Biomedical subjects

E C Reynolds

Publications and source records attributed to E C Reynolds.

At least 55 records · Page 3Linked to original sources

PubMed1996

Characterization of a Porphyromonas gingivalis gene prtR that encodes an arginine-specific thiol proteinase and multiple adhesins.

Cysteine proteinases of Porphyromonas gingivalis have been implicated as major virulence factors in the development of periodontitis. Several groups have reported the characterisation of similar genes encoding the same arginine-specific thiol proteinase from P. gingivalis; however, the reported size and structure of the genes have varied. We report here the complete nucleotide sequence of the gene prtR that encodes a polyprotein containing the Arg-specific proteinase and multiple haemagglutinins/adhesins. The nascent polyprotein consists of a putative leader sequence and a prosequence followed by the 45 kDa Arg-specific proteinase and 44, 15, 17 and 27 kDa sequence-related adhesins in that order. The size and structure of the prtR are consistent with the size of the mRNA transcript (5.3 kb) and the size and sequences of the individual protein components purified from P. gingivalis.

Adhesins, Bacterial↗

PubMed1996

Fluoride content of infant formulae in Australia.

The prevalence of dental fluorosis in Australia and the United States of America has increased in both optimally fluoridated and non-fluoridated areas. This has been attributed to an increase in the fluoride level of food and beverages through processing with fluoridated water, inadvertent ingestion of fluoride toothpaste, and the inappropriate use of dietary supplements. A major source of fluoride in infancy is considered to be infant formula which has been implicated as a risk factor for fluorosis in a number of studies. In this study the fluoride content of the infant formulae commonly used in Australia was determined. The acid diffusible fluoride of each powdered formula was isolated by microdiffusion and measured using a fluoride ion-specific electrode. The fluoride content of milk-based formulae ranged from 0.23 to 3.71 micrograms F/g and for soy-based formulae from 1.08 to 2.86 micrograms F/g. When reconstituted, according to the manufacturer's directions, with water not containing fluoride, the formulae ranged in fluoride content from 0.031 to 0.532 ppm, with the average fluoride content 0.240 ppm. Using average infant body masses and suggested volumes of formula consumption for infants 1-12 months of age, possible fluoride ingestion per kg body mass was estimated. None of the formulae, if reconstituted using water containing up to 0.1 ppm F, should provide a daily fluoride intake above the suggested threshold for fluorosis of 0.1 mg F/kg body mass. However, if reconstituted with water containing 1.0 ppm F they should all provide a daily fluoride intake of above the suggested threshold for fluorosis with intakes up to 2-3 times the recommended upper 'optimal' limit of 0.07 mg/kg body mass. Under these conditions the water used to reconstitute the formulae would provide 65-97 percent of the fluoride ingested. These figures are likely to be overestimates due to the intake of nutrients from other sources reducing formulae consumption and also due to the lower bioavailability of fluoride from milk-based formulae. Further, it is generally believed that the maturation stage of enamel formation is the critical period for fluorosis development by chronic, above-threshold fluoride exposure. The maturation stage for the anterior permanent teeth, however, is after the first twelve months of life where fluoride intake from infant formula consumption per kg body mass is highest. The level of fluoride in the commonly used Australian formulae would suggest that infant formula consumption alone is unlikely to be a risk factor for dental fluorosis in a non-fluoridated community, but could make a major contribution to an infant's daily fluoride intake. However, prolonged consumption (beyond 12 months of age) of infant formula reconstituted with optimally-fluoridated water could result in excessive amounts of fluoride being ingested during enamel development of the anterior permanent teeth and therefore may be a risk factor for fluorosis of these teeth.

Acids↗

PubMed1995

Cloning, expression and sequence analysis of the genes encoding the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50.

Two genes that encode methylmalonyl-CoA mutase (MCM) have been characterised in Porphyromonas gingivalis W50 (Pg). The genes, designated mcmA and mcmB are transcribed in an operon and encode the MCM small subunit (SS, 68,626 Da) and the MCM large subunit (LS, 78,703 Da), respectively. A recombinant Escherichia coli (Ec) clone harbouring the Pg mcmA and mcmB genes expressed MCM activity 280-times higher than that of the Ec control. The C terminus of the MCM LS has sequence homology to domains of a variety of enzymes that consume or produce methylmalonyl-CoA, suggesting that the MCM LS C-terminal domain is involved in substrate binding. The MCM LS C-terminal region also exhibits homology to other enzymes that have cobalamin-containing cofactors. It is likely, therefore, that the C terminus of the MCM LS is an important MCM domain involved in both substrate and cofactor binding.

Amino Acid Sequence↗

PubMed1995

A 1H-NMR study of the casein phosphopeptide alpha s1-casein(59-79).

Complete sequence-specific resonance assignments have been determined for a calcium phosphate sequestering, phosphoseryl-containing, tryptic peptide alpha s1-casein(59-79) containing the phosphorylated motif -SSSEE-. Spectra have been recorded in the presence of excess Ca2+ and at three different values of sample pH to characterize the changes in peptide conformation as calcium binds to the phosphorylated residues. The secondary structure of the peptide was characterized by sequential (i,i + 1), medium-range (i,i + 2/3/4), and long-range (i,i + 5) NOE connectivities, C alpha H chemical shifts, NH to C alpha H coupling constants and the observation of slowly exchanging amide protons. Two structured regions have been identified: residues P73 to V76 implicated in beta-turn conformations, and residues E61 to sigma 67 involved in a loop-type structure.

Amino Acid Sequence↗

PubMed1995

Complete nucleotide sequence of a gene prtR of Porphyromonas gingivalis W50 encoding a 132 kDa protein that contains an arginine-specific thiol endopeptidase domain and a haemagglutinin domain.

We have purified from Porphyromonas gingivalis W50 a 45 kDa arginine-specific, thiol-activated, EDTA-sensitive endopeptidase, designated prtR. Oligonucleotide probes based on the N-terminal amino acid sequence were used to isolate a genomic fragment containing an open reading frame (3654 bp) with the potential to encode a 132 kDa protein including the prtR N-terminus. Analysis of this prtR gene revealed that the predicted nascent product contains a protease domain followed by a haemagglutinin domain and is post-translationally processed by proteolytic (possibly autolytic) events to produce a 43-54 kDa arginine-specific, thiol protease and a 41-53 kDa haemagglutinin. Comparison of the prtR with the P. gingivalis prtH gene suggests that the prtH gene product also contains protease and haemagglutinin domains but in the reverse order to that in the prtR. An overlapping but shifted reading frame at the 3' end of the prtR encodes the 5' region of the prtH.

Amino Acid Sequence↗

PubMed1995

High performance capillary electrophoresis of casein phosphopeptides containing 2-5 phosphoseryl residues: relationship between absolute electrophoretic mobility and peptide charge and size.

Multiple phosphoseryl-containing peptides from casein containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- stabilize amorphous calcium phosphate at neutral and alkaline pH and have been shown to be anticariogenic in various in vitro, animal and human experiments. In an approach to obtain insight into the structure and function of these peptides, we previously developed a method for their analysis using high-performance capillary electrophoresis (HPCE). A linear relationship was obtained between absolute electrophoretic mobility and q/M2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M is the molecular mass. M2/3 is a measure of the surface area of a sphere that has a volume proportional to the M of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. To examine this relationship further, we have analyzed a range of high-performance liquid chromatography (HPLC)-purified cluster, di- and triphosphorylated peptides from casein enzymic (trypsin, alcalase and pancreatin) digests using HPCE and attempted to relate their absolute electrophoretic mobility to various models incorporating peptide size and charge. Absolute electrophoretic mobility of casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- was most closely correlated with q/M2/3. Di- and triphosphorylated peptide absolute electrophoretic mobility correlated with both q/M2/3 and ln(q+l)/n0.43. However, for both the q/M2/3 and In(q+l)/n0.43 relationships with absolute electrophoretic mobility, the di- and triphosphorylated peptides formed a separate linear relationship to that of the cluster peptides. From these relationships, a di- or triphosphorylated peptide exhibited a greater absolute electrophoretic mobility than a corresponding cluster peptide with sam q/M2/3 or In(q+l)/n0.43 value.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Characterization of glutamine transport in Streptococcus mutans.

Glutamine transport in glucose-energized cells of Streptococcus mutans Ingbritt exhibited Michaelis-Menten-type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a Kt of 4.1 microM. Diffusion of glutamine into de-energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 microM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force-driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed-phase high-pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.

ATP-Binding Cassette Transporters↗

PubMed1995

Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat.

Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 microL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals' molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Characterization of multiply phosphorylated peptides selectively precipitated from a pancreatic casein digest.

Anticariogenic phosphopeptides, released during the hydrolysis of casein with trypsin, contain the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu and have commercial potential as toothpaste, mouthwash, and food additives for the prevention of dental caries. To develop a commercial-scale process for the production of these peptides, we have comprehensively characterized casein phosphopeptides that were selectively precipitated using Ca2+ and ethanol from an acid-clarified (pH 4.6) pancreatic casein hydrolysate. Casein was hydrolyzed using pancreatin at 50 degrees C for 2 h. The precipitate contained a series of casein phosphopeptides that were slightly truncated relative to tryptic casein phosphopeptides. The major casein phosphopeptides released by pancreatin were beta-CN-4P(f7-24), alpha s1-CN-5P(f61-78), and alpha s1-CN-5P(f59-78), all containing the cluster sequence. The truncation of the tryptic peptides beta-CN-4P(1-25) and alpha s1-CN-5P(f59-79) resulted from the chy-motryptic and carboxypeptidase activities of the pancreatin. The peptides containing the cluster sequence constituted 77.8 +/- 6.7 mol/100 mol of the total peptides that were selectively precipitated. This composition was not significantly different from that of casein phosphopeptides produced under identical conditions using trypsin. In conclusion, pancreatin should be a suitable enzyme preparation for the production of anticariogenic casein phosphopeptides on a commercial scale.

Amino Acid Sequence↗

PubMed1994

Complete amino acid sequence and comparative molecular modelling of HPr from Streptococcus mutans Ingbritt.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Amino Acid Sequence↗

PubMed1994

A selective precipitation purification procedure for multiple phosphoseryl-containing peptides and methods for their identification.

Multiple phosphoseryl-containing sequences of proteins stabilize amorphous calcium phosphate and have been implicated in the regulation of biomineralization, protein structure, and enzyme activity. To facilitate studies on the identification and characterization of multiple phosphoseryl-containing sequences of proteins we have developed a simple and efficient purification procedure involving precipitation of Ca2+/ethanol-induced aggregates of the multiple phosphoseryl-containing peptides from enzymic digests. The multiple phosphoseryl-containing peptides of a tryptic digest of casein were selectively precipitated using Ca2+ (20 mol/mol protein) and 50% (v/v) ethanol at pH 3.5, 4.6, and 8.0. The individual peptides of the precipitates were purified using anion-exchange fast-performance liquid chromatography and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapor-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalyzed beta-elimination in the presence of ethanethiol. The modified peptides were sequenced using an Applied Biosystems Inc. automated protein sequencer fitted with a membrane cartridge. Only peptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- were precipitated by Ca2+/ethanol at pH 3.5. The pH 4.6 precipitate contained all the cluster peptides plus two diphosphorylated peptides containing -Ser(P)-Glu-Ser(P)- and -Ser(P)-Thr-Ser(P)-. At pH 8.0, a monophosphorylated peptide containing -Ser(P)-Glu-Glu- was also present in the precipitate with the diphosphorylated and cluster peptides. The recoveries of the peptides in the pH 8.0 selective precipitate ranged from 83 to 95% of that present in the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Hydroxyapatite mineralization and demineralization in the presence of synthetic phosphorylated pentapeptides.

A constant composition (CC) method was used to compare the influence of statherin-like N-terminal 5-residue fragments having different amino acids in the terminal position on hydroxyapatite (HAP) growth and dissolution. The CC experiments were done in solutions containing 4.00 x 10(-4) mol/l calcium and 2.40 x 10(-4) mol/l phosphate. The solutions used in the crystallization studies were supersaturated only with respect to HAP (pH = 7.40, sigma HAP = 3.60). The CC dissolution studies were done in solutions undersaturated with respect to HAP (pH = 6.00; sigma HAP = -0.39). The HAP mineralization and demineralization processes were markedly inhibited by the negatively charged pentapeptides. Those containing a phosphorylated terminal residue inhibited dissolution to a greater extent than the native statherin fragment having aspartate as the N-terminal residue. Strong dependencies of the degree of inhibition of growth and dissolution reaction rates on the extents of reaction were noted. As the reactions proceeded, the rate inhibition decreased in the case of crystal growth and increased for dissolution.

Adsorption↗

PubMed1994

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates.

A general synthetic method for the efficient preparation of Tyr(P)-containing peptides is described by the use of Fmoc-Tyr(PO3tBu2)-OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P)-Tyr(P)-OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8-13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using 32P-radiolabeled [Tyr(P)]4-angiotensin II, both unlabeled synthetic [Tyr(P)]4-angiotensin II and Ser-Ser-Ser-Tyr(P)-Tyr(P) reduced the release of 32P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]4-neurotensin 8-13 was ineffective as a competitive substrate and indicated that this particular Tyr(P)-containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P)-Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P)-Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.

Amino Acid Sequence↗

PubMed1993

The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis.

Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography. Purified fractions were then analyzed by CZE. Absolute electrophoretic mobilities of 13 peptides were determined by measurement of migration times relative to that of a neutral marker, mesityl oxide. A linear relationship (r2 = 0.993) was obtained between absolute electrophoretic mobility and q/M(r)2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M(r) is the molecular mass. M(r)2/3 is a measure of the surface area of a sphere that has a volume proportional to the M(r) of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. As absolute electrophoretic mobility is influenced by charge and size CZE can be used to monitor peptide phosphorylation, dephosphorylation, deamidation and truncation. This technique therefore would be suitable for quantitative analysis of peptide substrates in kinase and phosphatase studies. In conclusion CZE is a rapid and efficient technique for the resolution of multiple phosphoseryl-containing peptides from enzymic digests of casein.

Amino Acid Sequence↗

PubMed1993

The synthesis and use of pp60src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies.

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60src, -Asn-Glu-Tyr416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO3Bzl2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF3CO2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group (i.e. Tyr(Me) and Tyr respectively) but enhanced by the introduction of the hydrophilic phosphate group (i.e. as Tyr(P)).

Amino Acid Sequence↗

PubMed1993

Efficient solution phase synthesis and use of multiple O-phosphothreonyl-containing peptides for calcium phosphate binding studies.

The protected phosphothreonine derivative Boc-Thr(PO3Ph2)-OH was prepared in high yield from Boc-Thr-OH by a simple three-step procedure which involved 4-nitrobenzylcarboxyl protection, either phosphorotriester (diphenyl phosphorochloridate) or "phosphite-triester" (diphenyl N,N-diethylphosphoramidite) phosphorylation of the threonine hydroxyl group of Boc-Thr-ONb followed by hydrogenolytic carboxyl deprotection. The three Thr(P)-containing peptides, H-Thr(P)-Glu-Glu-NHMe.TFA, H-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA and H-Thr(P)-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA, were prepared in high yield by the use of Boc-Thr(PO3Ph2)-OH in the Boc mode of peptide synthesis (mixed anhydride method) followed by platinum-mediated hydrogenolytic deprotection of the Thr(PO3Ph2)-containing peptides. The use of the phosphopeptides in calcium phosphate binding studies showed that the triple Thr(P)-cluster was a basic structural requirement, since only the pentapeptide was able to bind calcium phosphate efficiently.

Amino Acid Sequence↗

PubMed1993

Branched-chain amino acid transport in Streptococcus mutans Ingbritt.

Leucine transport in glucose-energized cells of Streptococcus mutans exhibited Michaelis-Menten-type kinetics at low extracellular concentrations, with a K1 of 15.3 microM and a Vmax of 6.1 nmol/mg dry weight/min. At high extracellular leucine concentrations, the transmembrane diffusion of leucine was not saturable, indicating that passive diffusion becomes a significant mechanism of leucine transmembrane movement under these conditions. The proton motive force (PMF) was measured in glucose-energized cells of S. mutans and was found to have a maximum value of 126 mV at an extracellular pH (pH0) of 5.0; this decreased to 45 mV at pH0 8.0. The intracellular accumulation of leucine was significantly correlated with the magnitude of the PMF. The addition of excess isoleucine or valine caused a marked decrease in the leucine transport rate. Maximal rates of leucine transport occurred at pH0 6.0, and the rate of leucine transport was independent of the growth medium. The results suggest that there is a PMF-driven, branched-chain amino acid carrier in S. mutans with a proton: substrate stoichiometry of 1.

Amino Acids, Branched-Chain↗

PubMed1992

Role of phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1.

Casein kinase-2 (CK-2) is a ubiquitous Ser/Thr specific protein kinase that recognizes phosphorylatable residues located upstream of acidic determinants, its consensus sequence being Ser(Thr)-Xaa-Xaa-Acidic. Here we show that the phosphotetrapeptide AcSer(P)-Ser(P)-Ser-Ser(P), which is devoid of the canonical consensus sequence, is nevertheless phosphorylated by CK-2 with rates comparable to that of typical peptide substrates Ser-Glu-Glu-Glu-Glu-Glu and Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu routinely employed for assaying CK-2 activity. The phosphopeptide AcSer(P)-Ser-Ser(P) [but not Ac-Ser-Ser(P)-Ser(P) or AcSer(P)-Ser(P)-Ser] is also phosphorylated albeit less efficiently than AcSer(P)-Ser(P)-Ser-Ser(P). Further N-terminal elongation with additional phosphoseryl residues to give the peptides AcSer(P)-Ser(P)-Ser(P)-Ser-Ser(P) and AcSer(P)-Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P) does not improve but rather slightly decreases the phosphorylation efficiency by CK-2. These two peptides are conversely excellent substrates for CK-1, which does not appreciably phosphorylate either AcSer(P)-Ser-Ser(P) or AcSer-(P)-Ser(P)-Ser-Ser(P). Either individual or multiple replacement of the phosphorylated residues with glutamic acid in the peptide AcSer(P)-Ser(P)-Ser-Ser(P) drastically reduces the phosphorylation efficiency by CK-2, the phosphoseryl residue at position -2 playing an especially crucial role which cannot be surrogated by glutamyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗