On the Adsorption of Human Acidic Proline-rich Proteins (PRP-1 and PRP-3) and Statherin at Solid/liquid Interfaces

The objective of the present study was to investigate the adsorption of PRP-1, PRP-3 and statherin to solid surfaces in terms of dependence on concentration, the presence of electrolyte and surface wettability. Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates of pure PRP-1, PRP-3 and statherin onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. The initial film build-up was fast and plateaus were reached within 10 min at all concentrations for both types of surfaces and all proteins. The observed adsorption and calculated diffusion rates of PRP-1, PRP-3 and statherin, respectively, indicated that the initial adsorption was mass transport controlled at low concentrations. At hydrophobic surfaces, isotherm shapes and adsorbed amounts were similar for PRP-1 and PRP-3, while statherin adsorbed to a higher extent. At hydrophilic surfaces only PRP-1 adsorbed substantially, while for PRP-3 and statherin adsorbed amounts were low. The presence of Ca 2+ ions in the phosphate buffer solution increased the adsorption of statherin and PRP-3 on hydrophobic surfaces, while PRP-1 was unaffected. On hydrophilic surfaces, all three proteins adsorbed in higher amounts in NaCl, compared to CaCl 2 at similar ionic strength. It is concluded that acidic PRPs (PRP-1 and PRP-3) and statherin readily form films on a variety of materials and solution conditions, showing that their functions may be fulfilled under a wide range of conditions.     

Complete covalent structure of a proline-rich phosphoprotein, PRP-2, an inhibitor of calcium phosphate crystal growth from human parotid saliva

Human salivary secretions contain many proteins in which proline forms an unusually large fraction of the amino-acid residues present, typically from 20% to over 40%. These proteins are also unusually rich in glycine and glutamine, generally account for over half the total protein in saliva, and include acidic, basic and glycosylated molecules. The functions of most of these are not clearly defined. One group, however, the acidic proline-rich phosphoproteins (PRP), have been shown to be potent inhibitors of secondary precipitation (crystal growth) of calcium phosphate salts. Acting together with a salivary protein inhibitor of primary precipitation of calcium phosphates, statherin, the PRP stabilize saliva which is supersaturated with respect to the calcium phosphate salts which form dental enamel. These inhibitory activities act to provide a protective, reparative, but stable environment for dental enamel, which is important for maintaining the health of the teeth. The PRP are a complex group of phosphoproteins which include four major and at least eight minor members. The primary structures of three of the major proteins have been determined. These are PRP-1, also designated Protein-C, PRP-3, also designated Protein-A (17), and PRP-4. The designations PRP-1,-2,-3 and -4 will be used here. The purpose of this paper is to report the complete primary structure of PRP-2 as a further step towards establishing the structural basis of the biological activity of the PRP, and clarifying the genetic and biosynthetic relationships of these closely related proteins.     

Intraoral lubrication of PRP-1, statherin and mucin as studied by AFM

The aim of this paper was to elucidate the mechanisms behind salivary lubrication with special emphasis on the lubricity of three key components of the pellicle, viz human acidic proline-rich protein 1 (PRP-1), human statherin and bovine submaxillary mucin (BSM). The lubricating properties of the proteins have been assessed by means of colloidal probe atomic force microscopy, and are discussed in relation to their adsorption behaviour. To various extents, the proteins investigated all showed a lubricating effect when adsorbed to silica surfaces. For comparable concentrations, PRP-1 was found to have a more pronounced lubricating effect than BSM, which in turn showed a higher lubricity than statherin. The relative lubricity is in accordance with previously reported relative adsorbed amounts of the three proteins, within the investigated concentration interval. It is concluded that PRP-1 has the highest lubricating capacity as a pure fraction among the preparations investigated, and that the lubricating effect of PRP-1 as a pure fraction is notably large as compared to the lubricity of human whole saliva.     

Effect of Hypothalamic Proline-Rich Peptide (PRP-1) on Neuronal and Bone Marrow Cell Apoptosis

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we investigated the influence of PRP-1 on staurosporine-induced apoptosis of postnatal hippocampal cells and on doxorubicin-induced bone marrow granulocyte- and monocyte apoptosis. The intention was to further characterize the effect of PRP-1 on the survival rate of neurons and in context with myelopoiesis. We demonstrate that PRP-1 significantly reduced apoptosis of postnatal hippocampal cells induced by staurosporine. The protective effect of PRP-1 against apoptotic cell death was shown to be both time- and dose-dependent. Neuroprotection was more pronounced after prolonged pretreatment of the cells with PRP-1 before the induction of apoptosis with staurosporine. The related peptide [arg⁸]vasopressin did not reveal neuroprotection. PRP-1 also significantly reduced apoptosis of bone marrow monocytes and granulocytes induced by doxorubicin. This protective effect lasted for 2-4 h and was not detectable anymore after 24 h when PRP-1 and doxorubicin were added simultaneously. Previously obtained data and results of the current studies suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate myelopoiesis and neuron survival as we provide evidence that PRP can differentially reduce both staurosporine- and doxorubicin-induced hippocampal and bone marrow cell apoptosis.     

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.     

Isoaspartate residues dramatically influence substrate recognition and turnover by proteases

Posttranslational modifications influence the structure, stability and biological activity of proteins. Most of the reactions are enzyme-catalyzed, but some, such as asparagine (Asn) and glutamine (Gln) deamidation and the isoaspartate (isoAsp) formation within peptide chains, occur spontaneously. It has been previously shown that certain peptide sequences form isoAsp quite fast if the Asp stretches are exposed to the protein surface, thereby potentially changing susceptibility to proteolysis at these sites. This tempted us to investigate the activity of exo- and endopeptidases against Asp- or isoAsp-containing substrates. Members of the prolyl oligopeptidase family were unable to cleave substrates after proline if isoAsp was placed in the P2-position. Caspases, usually accepting Asp at P1-position of their substrates, did not cleave isoAsp-containing sequences. Similarly, the metal-dependent aminopeptidase amino peptidase N did not turnover N-terminal isoAsp-containing substrates, nor could the endopeptidase matrix metalloproteinase 3 (MMP 3) hydrolyze a serum amyloid A protein-like substrate if the sequence contained isoAsp instead of Asp. Also, the highly specific enterokinase, usually clipping after a stretch of four Asp residues and a lysine in the P1 position, could not turnover substrates if the P2 amino acid was replaced by isoAsp. In contrast, acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2 and 4 hydrolyzed substrates containing the isoAsp-Ala motif.     

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.     

A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.     

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.     

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.     

Myc-oncogene Inactivating Effect by Proline Rich Polypeptide (PRP-1) in Chondrosarcoma JJ012 Cells

Proline rich polypeptide (PRP-1) produced by NPV and NSO cells is released into the general circulation and exerts its effect on the activity of immunocompetent and neuronal cells. PRP-1 is a unique regulator of hematopoiesis, stimulator of bone-marrow hematogenesis. Taking into consideration our preliminary data on antitumor and unique diverse biological properties of PRP-1 previously described by Galoyan et al., we proceeded with investigation of the PRP-1 effect on chondrosarcoma, the second most common malignancy in bone, which tends to be locally invasive and then metastatic. Currently it does not have any effective treatment and does not respond either to radiation or chemotherapy, leaving surgical resection as the only option. Our experimental results of PRP-1 action on human chondrosarcoma JJ012 cells demonstrated inactivation, abolishment of Myc oncogene activity usually upregulated in chondrosarcoma cells and other malignancies. The fact that addition of PRP-1 caused drastic inactivation of Myc–luc response element to the control level in human chondrosarcoma JJ012 cell line prompts to investigate further this neuropeptides powerful antioncogenic potential, opening up possibilities to consider PRP-1 as a potential therapeutic tool for chondrosarcoma treatment.     

Amino acid structures of multiple forms of amyloid-related serum protein SAA from a single individual

Multiple forms of the acute-phase serum protein SAA were isolated from the lipoprotein fraction of plasma from a single individual. These protein forms were purified by size-exclusion, ion-exchange, and reverse-phase high-pressure liquid chromatography, and then the tryptic peptides were subjected to amino acid sequence analysis. A total of three distinct 104-residue proteins were identified. Two of these proteins differed only by having either an arginine or a histidine at position 71 while the third protein had seven amino acid differences. Each of these proteins has a 103-residue companion protein where the amino-terminal arginine has been removed. Two of these protein sequences match the two human SAA cDNA structures reported in the literature. The presence of three unique amino acid sequences in one individual is proof that there must be a minimum of two genes for SAA in humans.     

Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis>

The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of the Azotobacter vinelandii nitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.     

Restricted variable residues in the C-terminal segment of HIV-1 V3 loop regulate the molecular anatomy of CCR5 utilization

The V3 loop of the HIV-1 envelope glycoprotein (Env) is the major determinant for coreceptor utilization, but the structural basis for this specificity remains to be defined. By characterizing a set of naturally occurring R5 Env variants, we demonstrate that Asp324 in the conserved IIGDIR motif of the V3 loop (CTRPN(300)NNTRKSIHIGP(311)GRAFYTTGEIIGD(324)IRQAHC) C-terminal segment regulates the molecular anatomy of CCR5 utilization. Whereas gp120 subunits with Asp or Asn at position 324 were fusogenic with coreceptor chimeras containing either the N-terminal domain or the body of CCR5, substitution of charged (Glu, Lys) or small hydrophobic (Gly, Ala) residues resulted in complete loss of fusogenic activity with the N terminus and markedly reduced utilization of the body of CCR5, although their ability to use wild-type CCR5 was unchanged. This phenotypic conversion was confirmed in both gain and loss of function experiments using Env from multiple subtypes. Alignment of sequences of R5 V3 loops (n=599) from the HIV database revealed that the mutation of Asp324 in the conserved IIGDIR motif is restricted to Asn324, with proportions of 71.5% and 28%, respectively. Infection of primary CD4(+)T cells demonstrated that Env bearing Asp324 was less sensitive to RANTES, suggesting that Asp or Asn in this position may be crucial for viral fitness. The CD4-dependent gp120 binding to CCR5 was decreased when Asp324 was replaced with a charged or hydrophobic residue, but unchanged when replaced with Asn. Molecular modeling analyses predicted that Asp/Asn324 forms a critical H-bond with Asn300. These findings indicate that Asp or Asn at position 324 of the V3 stem stabilizes the conformation of V3 loop and hence influences the intensities of interaction between CD4-activated gp120 and CCR5 which results in viral entry.     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.     

Cell communication using intrinsically disordered proteins: what can syndecans say?

Because intrinsically disordered proteins are incapable of forming unique tertiary structures in isolation, their interaction with partner structures enables them to play important roles in many different biological functions. Therefore, such proteins are usually multifunctional, and their ability to perform their major function, as well as accessory functions, depends on the characteristics of a given interaction. The present paper demonstrates, using predictions from two programs, that the transmembrane proteoglycans syndecans are natively disordered because of their diverse functions and large number of interaction partners. Syndecans perform multiple functions during development, damage repair, tumor growth, angiogenesis, and neurogenesis. By mediating the binding of a large number of extracellular ligands to their receptors, these proteoglycans trigger a cascade of reactions that subsequently regulate various cell processes: cytoskeleton formation, proliferation, differentiation, adhesion, and migration. The occurrences of 20 amino acids in syndecans 1–4 from 25 animals were compared with those in 17 animal proteomes. Gly?+?Ala, Thr, Glu, and Pro were observed to predominate in the syndecans, contributing to the lack of an ordered structure. In contrast, there were many fewer amino acids in syndecans that promote an ordered structure, such as Cys, Trp, Asn, and His. In addition, a region rich in Asp has been identified between two heparan sulfate-binding sites in the ectodomains, and a region rich in Lys has been identified in the conserved C1 site of the cytoplasmic domain. These particular regions play an essential role in the various functions of syndecans due to their lack of structure.     

Molecular cloning of human haptoglobin cDNA: evidence for a single mRNA coding for alpha 2 and beta chains

Human haptoglobin (Hp) is a plasma glycoprotein composed of alpha and beta polypeptide chains that are covalently associated by disulfide bonds. It had been suggested that alpha and beta polypeptides could be synthesized via a common precursor polypeptide. We report the molecular cloning of DNA complementary to human Hp mRNA. One of the clones, pULB1148, carries a full length copy coding for both alpha 2 and beta polypeptides. In vitro translation of human liver mRNA hybridizing with this cDNA gives a protein mol. wt. of 49000 daltons. The sequence of the alpha 2 beta cDNA shows the presence of a single Arg residue between Gln 142 of the alpha 2 chain and Ileu 1 of the beta chain. With a few minor exceptions, the DNA sequence fits the previously published amino acid sequences. The differences are the presence of an Asp residue at position 52 of alpha 2 instead of Asn, the existence in beta of only one Lys residue between Gly 65 and the following Gln, the presence of Ser and Cys at positions 218-219 instead of Cys-Ser, and of Asp residues at positions 205 and 235 instead of Asn.     

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.