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PubMed1992

Measurement of protein phosphatase activity in biological samples using synthetic phosphopeptides.

A method has been developed for measuring specific protein phosphatase activity in biological samples using synthetic, phospho-Kemptide and phospho-GS-peptide. This method uses ion-exchange chromatography to determine phosphatase activity by quantifying the release of [32P]phosphate directly. The method was used to measure phosphatase activity of rat kidney, adrenals, heart, and liver cytosol and the activity of purified alkaline phosphatases, protein phosphatase 1, and protein phosphatase 2A. Ion-exchange chromatography was also used for the preparation of the radiolabeled phosphopeptide substrates. This method results in high recovery and specific activity of the labeled peptides. These techniques should be useful in isolating and characterizing specific protein phosphatases found in cells.

Animals↗

PubMed1990

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA.

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.

Animals↗

PubMed1987

Phosphopeptides in highly purified calf thymus DNA.

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.

Amino Acids↗

PubMed1981

[Search for phosphopeptides in the feces of axenic rats fed radioactive ovine casein].

Radioactive ovine casein was obtained by injecting 100 microCi of 14C-Ser into the jugular vein of an ewe. The milk collected 17 and 24 h after this injection contained 12 p. 100 of the radioactivity injected in protein form. The seryl residues were specificially labelled. This casein was used as the only protein source fed to axenic rats; 0.30 p. 100 of the tracer ingested was found in the feces of those rats. Since phosphoserine represented 25 p. 100 of the total casein seryl residues, the phosphopeptides may not be selectively unabsorbable.

Animals↗

PubMed1982

In vivo thiophosphorylation of chromosomal proteins. Recovery and analysis of HeLa histones and derivative phosphopeptides.

HeLa cells are shown to incorporate [35S]thiophosphate (added to the medium as sodium thiophosphate) into the gamma-positions of ATP and GTP, and into other nucleotides. Under these conditions there is a transfer of radioactive thiophosphoryl groups to histones H1, H2A, H3, and H4. The newly thiophosphorylated chromosomal proteins can be recovered selectively by affinity chromatography on organomercurial-Sepharose columns. The thiophosphorylated histone H1 and its NH2-terminal and COOH-terminal fragments were subjected to tryptic digestion and the sulfur-derivatized phosphopeptides were isolated by Hg-affinity chromatography prior to electrophoretic separation of different sites of modification. Thiophosphate was found to be incorporated into both serine and threonine residues of histone H1 in HeLa cells during logarithmic growth. When [3H]threonine and nonradioactive thiophosphate were employed simultaneously as precursors, the thiophosphorylated H1 molecules retained on the mercury column also showed the presence of the [3H]threonine label. It follows that newly synthesized H1 molecules are subject to thiophosphorylation in the growing cell cultures.

Adenosine Triphosphate↗

PubMed1995

A new approach to phosphoserine, phosphothreonine and phosphotyrosine synthons and to thiophospho analogs. Stepwise synthesis of mono- and multiphosphorylated phosphopeptides related to src-protein kinase.

Several phosphoserine, phosphothreonine and phosphotyrosine synthons suitable for the stepwise synthesis of phosphopeptides were prepared. Treatment of methylthiomethyl (MTM) esters of either Z-, Boc-, Allocserine and threonine with phosphochloridate in pyridine followed by MgBr2 cleavage of MTM in diethyl ether afforded the title compounds in good yield. Thiophosphoserine and phosphotyrosine synthons were also obtained by the phosphoramidite method using di-(2,2,2-trichloroethyl)-N,N-diisopropylphosphoramidite and MCPBA as oxidizing reagent. Trichloroethyl proved valuable as phosphate protecting group especially in phosphotyrosine derivatives owing to its stability in acidic conditions. These synthons were involved in the liquid-phase synthesis of several phospho and/or thiophosphopeptides related to either src-protein kinase or rat liver pyruvate kinase.

Amino Acid Sequence↗

PubMed1998

Minimum effective dose of casein phosphopeptides (CPP) for enhancement of calcium absorption in growing rats.

We examined the effect of ingested casein phosphopeptides (CPP) on intestinal calcium (Ca) absorption and determined the minimum effective dose for enhancement of Ca absorption under conditions of marginal dietary Ca levels. Male Sprague-Dawley rats, divided upon weaning into five groups, were fed a control diet (isolated soyprotein, ISP; 20%) or a CPP diet (ISP + CPP; 20%, CPP/Ca: 0.1, 0.2, 0.35 and 1.0) for 4 weeks. All diets contained the same amounts of Ca (0.35%) and phosphorus (0.70%). The apparent Ca absorption, the retention of Ca, and the luminal soluble Ca content in the small intestine as determined at 4 weeks in the rats fed CPP diet with a weight ratio of CPP/Ca of more than 0.2 were significantly (p < 0.05) higher than those in the rats fed control diet. The wet weight, length and Ca content of the femur were not significantly different among the groups. These results indicate that the minimum effective dose of CPP for enhancement of Ca absorption is 0.7 g/kg or a weight ratio of CPP/Ca of 0.2 in the ISP diet and that CPP supplementation has the effect of significantly increasing Ca absorption at least under conditions of marginal dietary Ca levels.

Administration, Oral↗

PubMed1994

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture.

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozen-thawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca2+. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca2+ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca2+ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca2+ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.

Amino Acid Sequence↗

PubMed1987

Evidence for covalent binding between phosphopeptides and terminal deoxynucleotides in highly purified calf thymus DNA.

Highly purified DNA from calf thymus nuclei (N-DNA) was found to cleave after reaction with a chelating agent and subsequent dialysis. During the cleavage phosphopeptides (PPs) were released into the dialysates. At the end of the cleavage, approximately one half of the PP material remained with the DNA. Since it was so strongly bound, it was considered to be retained in the DNA structure by covalent bonding. In order to confirm this, a commercial DNA (S-DNA) was ultrasonicated and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. DEAE Sephacel chromatography of the digested material yielded 5 fractions. The fraction 2, having the highest proportion of proteinaceous material, was digested with Pronase. Amino acid analysis of the hydrolysis mixture yielded phosphoserine (Pser), asp, thr, ser, glu, gly, ala, val, ile, leu, and arg. The mixture was chromatographed again on DEAE Sephacel. From this a single fraction, number 5, was found to contain both deoxynucleotides and the amino acids, Pser, asp, ser, glu, and gly in a molar ratio of greater than 7:3:2:2:5. The mixture obtained by hydrolysis of this fraction with snake venom diesterase was again chromatographed on DEAE Sephacel. This fractionation gave two main peaks, one corresponding to the same 5 amino acids and the other to deoxynucleotide material. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between the deoxynucleotide and amino acid portions.

Amino Acids↗

PubMed1993

Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase. A study with src and cdc2 derived phosphopeptides.

Spleen TPK-IIB is an acidophilic protein tyrosine kinase, devoid of autophosphorylation activity, whose phosphorylation of the src-peptide NEYTA is crucially specified by Glu-2[(1991) J. Biol. Chem. 266, 17798-17803]. We show that phosphothreonine, phosphotyrosine and phosphoserine are, in this order, specificity determinants even more effective than glutamic acid if they are replacing Glu-2, to give the phosphopeptides NTpYTA, NYpYTA, NSpYTA, respectively. Non-phosphorylated threonine, tyrosine and serine are conversely ineffective. Consequently also the heptapeptide GEGTYGV reproducing the phosphoacceptor and inhibitory site of p34cdc2 is not appreciably affected by TPK-IIB, unless its threonyl residue is previously phosphorylated, the phosphoderivative GEGTpYGV being readily phosphorylated at its tyrosyl residue. Such a behaviour is unique for TPK-IIB among the protein tyrosine kinases tested (lyn-TPK, fgr-TPK and EGF-receptor, besides TPK-IIB). These data provide the first evidence that, in some instances, the targeting by protein tyrosine kinases can be specifically determined by the previous phosphorylation of the peptide substrate, thus extending the concept of 'hierarchal phosphorylation' [(1991) J. Biol. Chem. 266, 14139-14142] to tyrosyl residues as well.

Amino Acid Sequence↗

PubMed2001

Dephosphorylation of sodium caseinate, enzymatically hydrolyzed casein and casein phosphopeptides by intestinal alkaline phosphatase: implications for iron availability.

Clusters of phosphoserine residues in casein bind iron with high affinity. Casein inhibits iron absorption in humans but partial hydrolysis of casein prior to ingestion diminishes this inhibition. The objective of this study was to test two hypotheses: 1. Partial hydrolysis of the peptide bonds in casein exposes phosphoserine residues to attack by intestinal alkaline phosphatase (IAP). 2. Hydrolysis of the phospho-ester linkage in phosphoserine residues in casein by IAP releases bound iron or inhibits iron chelation, thereby allowing its absorption. Test of hypothesis 1: Suspensions of sodium caseinate (SC), enzymatically hydrolyzed casein (EHC), and casein phosphopeptides (CPP) were subjected to an in vitro pepsin/pancreatin digestion and subsequently incubated in the presence of calf IAP. The rate of release of inorganic phosphate was measured with the following results (expressed as &mgr;mol phosphate released/unit of IAP/min): 0.081, 0.104, 0.139 for SC, EHC, and CPP, respectively. These results are consistent with hypothesis 1. Test of hypothesis 2: (59)Fe-citrate or (59)Fe-citrate + CPP in minimum essential media were spiked with a Na(2)WO(4) solution or water (Na(2)WO(4) is a known inhibitor of IAP) and placed on Caco-2 cell monolayers. Uptake of (59)Fe by the cells was used as an index of iron bioavailability. Na(2)WO(4) did not affect (59)Fe uptake from samples containing only iron but did slightly inhibit (by 10%) uptake from samples containing iron + CPP. These results are consistent with hypothesis 2 and provide a possible explanation for the observation that partial hydrolysis of casein improves iron bioavailability.

Journal Article↗

PubMed2004

Structure of the BRCT repeats of BRCA1 bound to a BACH1 phosphopeptide: implications for signaling.

The recognition of the phosphorylated BACH1 helicase by the BRCA1 C-terminal (BRCT) repeats is important to the tumor suppressor function of BRCA1. Here we report the crystal structure of the BRCT repeats of human BRCA1 bound to a phosphorylated BACH1 peptide at 2.3 A resolution. The phosphorylated serine 990 and phenylalanine 993 of BACH1 anchor the binding to BRCA1 through specific interactions with a surface cleft at the junction of the two BRCT repeats. This surface cleft is highly conserved in BRCA1 across species, suggesting an evolutionarily conserved function of phosphopeptide recognition. Importantly, conserved amino acids critical for BACH1 binding are frequently targeted for missense mutations in breast cancer. These mutations greatly diminish the ability of BRCA1 to interact with the phosphorylated BACH1 peptide. Additional structural analysis revealed significant implications for understanding the function of the BRCT family of proteins in DNA damage and repair signaling.

BRCA1 Protein↗

PubMed2006

Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate.

Casein phosphopeptide amorphous calcium phosphate nano-complexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to re-mineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to re-mineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2.0 or 5.0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples re-mineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The re-mineralizing effect of CPP-ACP in milk was dose-dependent with 2.0 and 5.0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0.001). In conclusion, this study shows that the addition of 2.0-5.0 g CPP-ACP/l to milk substantially increases its ability to re-mineralize enamel subsurface lesions.

Administration, Buccal↗

PubMed1999

Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases.

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.

Amino Acid Sequence↗

PubMed1995

Phosphotryptic peptide analysis of the human androgen receptor: detection of a hormone-induced phosphopeptide.

Phosphorylation of the androgen receptor (AR) in human prostate tumor cells (LNCaP) is increased by androgens. The AR is expressed as two isoforms with apparent molecular masses of 110 and 112 kDa. Metabolic labeling experiments with [32P]orthophosphate revealed that only the 112 kDa isoform is radioactively labeled. Phosphoamino acid analysis revealed only phosphorylation on serine residues. Phosphotryptic peptide analysis of human AR protein by two-dimensional peptide mapping and by reverse-phase HPLC showed phosphorylation at multiple sites. Comparison of phosphopeptide maps of AR protein from cells incubated in the absence or presence of the synthetic androgen R1881 indicated that the ligand-stimulated phosphorylation is probably due to induction of phosphorylation at a new site rather than increased phosphorylation at an existing site. This result suggests that hormone-dependent AR phosphorylation might play a role in the signal transduction pathway of androgens.

Androgens↗

PubMed2006

Bovine beta-casein (1-28), a casein phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via Toll-like receptor 4.

This study was conducted to elucidate the target cells and receptors which participate in the mitogenic and interleukin (IL)-6-enhancing effect of bovine beta-casein (1-28), a casein phosphopeptide. When the spleen lymphocyte subset (CD4+, CD8+, and CD19+ cells) from C3H/HeN mice was cultured with the beta-casein (1-28), it exerted a dose-dependent mitogenic effect on CD19+ cells. The effect of beta-casein (1-28) was not apparent in the case of CD19+ cells from C3H/HeJ mouse. In addition, the effect was significantly inhibited by treating the C3H/HeN mouse-derived CD19+ cells with neutralizing antibody for toll-like receptor 4 (TLR4). Reverse transcription-polymerase chain reaction analysis showed that the beta-casein (1-28) exerted an IL-6-enhancing effect on the CD19+ cells. The effect was also abrogated in either C3H/HeJ mouse-derived CD19+ cell culture or the anti-TLR4 antibody-added culture. These results suggest that the beta-casein (1-28) stimulates both proliferation and IL-6 expression of CD19+ cells via TLR4.

Animals↗

PubMed2002

General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways.

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.

Peptides↗