The presence and origin of phosphopeptides in human saliva.

The presence of phosphopeptides in whole saliva (saliva expectorated from the mouth) was demonstrated and their origin was evaluated. Whole saliva contained much larger numbers of small phosphopeptides than are found in the glandular secretions. Most of these originated from the acidic proline-rich proteins (PRPs) in the major salivary glands and were formed, after secretion into the oral cavity, as a result of rapid degradation by proteolytic enzymes from extraglandular sources contained in sediment from whole saliva. Some peptides may have been formed by cleavage of basic PRPs, but other phosphoproteins apparently contributed little to the observed phosphopeptides. Most of the enzymes that produced phosphopeptides are serine proteinases. The gel-electrophoretic band patterns of the phosphopeptides obtained from 26 individuals of various acidic-PRP phenotypes were remarkably similar, demonstrating that the enzymes responsible were generally present in the population surveyed and that similar cleavages occur regardless of the nature of the acidic PRPs. Many of these peptides were N-terminal proteolytic cleavage products of acidic PRPs. The N-terminal phosphorylated region of acidic PRPs contains various biological activities, such as inhibition of hydroxyapatite formation, calcium binding and binding to hydroxyapatite, the major mineral of teeth. The demonstration of these phosphopeptides in the saliva that is in contact with the oral surface may therefore be of biological importance.     

Tests of potential adipokinetic hormone precursor related peptide (APRP) functions: lack of responses.

The adipokinetic hormone (AKH) precursor related peptides (APRPs) are end products of the synthesis of the well-conserved AKHs. The large amount of metabolic energy devoted to APRP synthesis suggests they have an important function(s) in the insects. Several functions have been proposed, but currently none are known. We tested whether the APRPs stimulate hyperlipemia, hypertrehalosemia, fat body glycogen phosphorylase activation, Malpighian tubule secretion, and hindgut myotropia. Surprisingly, none of these responses were stimulated by APRPs isolated from the lubber grasshopper, Romalea microptera (= guttata). In addition, the APRPs delivered in concert with AKHs did not significantly increase hyperlipemia, hypertrehalosemia, or phosphorylase activation over the AKHs alone. Our data discount several proposed functions for the APRPs. Arch.     

Purification and characterization of a glycosylated proline-rich protein from human parotid saliva.

1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva.     

Isolation and identification of the AKH III precursor-related peptide from Locusta migratoria

We have isolated an 8770Da peptide from extracts of corpora cardiaca of adult male and female Locusta migratoria. The N-terminal amino-acid sequence as partially established by Edman degradation is Ala-Leu-Gly-Ala-Pro-Ala-Ala-Gly-Asp. These nine amino acids correspond to the first nine N-terminal amino acids of the adipokinetic hormone precursor-related peptide gamma-chain (APRP-gamma), a peptide that is predicted from the gene encoding the adipokinetic hormone III precursor. The APRP-gamma chain has a monoisotopic mass of 4387Da and contains two cysteine residues. It is known that both AKH I and AKH II precursors occur as dimers. After processing they give rise to the active hormones and three dimeric (two homodimers and one heterodimer) adipokinetic hormone precursor related peptides (APRPs). Based on the mass of 8770Da and the established N-terminal sequence tag, we conclude that the isolated peptide is a homodimer consisting of two APRP-gamma units, covalently linked to each other by two disulphide bounds. In analogy with the previous identified APRPs (APRP-1, APRP-2, and APRP-3), this APRP will be designated as APRP-4.     

Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern

In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.     

Salivary proline-rich proteins

Summary Proline-rich proteins are major components of parotid and submandibular saliva in humans as well as other animals. They can be divided into acidic, basic and glycosylated proteins. The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins. The proline-rich proteins are apparently synthesized in the acinar cells of the salivary glands and their phenotypic expression is under complex genetic control.The acidic proline-rich proteins will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva. Moreover they can inhibit formation of hydroxyapatite, whereby growth of hydroxyapatite crystals on the tooth surface in vivo may be avoided. Both of these activities as well as the binding site for hydroxyapatite are located in the N-terminal proline-poor part of the protein. Little is known about the functions of the glycosylated and basic proline-rich proteins.     

The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.

An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE1 or PGE2, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva; PGF2 alpha was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 micrograms/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE1, PGE2, PGF2 alpha or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF2 alpha, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion, Endogenous prostaglandins themselves may not play a role in secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, and inhibitor of prostaglandin biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion, The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Hormone-dependent phosphorylation of the avian progesterone receptor

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.     

Characterization of tryptic casein phosphopeptides prepared under industrially relevant conditions

Anticariogenic casein phosphopeptides (ACPP) 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. In an approach to develop a commercial-scale process for the production of ACPP we have comprehensively characterized casein phosphopeptides (CPP) produced under industrially relevant conditions. Sodium caseinate (10% w/v) was hydrolyzed by Novo trypsin (commercial grade) at 50 degrees C for 2 h and CPP were purified from the acid clarified hydrolysate by a single-step selective precipitation procedure involving Ca(2+) (20 mol/mol casein) and ethanol (50% v/v) at pH 4.6 or 8.0. The individual peptides of the CPP preparations were purified by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid composition and sequence analyses. The yield of the pH 8.0 precipitate (13.85 +/- 0.48 wt % of the caseinate) was slightly higher than that of the pH 4.6 precipitate (11.04 +/- 0.30 wt % of the caseinate). However, the pH 4.6 precipitate contained predominantly (86.4 mol %) ACPP cluster peptides with small amounts of the diphosphorylated peptides (13.6 mol %), alpha(s1)(43-58) and alpha(s2)(126-136). In the pH 8.0 precipitate the cluster peptides represented a smaller proportion of the total peptides (61.9 mol %) due to increased recoveries of the diphosphorylated peptides (24.4 mol %) as well as the additional recovery of the monophosphorylated peptide beta(33-48) (13.7 mol %) indicating increased cross-linking by Ca(2+) at the higher pH. The recovery of the ACPP from the original caseinate was similar for both the pH 4.6 and 8.0 precipitates. Slight chymotryptic activity was detected in the industrial-grade enzyme, resulting in minor truncation of some peptides. Also some deamidation and methionine oxidation of one peptide, alpha(s1)(59-79), were detected. In conclusion, ACPP can be produced under industrially relevant conditions with only minor modifications such as slight truncation, deamidation, and methionine oxidation. However, in order to prepare casein phosphopeptides predominantly containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, the single-step selective precipitation with Ca(2+)/ethanol should be performed at pH 4.6 rather than pH 8.0. (c) 1995 John Wiley & Sons, Inc.     

Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells.

There is evidence that the rab class of low molecular weight GTP-binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP-binding proteins on catecholamine secretion from digitonin-permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector-binding domains of the rab, ras and ral classes of low molecular weight GTP-binding proteins and correspond to ras(33-48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca(2+)-dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19-31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced GppNHp-induced secretion 30-70%. The peptides had little or no effect in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.     

Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT

Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100-500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ～80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ～1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200-500 μg), identifying up to 4000 phosphopeptides per run.     

Extraction and purification of proteoglycans from mature bovine bone.

Proteoglycans were extracted in good yields from the mineralized matrix of ground bovine bone, by using a two-step extraction procedure. Proteoglycans (8% of total), not associated with the bone mineral, were extracted at - 20 degrees C with 4M-guanidinium chloride containing proteinase inhibitors. Proteoglycans associated with the mineral, which accounted for 60% of the total, were then solubilized when EDTA was added to the extraction solvent. They were fractionated and purified in the presence of 4M-guanidinium chloride by CsCl-density-gradient centrifugations followed by chromatography on Sepharose CL-4B. Further purification was obtained by chromatography on DEAE-cellulose and hydroxyapatite in the presence of 7 M-urea. Three populations of proteoglycans and additional glycosaminoglycan peptides were obtained. The molecular dimensions of both intact molecules and of their side chains as well as their amino acid composition were different, indicating that they represent separate molecular entities. The main proteoglycan self-aggregated in the absence of 4M-guanidinium chloride or 7 M-urea, a property that was abolished when the proteoglycan core protein was fragmented.     

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.     

Characterization of porcine osteonectin extracted from foetal calvariae.

Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described 'culture shock', SPARC and BM-40 proteins, indicating that these are homologous proteins.     

Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana: a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.     

Collagen-induced platelet activation mainly involves the protein kinase C pathway.

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.     

Isolation and characterization of histamine-releasing peptides from human parotid saliva

Peptides responsible for releasing histamine were purified from human parotid saliva. The amino acid composition of the peptides showed a high proportion of histidine, lysine and arginine. Molecular weights of these peptides were between 3000 and 5000 as determined by SDS-acrylamide gel electrophoresis. These peptides induced histamine release from rat-isolated mast cells accompanied with degranulation in a dose-dependent manner over the concentration range 5-50 micrograms/ml.     

ATP depletion in human platelets caused by permeabilization with saponin does not prevent serotonin secretion induced by collagen

Saponin (5 to 25 micrograms/ml) produced a concentration-dependent decrease in the cellular content of total ATP and [32P]ATP in 32P-labeled human platelets. In platelets whose ATP had been profoundly decreased by saponin, Ca2+ produced phosphomonoesteratic cleavage of the polyphosphoinositides with a concomitant accumulation of phosphatidylinositol. Collagen still induced secretion of serotonin in platelets that had been treated with saponin in the presence or absence of Ca2+. This effect of collagen occurred in the absence of the formation of cyclooxygenase metabolites. In platelet permeabilized with saponin, agonist-induced secretion and aggregation seems to be unrelated to protein phosphorylation, breakdown of the inositol phospholipids by phospholipase C and formation of cyclooxygenase metabolites.     

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins.

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.