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.     

Two-dimensional separation of phosphoamino acids from nucleoside monophosphates

A comparative analysis of the migration of phosphoamino acids and nucleoside monophosphates in three different two-dimensional systems is presented. The three phosphoamino acids studied are phosphoserine, phosphothreonine, and phosphotyrosine, which are the residues most commonly occurring in proteins phosphorylated by protein kinases. Their migration properties have been compared to those of UMP, AMP, CMP, GMP, and TMP, which are the basic components of nucleic acids. Special attention has been paid to the behavior of UMP, which has previously been reported to often co-migrate with phosphoamino acids. Also, the migration of inorganic orthophosphate and ribose monophosphate, which are frequently present in samples derived from macromolecule hydrolysis, has been analyzed. The following separating systems have been used: double chromatography, electrophoresis followed by chromatography, and double electrophoresis. The latter is shown to have the best resolving power and to be the most convenient system.     

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.     

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.     

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.     

Phosphorylation of histidine in proteins by a nuclear extract of Physarum polycephalum plasmodia

A high salt nuclear extract from the true slime mold Physarum polycephalum was used as a source of kinase activity for the incubation of calf thymus histones with [gamma-32P]ATP. A major proportion of the 32P incorporated into histones was acid-labile and alkali-stable. The nature of the alkali-stable phosphorylated component was analyzed by subjecting the phosphorylated protein to total alkaline hydrolysis and separating the resultant phosphoamino acids by anion exchange chromatography. The 32P-labeled material co-chromatographed with phosphohistidine standards and did not co-chromatograph with phosphoserine, phosphothreonine, or phosphotyrosine standards. In similar experiments using reversed phase high-performance liquid chromatography to separate the phosphoamino acids, the 32P-labeled phosphoamino acid behaved like the 1-isomer of phosphohistidine, in not being retained by the column, and unlike 3-phosphohistidine, phosphoserine, phosphothreonine, phosphotyrosine, and phosphoarginine, which were all retained on the column. Histone H4 was a good substrate for the histidine kinase activity and the location of the phosphorylated histidine residue was probed by peptide mapping using chymotrypsin or V8 protease. Both maps were consistent with labeling of histidine 75 and inconsistent with labeling of histidine 18. The data show that Physarum nuclei contain a major kinase activity which produces phosphohistidine. The methods we have developed for studying this kinase activity provide the basis for a complete characterization of the structure and function of the Physarum enzyme and can be applied to the study of similar kinase activities in other systems.     

Suppressive role of endogenous regucalcin in the regulation of protein phosphatase activity in rat renal cortex cytosol

The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.     

Use of electroblotting to detect and analyze phosphotyrosine containing peptides separated by two-dimensional gel electrophoresis

A technique to detect and analyze phosphotyrosine containing peptides after separation of total cellular proteins by two-dimensional gel electrophoresis is described. This is achieved by electroblotting of proteins on nylon membranes followed by alkali treatment. In comparison with direct alkali treatment of the polyacrylamide gel, this procedure is easier to perform; avoids the diffusion of proteins out of the gel during alkali treatment; allows a more precise localization of phosphotyrosine containing peptides on the untreated membrane; and is less time consuming with respect to extraction of proteins for phosphoamino acid analysis.     

Capillary electrophoresis of phosphorylated amino acids with fluorescence detection

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5mM NDA, 40 mM NaCN and 20mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0mM beta-cyclodextrin (beta-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 x 10(-9)M, 5.6 x 10(-9)M and 7.2 x 10(-9)M, respectively (S/N=3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With beta-casein as the analysis protein, this method was successfully validated.     

Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase.

The motif Ser-Ser-Ser-Glu-Glu is readily phosphorylated by casein kinase-2 (CK-2), a growth-related protein kinase whose consensus sequence is Ser(Thr)-Xaa-Xaa-Glu(Asp) [(1990) Biochim. Biophys. Acta 1054, 267-283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK-2 phosphorylation, the phosphotyrosyl peptide Ser-Ser-Ser-TyrP-TyrP actually being a substrate more efficient than Ser-Ser-Ser-Glu-Glu itself both in terms of Km (0.69 vs 2.43 mM) and Vmax. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK-2. While Ser-Ser-Ser-TyrP-TyrP is a better substrate than Ser-Ser-Ser-SerP-SerP, which in turn is better than Ser-Ser-Ser-Glu-Glu, Ser-Ser-Ser-ThrP-ThrP is a less efficient substrate than Ser-Ser-Ser-Glu-Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK-2 appears to be TyrP greater than SerP much greater than ThrP.     

Autophosphorylation of the insulin-like growth factor I receptor cytoplasmic domain

The cytoplasmic domain of the beta subunit of the insulin-like growth factor I receptor (amino acids 936-1337) was overexpressed in Sf9 insect cells using a baculovirus expression system, and the 6-His tagged receptor was purified by metal-affinity chromatography. Autophosphorylation of the receptor was concentration dependent, consistent with a trans phosphorylation mechanism. Phosphoamino acid analysis of the autophosphorylated receptor showed predominantly phosphotyrosine, but phosphoserine and phosphothreonine were also present. However, when the receptor was further purified by gel filtration on Sephadex G-100 and then autophosphorylated, phosphoamino acid analysis showed only phosphotyrosine. We conclude that the IGF-I receptor tyrosine kinase is not a dual-specificity kinase and that autophosphorylation of the beta subunit is by a trans mechanism.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Molecular heterogeneity of creatine kinase isoenzymes

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Phosphorylation of tyrosine in cultured human placenta

The [³²P]phosphoamino acids in proteins of first-trimester and term-cultured human placentas have been separated and their relative amounts have been measured. Significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2–4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [³²P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first-trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.     

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.     

Phosphorylation of tyrosine in cultured human placenta

The [³²P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2–4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [³²P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Role of nuclear PKC delta in mediating caspase-3-upregulation in Jurkat T leukemic cells exposed to ionizing radiation

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.     

Induction of acid phosphatase activity during germination of maize (Zea mays) seeds.

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.