy.

Angiotensin-Converting Enzyme Inhibitors↗

PubMedDate not supplied

[Aminoacid levels in Werding-Hoffmann and Kugelberg-Welander diseases (author's transl)].

The concentration of free aminoacids in plasma and urine were estimated in 10 patients suffering from Werdnig-Hoffmann's disease of long duration. The age of the patients was between 5 and 14 years. Estimations were also made in 10 patients with Kugelberg-Welander's disease aged between 11 and 34 years. The aminoacid concentrations were estimated on samples of plasma and 24 hours samples of urine by means of chromatography on ion-exchange resins. The data obtained were compared respectively with groups of thirty and ten healthy subjects of the same age. In the group of patients with Werdnig-Hoffmann's disease a significant increase of taurine (p less than 0.001) and of glutamic acid (p less than 0.001) was found in the plasma. The urinary excretion of glutamine was increased in the same group of patients (p less than 0.001) and in the group with Kugelberg-Welander's disease (p less than 0.005). These aminoacid levels are interpreted as an expression of a reduced oxygen metabolism and increased proteolysis in the skeletal muscles in conditions of chronic denervation.

Adolescent↗

PubMed1981

Differential assay for urinary catecholamines by use of liquid chromatography with fluorescence detection.

I describe a relatively simple and rapid procedure for quantitative analysis for unconjugated urinary norepinephrine, epinephrine, and dopamine. Catecholamines are isolated from urine on a carboxylic acid ion-exchange resin (Bio-Rex 70) and eluted with 0.8 mol/L boric acid. The eluates are assayed for the individual catecholamines by "high-performance" liquid chromatography with an octadecylsilane reversed-phase column and 10 mmol/L perchloric acid-acetonitrile (99/1) as the mobile phase. The catecholamines are detected by measuring their intrinsic fluorescence on excitation at 200 nm. The method is highly reproducible, the coefficient of variation for normal concentrations in urine being 4.4% for norepinephrine, 8.8% for epinephrine, and 4.8% for dopamine. Moreover, it appears to be practically free of interference by drugs or endogenous compounds in urine. The range of catecholamine concentrations found in 143 hypertensive patients agrees well with previous studies.

Catecholamines↗

PubMed1997

Fully automated measurement of total iron-binding capacity in serum.

We established a method for fully automated measurement of total iron-binding capacity (TIBC) in serum without separation of the unbound excess iron after saturating serum transferrin. After saturation of serum transferrin with an excess amount of iron (first step), the unbound iron was eliminated by formation of a complex with ferrozine, which was used as a chromogenic reagent (second step). For the TIBC assay, iron dissociated from transferrin by shifting the pH to acidic was reacted with ferrozine, and the increase in the absorbance at 570 nm was measured (third step). Because the iron used as a calibrator, which was added to saturate transferrin, reacted completely with ferrozine in the second step (elimination of unbound iron), the change in the absorbance to generate a calibration factor could not be monitored in the third step. To solve this problem, we used N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA) to complex with the iron added to saturate transferrin in the second step. This made it possible to form an iron-ferrozine complex at acidic pH because iron was dissociated from HEDTA at acidic pH. The within-run CVs of this method were 0.66-2.43% at 17.7-77.0 mumol/L, and the day-to-day CVs were 1.06-1.57% at 29.9-60.4 mumol/L (n = 10). The correlation between the values obtained with this method (y) and those from the direct TIBC assay, which involved removal of unbound iron by ion-exchange resin (x), was: y = 0.963x + 0.29 mumol/L (r = 0.973, Sy/x = 2.83, n = 59), and with the TIBC values calculated from the serum iron concentrations and the unbound iron-binding capacities measured by a direct colorimetric method (x) was: y = 1.01x - 1.06 mumol/L (r = 0.994, Sy/x = 1.66, n = 51).

Blood Proteins↗

PubMed1990

Metabolic fate of [13N]ammonia in human and canine blood.

Nitrogen-13- ([13N]) ammonia is a widely used tracer for PET myocardial blood flow studies. Quantification of blood flow using tracer kinetic principles requires accurate determination of [13N]ammonia activity in blood. Since [13N] ammonia is rapidly metabolized, the arterial input function may be contaminated by labeled metabolites. We, therefore, characterized the 13N-labeled metabolites in blood after intravenous (i.v.) injection of 20 mCi [13N]ammonia in nine healthy volunteers. Utilizing a series of ion exchange resins, 13N-labeled compounds were separated into four groups: ammonia, neutral amino acids, acidic amino acids, and urea. Analysis of the metabolic fate of [13N]ammonia indicates that over 90% of the blood activity within the first two minutes after injection is present as [13N]ammonia. However, there is considerable contamination of the blood activity at 3-5 min by [13N]glutamine (amide) and urea, which collectively represent 18%-50% of the blood activity. Thus, correction of the arterial input function for 13N-metabolites is required to accurately quantify the arterial input function of [13N]ammonia in myocardial blood flow studies.

Adult↗

PubMed1973

Studies on mono- and diiodohistidine. I. The identification of iodohistidines from thyroidal iodoproteins and their peripheral metabolism in the normal man and rat.

The problem as to whether iodohistidines are normally biosynthetized in thyroglobulin and thyralbumin has been examined both in man and the rat. Evidence has been obtained for the first time that diiodohistidine (DIH) is present in both species in these two iodoproteins. The biosynthesis of monoiodohistidine (MIH) in the thyroglobulin of the normal rat has been confirmed and extended to rat thyralbumin and to human thyroid iodoproteins. THE IODOHISTIDINE IDENTIFICATION IS BASED ON FIVE ORIGINAL METHODS INCLUDING: (a) the preparation of stable and radioiodine-labeled iodohistidines; (b) the protection of the labile iodohistidines during the iodoprotein enzymatic hydrolysis; (c) the isolation of iodohistidines by ion-exchange resin chromatography; (d) their separation from each other and from iodinated cationic butanol-insoluble compounds by Sephadex G-10 chromatography; and (e) their purification by successive crystallizations to a constant specific activity. Iodohistidine levels (in percent of protein radioactivity from iodide given in vivo) were found comparable in man and the rat. However, the values (mean +/-SE) for thyroglobulin (MIH, 0.61+/-0.10%; DIH, 0.050+/-0.015%) and for thyralbumin (MIH, 2.61+/-0.57%; DIH, 0.28+/-0.09%) differ significantly (P < 0.05). Iodohistidines are stable during in vitro exposure to iodotyrosine dehalogenase preparations. In contrast to iodotyrosines the iodohistidines when given in vivo to man either orally or intravenously were in large part recovered in 24-h urines.

Administration, Oral↗

PubMed1989

Screening tests for porphobilinogen are insensitive. The problem and its solution.

Standard screening tests for porphobilinogen (PBG) do not turn positive until the concentration of PBG exceeds 10-20 times the upper limit of normal. The authors have developed a screening procedure that uses a new ion-exchange resin to separate PBG from interfering substances in urine. On addition of Ehrlich's reagent, the color is more intense than that produced in the Watson-Schwartz test, and its spectrum more closely resembles that of pure PBG. By measuring the absorbance of this solution at 555 nm, it is possible to discriminate between urine samples with 9 mumol/L (2 mg/L) of PBG and those with no detectable PBG. The new screening test had positive results in two patients with latent acute intermittent porphyria; the Watson-Schwartz test had negative results in both cases. This procedure is easy to perform, has much greater sensitivity than the Watson-Schwartz test, and uses objective spectrophotometric data to separate positive from negative results.

Benzaldehydes↗

PubMed1981

Transplantation in miniature swine. IX. Swine histocompatibility antigens: isolation and purification of papain-solubilized SLA antigens.

Histocompatibility antigens have been purified by papain treatment of crude cellular membranes obtained from spleen and mesenteric lymph nodes of individual miniature swine that are homozygous at the major histocompatibility complex. The purification protocol included ultracentrifugation, gel filtration, ion-exchange chromatography, and in some instances preparative isoelectric focusing. This procedure was applied to the preparation of sLA antigens from the 3 haplotypes available in our partially inbred miniature swine herd. The purity of the SLAdd molecules was evidenced by: 1) a single activity peak of approximately 50,000 daltons on gel-permeation chromatography; 2) the antigen elution as a symmetrical peak from ion-exchange resins and from isoelectric focusing columns; 3) the presence of 2 predominant polypeptide chains on SDS-PAGE at apparent m.w. 43,000 and 13,000 daltons; and 4) specific immunoprecipitations by alloantisera and by anti-beta 1 microglobulin antisera. The antigenic activity of SLA products was analyzed by inhibition of complement-mediated cytotoxicity and by removal on anti-beta 2-microglobulin affinity columns. Heavy and light chains were separated by gel-filtration on Sephacryl S-200 in 6 molar guanidine. These SLA products, which have thus been shown to be pure by a variety of criteria, are now readily available for structural analysis and for analysis of biologic activity.

Animals↗

PubMed1993

In patients receiving dopamine infusions for treatment of shock do free radicals convert dopamine to 6-hydroxydopamine?

Free hydroxyl radicals react with dopamine in vitro to produce the neurotoxin 6-hydroxydopamine (6-OHDA). If 6-OHDA were produced in vivo it could destroy central and/or peripheral neurons. Free radicals are produced in patients with cardiovascular or toxic shock. These patients are often treated with intravenous dopamine infusions. Using a newly developed method for measuring 6-OHDA in biological samples, we have examined blood from 15 patients receiving dopamine infusions for treatment of shock. 6-OHDA neither binds to nor elutes from either alumina or ion-exchange resins; therefore we used ODA-silica (Sep-Pak) to prepare samples for HPLC analysis with electrochemical detection. 6-OHDA disappeared rapidly from whole blood or serum at room temperature but was stable at -70 degrees C in an HClO4 extract. Recovery from Sep-Pak was 95% and detection limit 10 nmol/L. We repeated the experiments that showed in vitro hydroxyl radical conversion of dopamine to 5- and 6-OHDA. We detected neither 5- nor 6-OHDA in plasma samples from patients receiving dopamine infusions at 10-28 micrograms kg-1 min-1. Plasma dopamine in these patients was 1-5 mumol/L. We conclude that any 6-OHDA produced by hydroxyl radical attack in vivo is rapidly scavenged by endogenous substances containing sulfhydryl groups.

Chromatography, High Pressure Liquid↗

PubMed1983

The intracellular chloride activity of rat kidney proximal tubular cells.

The intracellular Cl- activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl- sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measure VCl and using - in separate experiments - conventional KCl-filled microelectrodes to measure the membrane potential, Vm. After correction for interference from other anions on VCl the intracellular Cl- activity averaged 13.1 mmol X l-1 SD +/- 4.5 mmol X l-1 (n = 96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl- activity of 90-103 mmol X l-1 and Vm of -71.2 mV, SD +/- 4.9 mV (n = 23) to amount to 6.3 to 6.7 mmol X l-1. Since both cell membranes are permeable for Cl- ions, as concluded from luminal and/or peritubular Cl- substitution experiments, we conclude that the cellular Cl- accumulation above equilibrium results from transcellular active Cl- transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl- concentration after substitution of luminal Cl- by gluconate, however, we deduce that transcellular Cl- absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl- flux is passive and paracellular.

Animals↗

PubMed1998

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme.

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.

Amino Acid Sequence↗

PubMed1985

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms.

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.

Adenosine Triphosphate↗

PubMed1989

Multiple chromatographic forms of the formylpeptide chemoattractant receptor and their relationship to GTP-binding proteins.

The radiolabeled formylpeptide chemoattractant receptor, partially purified by wheat germ agglutinin-Sepharose chromatography, eluted in three distinct peaks when chromatographed on DEAE-Fractogel. Incubation of the lectin-Sepharose purified receptor with 100 microM guanosine 5-(3-O-thio) triphosphate markedly altered the distribution of the radiolabeled receptor when chromatographed on the ion exchange resin. Peaks 2 and 3 were reduced by approximately 50% while peak 1 was concomitantly increased. Western blot analysis revealed the presence of a 40 kDa GTP-binding protein alpha-subunit only in peak 3. Incubation of Western blots with [alpha-32P]GTP detected low molecular mass GTP-binding proteins (24 and 26 kDa) that coeluted with the receptor in peak 2. Incubation of the peak 1 receptor fractions with fractions containing a mixture of GTP binding proteins resulted in the generation of peaks 2 and 3 when chromatographed on DEAE-Fractogel. These results demonstrate that the chromatographic behavior of the formylpeptide receptor is dependent upon its association with GTP binding proteins and that more than one type of GTP-binding protein may be involved.

Cell Line↗

PubMed1973

Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methylthymidine.

1. DNA was treated with N-methyl-N-nitrosourea at pH7-8, 37 degrees C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-(14)C-labelled N-methyl-N-nitrosourea and use of [(14)C]thymine-labelled DNA. 2. The synthesis of O(4)-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O(4)-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed.

Animals↗

PubMed2005

The regeneration and recycle of chromium etching solutions using concentrator cell membrane technology.

The regeneration of chromium (VI) and the recovery of etched copper from chromium etching solutions by electrodialysis is improved by the addition of a concentrator cell in the catholyte chamber. The concentrator media used are ion-exchange resins or activated carbon cloth. The maximum percentages for the regeneration of chromium and recovery of copper in these systems is however less than 80% and 90% respectively because of the competition between the processes of oxidation of Cr(III) and electrodeposition of copper. A novel combination of electrolysis with electrodialysis and concentrator cell technology is developed that achieves 92% chromium regeneration and 90% copper recovery.

Chromium↗

PubMedDate not supplied

Some basic properties of sheep liver cAMP-dependent protein kinase.

Sheep liver cytosol (105,000 X g supernatant) yields two major peaks of protein kinase by using DEAE-Trisacryl M as an ion-exchange resin at pH 7.0. Peak I (Type-I), corresponding to 30-50% of the total activity, is not retained by the column at a starting ionic strength of ca. 0.06 M, while Peak-II (Type-II) is eluting at 0.17 M ionic strength. Both peaks are found to be dependent on cAMP and are active on histone (ATP: Protein phosphotransferase, EC 2.7.1.37). Kms apparents for histone and ATP are 1.5 +/- 0.5 mg/ml and 16 +/- 4 microM, respectively, for PrK-I while that of PrK-II are 1.8 mg/ml and 28.6 microM, respectively. Both enzymes are found to be stable for two weeks at 4 degrees C. Molecular weight determination of crude extract (105,000 X g supernatant) show three peaks corresponding to the molecular weights of 251,000; 131,800 and 43,600.

Adenosine Triphosphate↗

PubMed1983

Intracellular chloride in retinal neurons: measurement and meaning.

Intracellular chloride activity measurements were obtained from mudpuppy retinal neurons using dual microelectrodes, one of which was made chloride-selective by filling the tip with chloride liquid ion exchange resin. In addition ionic substitution experiments were carried out in the perfused retina-eyecup preparation of the mudpuppy. A comparison of the membrane potential and calculated chloride equilibrium potential shows that retinal neurons differ in relative transmembrane chloride distribution. Ganglion cells have an ECl more negative than the resting membrane potential, whereas amacrine cells have a passive distribution of chloride. Horizontal cells have chloride distributed such that an increase in chloride conductance is depolarizing. On-bipolars have a chloride distribution similar to that of horizontal cells whereas off-bipolars show either passive distribution or some chloride accumulation. These findings are consistent with the idea that chloride ions may play a role as a depolarizing driving force for electrogenic activity of horizontal cells and on-bipolars. Results with Cl substitution experiments are consistent with this interpretation.

Animals↗