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At least 1,153 records · Page 64Linked to original sources
PubMed1987

Fluorometric determination of streptomycin in serum by high-performance liquid chromatography using mobile phase containing fluorogenic reagent.

A new postcolumn derivatization method for the fluorometric determination of streptomycin in serum by high-performance liquid chromatography is described. The serum was treated with 3.5% perchloric acid to precipitate proteins and the supernatant was directly injected into the chromatograph. Streptomycin was separated by reversed-phase, ion-pair chromatography with a mobile phase containing ninhydrin as a fluorogenic reagent, octanesulfonate, and 1,2-ethanedisulfonate as counterions, and was detected by fluorescence using continuous-flow, postcolumn derivatization in an alkaline stream with ninhydrin in the mobile phase. This method is sensitive to 1.0 microgram/ml using only 100 microliter of serum. Comparison with a fluorescence polarization immunoassay gave a good correlation coefficient of 0.976.

Calibration↗
PubMed1988

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing.

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.

Amino Acid Sequence↗
PubMed1988

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components.

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.

Acetone↗
PubMed1988

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography.

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.

Animals↗
PubMed1988

Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography.

The status of the oxidative metabolism of L-tryptophan is usually evaluated by the determination of tryptophan metabolites in serum or urine and/or the activities of various oxidative enzymes in tissues. I have developed assays for serum kynurenine and hepatic tryptophan dioxygenase (TDO) activity based on the determination of kynurenine (KYN) by isocratic, reverse phase HPLC with spectrophotometric detection at 365 nm. Sample pretreatment prior to HPLC requires little more than perchloric acid precipitation of serum or a TDO incubation mixture. The analytical recovery for the serum assay was 101 +/- 2%, while the run-to-run coefficient of variation at normal KYN levels was approximately 8%. Serum KYN levels in 40 apparently healthy fasting humans were normally distributed and ranged from 0.27 to 0.69 microgram/ml (mean +/- SD: 0.47 +/- 0.1). Serum KYN in predialysis specimens from a group of 20 patients with chronic renal failure demonstrated a highly significant increase (mean +/- SD: 0.83 +/- 0.35 microgram/ml; P less than 0.001) as compared to the reference population. It is possible that such an increase might contribute to the pathophysiology of the uremic state. The analytical recovery of KYN from TDO incubation mixtures was approximately 90%. There was no evidence for the onward metabolism of KYN during the assay of whole liver homogenates. The mean (+/- SD) TDO activity of rat liver homogenates preincubated with ascorbate and hematin was 2.3 +/- 0.8 mumol/h/g wet wt (30 degrees C). The sensitivity, specificity, and convenience of these two methods suggest that they are suitable for routine use in the investigation of the biology and pathology of oxidative tryptophan metabolism.

Animals↗
PubMed1989

Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography.

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 M perchloric acid or 0.5 N sodium hydroxide containing 50% (v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 X 150 mm) column with 0.1 M potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.

Adenosine Diphosphate↗
PubMed1989

A spectrophotometric method for the estimation of amino groups on polymer supports.

A simple and sensitive method for the estimation of polymer-supported amino groups is reported. The polymer support is treated either with N-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate or 2,4-dinitrophenyl-4-O-(4,4'-dimethoxytrityl)-butyrate and a catalytic amount of 4-dimethylaminopyridine. After removal of the excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the solid support into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption (epsilon 498 = 70,000/M) at 498 nm, is determined spectrophotometrically. A comparative study of these reagents with N-succinimidyl-3-(2-pyridyldithio)-propionate, 4,4'-dimethoxytrityl chloride, and sodium 2,4,6-trinitrobenzenesulfonate methods is also included.

Acylation↗
PubMed1990

Analysis of aldehydic lipid peroxidation products in rat liver and hepatocytes by gas chromatography and mass spectrometry of the oxime-tert-butyldimethylsilyl derivatives.

A method for analysis of aliphatic aldehydes in biological samples is described. Cyclohexanone is added as internal standard and the samples are treated with hydroxylamine and perchloric acid. The oximes are extracted and converted to the oxime-tert-butyldimethylsilyl derivatives, which are quantitated by capillary gas chromatography and identified by mass spectrometry. The characteristic M-57 fragment ions in the mass spectra enabled a rapid identification of the derivatives of the aldehydes, alkanals, alk-2-enals, alka-2,4-dienals, and 4-hydroxyalk-2-enals, which in addition gave rise to characteristic double peaks in the gas chromatographic analysis. The method was applied to analysis of autoxidized arachidonic acid, ADP-Fe3(+)-treated rat hepatocytes, and rat liver given a single dose of ethanol, 5 g/kg. The amounts of hexanal and 4-hydroxynon-2-enal were not increased 6 h after the administration of ethanol.

Adenosine Diphosphate↗
PubMed1990

A spectrophotometric method for the estimation of polymer-supported sulfhydryl groups.

A sensitive and simple method is described for the quantitative determination of free sulfhydryl (-SH) groups on polymer supports. The method includes the reaction of 4,4'-dimethoxytrityloxy-S-(2-thio-5-nitropyridyl)-2-mercapto ethane (DTNPME) with polymer-supported sulfhydryl groups. After removal of excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the polymer support into the solution. The dimethoxytrityl cation (lambda max = 498 nm, epsilon 498 = 70,000/M) is then quantified spectrophotometrically. A comparative study of the reagent DTNPME with 2,2'-dithiobis(5-nitropyridine) is also described.

Chromatography, Affinity↗
PubMed1991

Fluorometric determination of plasma adenosine concentrations using high-performance liquid chromatography.

Methods are described for the fluorometric determination of plasma adenosine concentrations, using HPLC. Plasma obtained from blood of dogs treated with erythro-(2-hydroxy-3-nonyl)adenine hydrochloride and dipyridamole was deproteinized with perchloric acid and the neutralized sample was put sequentially onto a SepPak C18 and boronic acid affinity column. Subsequently, adenosine in the final elution was converted to 1,N6-ethenoadenosine and was quantitated by HPLC with a fluorescence detector. The percentage recovery of adenosine added to the deproteinized plasma was nearly 100%. In the adenosine deaminase treated plasma, the increase in adenosine concentration of even 4 nM can be accurately determined. The control renal venous plasma concentrations of adenosine in anesthetized dogs were 19.9 +/- 1.9 nM, a significantly higher value than the corresponding arterial concentrations (12.7 +/- 1.1 nM), thereby suggesting the renal release of adenosine. This release was markedly enhanced following the removal of the renal arterial occlusion. Thus, taken together with the in vivo results, the present method is sensitive, hence most useful for the determination of plasma adenosine concentrations.

Adenosine↗
PubMed1984

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cultured fibroblasts.

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/micrograms DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/micrograms DNA). After incubation of fibroblasts with [3H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [35S]sulfate resulted in the incorporation of [35S]sulfate into 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [35S]sulfate and [3H]glucosamine from 5 min to 16 h showed that [35S]PAPS was equilibrated in less than 10 min, while [3H]glucosamine required a longer time, 2-4 h, to attain a steady state with UDP-N-acetylhexosamine. [14C]Glucose required approximately the same time as [3H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.

Adenine Nucleotides↗
PubMed1986

Energy metabolism in rat brain in vivo studied by 31P nuclear magnetic resonance: changes during postnatal development.

The energy metabolism in rat brains during postnatal development was followed by in vivo 31P NMR. Using a small surface coil (from several to 10 mm in diameter) placed at the head of a conscious rat, high-energy phosphate compounds in the brain and the steady-state kinetics among them were measured. The cellular contents of some phosphate compounds changed widely during the period of postnatal cell growth from age 10 to 20 days. During the same period, the cellular activity of creatine kinase increased by a factor of more than 5 as measured by a saturation transfer technique. The in vivo value of the creatine/creatine phosphate ratio was estimated from the in vitro value (in perchloric acid extracts), assuming that the in vivo ratio of the creatine and creatine phosphate pool over the ATP and ADP pool was the same as the corresponding in vitro value. From the creatine/creatine phosphate ratio thus obtained, the value of the cytosolic ATP/ADP ratio was estimated for brains of adult rats and neonate rats. Unexpectedly the value in the latter was found to be smaller.

Adenosine Diphosphate↗
PubMed1989

Stability and mutual conversion of enamel apatite and brushite at 20 degrees C as a function of pH of the aqueous phase.

By calculation, apatite is more soluble than brushite at low pH and less soluble at high pH. Apatite, therefore should be able to transform spontaneously to brushite at low pH and brushite to apatite at high pH. The aim was to describe this mutual conversion as related to the aqueous phase composition. Powdered enamel, brushite or 1:1 mixtures of the two salts were suspended in distilled water for up to 12 weeks at 20 degrees C, pH was adjusted to 4, 5, 6, 7, 8, 9 or 10 by drops of perchloric acid or potassium hydroxide. The calcium and the phosphate concentrations and pH were determined, and the nature of the calcium phosphate powder was examined by X-ray diffraction. At pH 8 and above, brushite was invariably converted to apatite, whilst it was transformed to octacalcium phosphate at pH 7. In the pH range 6-4 brushite was not converted to apatite and enamel apatite was not transformed to brushite spontaneously within 2 months. In the enamel apatite suspensions, the apatite ion product altered with pH, which explained why apatite did not transform to brushite at pH 5-4. At pH 3.7, however, the enamel apatite was converted to brushite. No transformation of apatite to brushite was identified in apatite-brushite mixtures at pH 6-4. Supplementary experiments showed that ethanol, used as an agent for removal of water, salted out the water-free dicalcium phosphate, monetite.

Apatites↗
PubMed1975

Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase.

The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA.

Adenosine Triphosphate↗
PubMed1977

Staining of proteins after isoelectric focusing in gels by new procedures.

Four procedures are described for staining of protein bands after isoelectric focusing in polyacrylamide gels. In one procedure, Coomassie Brilliant Blue R 250 is used whereas in the others the G 250 form is used in an aqueous solution of perchloric acid. By using isoelectric focusing and the new staining procedures it is possible to detect less than 0.2 microng of many proteins. One of the procedures is especially rapid, allowing detection of proteins without destaining. Procedures for preservation of the protein patterns and determination of proteins by scanning in a densitometer are also described.

Evaluation Studies as Topic↗
PubMed1980

The isolation of three new high mobility group nuclear proteins.

In addition to the four high mobility group non-histone chromosomal proteins HMG 1, 2, 14 and 17 and histone H1, perchloric acid extracts of nuclei contain a number of other smaller low molecular weight proteins. Three of these proteins (HMG 18, 19A, and 19B) have been purified and characterized. Protein HMG 18 has high lysine and alanine contents, resembling histone H1. Proteins HMG 19A and 19B have high contents of basic and acidic amino acids and resemble HMG proteins 1, 2, 14 and 17. N-terminal sequence analyses of the proteins show that they are not degradation products of histones or the other HMG proteins. However, there are sequence similarities between HMG 18 and histone H5, and between HMG 19B and HMG 17, supporting the view that the HMG proteins and the lysine-rich histones are functionally related.

Amino Acid Sequence↗
PubMed1985

Thiophosphorylation and phosphorylation of chromatin proteins from calf thymus in vitro.

Thiophosphorylation and phosphorylation of 5% perchloric acid extractable proteins from calf thymus chromatin were studied using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. The phosphorylation reaction catalyzed by nuclear protein kinase II utilized [gamma -35S]ATP as a phosphate donor almost as efficiently as [gamma -32P]ATP, but the cGMP-dependent protein kinase mediated phosphorylation by [35S]ATP was about 20 times less effective than that by [32P]ATP. In addition, using [35S]ATP instead of [32P]ATP changed markedly the cGMP-dependent phosphorylation pattern of the PCA-extractable proteins as examined by gel electrophoresis. Thus, depending on the type of protein kinase, the results from thiophosphorylation and phosphorylation reactions may vary considerably.

Amino Acids↗
PubMed1991

A 31P-NMR study on the energy state of rat liver in an experimental model of chronic dietary iron overload.

31P-NMR spectroscopy of rat liver perchloric acid extracts was utilized to assess the hepatic energy state in an experimental model of chronic dietary iron overload. Oral administration of iron for a period of 65 days that induces a steady ten-fold increase in hepatic iron concentration causes a significant decrease in the hepatic ATP level not associated with appreciable modifications of ADP and Pi levels. The phosphorylation ratio appears on the average decreased. The values of the energy state parameters revert to the normal if the concentration of iron in the liver is reversed below the critical level upon withdrawal of iron treatment after 45 days for a period of 20 days. The implication of these energy modifications for the pathogenesis of cell damage in the siderosis is discussed.

Adenosine Diphosphate↗