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At least 19 recordsLinked to original sources
PubMed1980

High-performance liquid chromatography of acid-stable and acid-labile phosphoamino acids.

A high-performance liquid chromatographic system has been developed which permits the separation of both acid-stable and acid-labile phosphoamino acids. An anion-exchange resin and two buffers of different ionic strength and near neutral pH are used. A low-ionic-strength buffer is used for the separation of N-omega-phosphoarginine and N-epsilon-phospholysine, while the higher-ionic-strength buffer permits the clear separation of tau-phosphohistidine, omicron-phosphoserine and omicron-phosphothreonine. An in-stream fluorometric detection system using omicron-phthalaldehyde permits the rapid analysis of samples containing as little as 25 pmoles of phosphoamino acid. This method has been applied to the detection of tau-phosphohistidine from alkaline digests of chemically phosphorylated calf thymus histone 4 and bovine myelin basic protein.

Amino Acids↗
PubMed2004

Synthesis of fluorine-containing bioisosteres corresponding to phosphoamino acids and dipeptide units.

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine-containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF(2)) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF(2)-substituted pThr mimetics and development of a two-step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that gamma,gamma-difluoro-alpha,beta-enoates were reduced to gamma-fluoro-beta,gamma-enoates by organocopper reagents and then applied to the synthesis of (Z)-fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper-mediated reduction led us to development of a SmI(2)-mediated approach toward the synthesis of the fluoroalkene isosteres.

Biomimetic Materials↗
PubMed1985

Acid phosphatase and phosphoamino acid phosphatases in murine erythroleukaemic cells.

The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM).

Acid Phosphatase↗
PubMed1989

Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins.

Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1991

Distribution and contents of free O-phosphoamino acids in animal tissues.

This study was carried out to investigate the distribution and contents of O-phosphoserine (P-Ser), O-phosphothreonine (P-Thr), and O-phosphotyrosine (P-Tyr) as their free forms in animal tissues. After extraction of a tissue sample with trichloroacetic acid, these O-phosphoamino acids were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and then quantitated by GLC, with flame photometric detection, on a DB-1701 capillary column. Using this method, nanogram levels of O-phosphoamino acids in tissue samples could be accurately and precisely determined without any interference by coexisting substances. Free P-Ser and P-Thr were widely found in animal tissues, pancreas, spleen, stomach, kidney, liver, and lung containing considerable amounts of these O-phosphoamino acids. On the other hand, free P-Tyr was not detected in any of the tissues investigated in this study.

Animals↗
PubMed2005

Synthesis of nucleoside N-phosphoamino acids and peptide formation.

Nucleoside N-phosphoamino acids were synthesized through Atherton-Todd reaction of nucleoside H-phosphonate with amino acids, and their structures were confirmed by NMR and ESI-MS. After nucleoside N-phosphoamino acid was incubated in anhydrous methanol at 40 degrees C for 72 h, di- to tetra-peptide derivatives were detected by ESI-MS, and their structures were further identified by multistage mass spectrometry. These and previously published studies in aqueous solution suggest that nucleoside N-phosphoamino acids could have been prebiotic precursors of oligopeptides.

Magnetic Resonance Spectroscopy↗
PubMedDate not supplied

Analysis of free and bound O-phosphoamino acids in urine by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the analysis of free and bound forms of O-phosphoamino acids, such as O-phosphoserine, O-phosphothreonine and O-phosphotyrosine, in urine samples by gas chromatography (GC). For free O-phosphoamino acid analysis, the urine sample was extracted with trichloroacetic acid and run through an ion exchange column. For total (free plus bound) O-phosphoamino acid analysis, the urine sample was hydrolysed in acid and base in order to release the O-phosphoamino acid from peptides or proteins. O-Phosphoamino acids in these prepared samples were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and then measured by GC with flame photometric detection (FPD-GC). The calibration curve was linear in the range of 10-500 ng for each O-phosphoamino acid, and the detection limits were 30-80 pg as injection amounts. O-Phosphoamino acids in the urine samples could be selectively determined by the FPD-GC method without any influence from coexisting substances. The recoveries of O-phosphoamino acids added to urines and urine hydrolysates were 90-98% and relative standard deviations were 1.5-8.0%. By using this method, we investigated an age dependence of O-phosphoamino acid excretion in normal subjects.

Amino Acids↗
PubMed1986

Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives.

Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.

Amino Acids↗
PubMed2004

Determination of phosphoamino acids derivatized with 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein by micellar electrokinetic chromatography.

A sensitive analytical protocol for determining phosphoamino acids using capillary electrophoresis coupled with laser-induced fluorescence detection has been developed. The technique involved the derivatization of the phosphoamino acids with fluorescent reagent 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the analyses of the derivatives by micellar electrokinetic chromatography with laser induced fluorescence detection (MEKC-LIF). Different variables that affect derivatization (DTAF concentration, pH, temperature and time) and separation (kind of surfactant, pH and concentration of buffer) were studied. The baseline separation of three phosphoamino acids could be obtained in less than 11 min with good reproducibility. There was a linear relationship between the peak area of the analyte and its concentration, with correlation coefficients in the range of 0.9979-0.9997. The concentration detection limits (signal to noise = 3) with respect to each single phosphoamino acid were in the range of 0.5-1 nM. The developed method was successfully applied for the determination of phosphoamino acids in the hydrolyzed phosphorylated protein samples.

Buffers↗
PubMedDate not supplied

Phosphoamino acids in proteasome subunits.

Proteasomes, the major catalysts of the non-lysosomal proteolytic pathway in eukaryotic cells, were analyzed for their content of phosphoamino acids using polyacrylamide gel electrophoresis and subsequent detection on Western blots by phosphoamino acid antibodies. No specific binding to proteasome subunits was observed with phosphoserine or phosphothreonine antibodies, whereas phosphotyrosine antibodies were bound by a single proteasome subunit, which was identified in rat as well as in human proteasomes as subunit C7-1. Since dephosphorylation of the subunit by phosphatases was not possible, analysis of phosphoamino acid content of all proteasome subunits was performed using another method. All proteasome subunits were isolated from 2D-polyacrylamide gels and subjected to partial acid hydrolysis. Phosphoamino acids were subsequently detected by capillary electrophoresis after their derivatization with phenylisothiocyanate. This analysis revealed no phosphorylated amino acid in subunit C7-1, however, subunit C3 contained phosphotyrosine and phosphothreonine, and phosphoserine was detected in subunits zeta, C5, C8 and C9.

Amino Acids↗
PubMed1988

Determination of [32P]phosphoamino acids in protein hydrolysates by isocratic anion-exchange high-performance liquid chromatography.

We have developed a simple method for phosphoamino acid analysis of 32P-labeled phosphoproteins using anion-exchange high-performance liquid chromatography (HPLC). Phosphoproteins undergo partial acid hydrolysis and the resulting hydrolysate is injected directly onto a column. The sample is then isocratically eluted from the column by 35 mM phosphoric acid at pH 3.0 with collected fractions analyzed by Cerenkov counting. Phosphoamino acid identification is accomplished by the comparison of the retention times of 32P-labeled peaks to retention times of phosphoamino acid standards which had been monitored at 206 nm. This method has greater sensitivity and is more reliable than cellulose thin-layer electrophoresis and the results obtained by high-efficiency Cerenkov counting can be evaluated immediately, instead of waiting days or weeks for autoradiographic development of cellulose plates. This HPLC protocol is an improvement over other published HPLC protocols in that there is no need for pre- or post-column derivatization and the free [32P]phosphate elutes long after the phosphoamino acids. Thus sensitivity is increased as there is no interference from the free phosphate. Selection of an HPLC anion-exchange column is critical for this separation. Only two of the four columns that we tested performed well. We present data from several phosphoproteins including calcium-calmodulin dependent protein kinase, the beta-subunit of the insulin receptor, and phosphorylated calmodulin to demonstrate the utility of this procedure.

Buffers↗
PubMed1991

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.

Amino Acids↗
PubMed1985

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography.

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.

Chromatography, High Pressure Liquid↗
PubMed1998

Problems with phosphoamino acid analysis using alkaline hydrolysis.

Although a seemingly simple concept, sample volume and the reaction vessel size are important considerations when undertaking an alkaline hydrolysis of a phosphoprotein, particularly if the phosphoamino acid of interest is either phosphohistidine or phosphotyrosine. It should be noted that the experiments conducted in this article used large concentrations of both phosphotyrosine and the most stable form of phosphohistidine (8), which highlights the problems that may be faced during phosphoamino acid analysis of a protein sample that contains only small amounts of either phosphoamino acid. Although not tested, it is likely that similar hydrolysis effects may occur for phospholysine. If the reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane blot or in solution, then the use of a mineral oil overlay should be considered to prevent concentration of the alkali and hydrolysis of the phosphate moiety.

Alkalies↗
PubMed1985

Rapid separation of phosphoamino acids including the phosphohistidines by isocratic high-performance liquid chromatography of the orthophthalaldehyde derivatives.

Amino acids were derivatized with orthophthalaldehyde and separated by high-performance liquid chromatography on a polymer-based reverse-phase column (Hamilton PRP-1) at pH 7.2 using isocratic elution with 14.3 mM sodium phosphate, 1.1% tetrahydrofuran, 6.6% acetonitrile. Phosphorylated amino acids were eluted with baseline resolution in the following order: 1-phosphohistidine, phosphoserine, 3-phosphohistidine, phosphotyrosine, phosphothreonine, and phosphoarginine. Each of the phosphoamino acids was separated from its parent amino acid but aspartate and glutamate eluted in the same region as the phosphoamino acids. The sensitivity is in the picomole range and the separation time, injection to injection, is 15 min. The linearity for phosphothreonine extends at least from 30 pmol to 30 nmol. Quantitation by radioactivity is good for each of the phosphoamino acids except in the case of [1-32P]phosphohistidine, which coelutes with inorganic phosphate.

Aldehydes↗
PubMed1996

Analysis of the phosphoamino acid content of phosphoproteins.

A method has been developed for the analysis of phosphoserine, phosphothreonine and phosphotyrosine in 32P-phosphoprotein hydrolysates. The hydrolysates are treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After a clean-up using a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC aluminum sheets using a one-dimensional solvent system. The method is straightforward and permits the simultaneous analysis of numerous samples. Very clean chromatograms are obtained enabling the unambiguous identification of the well separated dabsylated phosphoamino acids with autoradiography. The phosphoamino acids can be quantified by simply cutting out the relevant spots from the aluminum sheets followed by 32P-quantification using liquid scintillation spectrometry.

Amino Acids↗
PubMed2003

Characteristic fragmentation behavior of phosphoamino acid conjugates with 3'-azido-3'-deoxythymidine by electrospray ionization tandem mass spectrometry.

The conjugates of phosphoamino acids with 3'-azido-3'-deoxythymidine were synthesized and their structures were determined by various spectral methods. In positive and negative ion electrospray mass spectrometry (ESI-MS), the fragmentation pathways were investigated in conjunction with tandem mass spectrometry (MS/MS). The results showed that there were very different characteristic fragment ions in the positive ion MS/MS spectra and the negative ion MS/MS spectra.

Molecular Structure↗
PubMed1989

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Animals↗