Development, analytical validation, and initial clinical evaluation of a radioimmunoassay for the measurement of soluble CD25 concentrations in canine serum.

During immune activation, CD25 is expressed by T cells, and its soluble form (sCD25) is released into the extracellular matrix and the bloodstream. In humans, serum sCD25 concentrations are used as a surrogate marker for autoimmune diseases, malignancies, and transplant rejection. However, a canine-specific assay for the measurement of sCD25 in dog serum has not previously been described. Therefore, the aims of this study were to develop and analytically validate a radioimmunoassay to measure sCD25 in canine serum, to establish a reference interval for canine sCD25, and to test the clinical utility of this assay with serum samples for dogs with various diseases. A competitive radioimmunoassay (RIA) was developed and analytically validated. Analytical validation consisted of lower limit of detection (LLOD), dilutional parallelism, spiking recovery, and intra- and inter-assay variability using pooled surplus canine serum samples. A reference interval was established in healthy dogs and serum samples from dogs with various types of neoplasia, IBD, liver disease, suspected pancreatitis, or suspected small intestinal disease and serum samples with an increased C-reactive protein concentration (CRP) were analyzed to test the clinical utility of the assay. LLOD was calculated to be 0.5 ng/mL. The mean (±SD) observed-to-expected ratio (O/E) for serial dilutions was 101.7 ±â€¯14.0%, and the mean (± SD) O/E for spiking recovery was 93.2 ±â€¯4.2%. Coefficients of variation (CVs) for intra-assay variability were ≤12.5% (mean ±â€¯SD: 7.5 ±â€¯4.2%), and inter-assay CVs were ≤15.7% (mean ±â€¯SD: 11 ±â€¯4.4%). A reference interval (RI) for canine sCD25 of 1.2-4.2 ng/mL was established from a population of 112 clinically healthy dogs. Dogs with neoplasia and dogs with suspected small intestinal disease had decreased concentrations of serum sCD25 when compared to healthy dogs (p < 0.0001, respectively). However, the majority of clinical samples used in this study were within the reference interval. Median concentrations of serum sCD25 were 1.9 ng/mL for healthy dogs. Dogs with cancer, IBD, liver disease, suspected pancreatitis, or suspected small intestinal disease, as well as sera with an increased serum CRP concentration, had median serum sCD25 concentrations of 1.6 ng/mL, 2.1 ng/mL, 2.2 ng/mL, 1.7 ng/mL, 1.5 ng/mL, and 1.8 ng/mL, respectively. Thus, the RIA described here is linear, accurate, precise, and reproducible for measuring sCD25 in canine serum. However, this assay shows little clinical utility of sCD25 as a biomarker for dogs with inflammatory, autoimmune, and/or neoplastic conditions.

Identification of albumin precursor protein, Phi AP3, and alpha-smooth muscle actin as novel components of redox sensing machinery in vascular smooth muscle cells.

Aerobic organisms are continually subjected to environmental stressors that compromise redox homeostasis and induce cellular injury. In vascular smooth muscle cells (vSMCs), the activation/repression of redox-regulated genes after environmental stress often involves protein binding to cis-acting antioxidant response elements (AREs). The present study was conducted to identify proteins that participate in redox-regulated protein binding to human c-Ha-ras and mouse glutathione S-transferase A1 AREs in vSMCs after oxidant injury. Challenge of vSMCs with 0.3 or 3 microM hydrogen peroxide, 3-methylcholanthrene, benzo[a]pyrene-7,8-diol, 3-hydroxy benzo[a]pyrene, and benzo[a]pyrene-3,6-quinone induced concentration-related increases in ARE protein binding. The profiles of ARE complex assembly were comparable, but exhibited chemical specificity. Pretreatment with 0.5 mM N-acetylcysteine inhibited activation of ARE protein binding in hydrogen peroxide-treated cells. Preparative electrophoretic mobility shift assays coupled to Western analysis identified NF-E2-related proteins 1 and 2 and JunD in complexes assembled on AREs. Polyethylenimine affinity and sequence-specific serial immobilized DNA affinity chromatography followed by N-terminal sequencing identified albumin precursor protein, phi AP3, and alpha-smooth muscle actin as members of the ARE signaling pathway. Sequence analysis of albumin protein revealed homology to the redox-regulated transcription factors Bach1 and 2, as well as cytoskeletal and molecular motor proteins. These results implicate albumin precursor protein, phi AP3, and alpha-smooth muscle actin as participants in redox sensing in vSMCs, and suggest that protein complex assembly involves interactions between leucine zipper and zinc finger transcription factors with cytoskeletal proteins.

Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: development and application to the study of a folding/unfolding intermediate of bacterial luciferase.

A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].

Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 +/- 0.06 m(-)(1) min(-)(1). The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated with N-[ethyl-1-(14)C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.

Folding, stability, and physical properties of the alpha subunit of bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels. The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain. Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the alpha subunit with those of the alphabeta heterodimer suggested that the unfolding pathway of the alpha subunit is the same, whether it is in the form of the free subunit or in the heterodimer.

Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase.

2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.

The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid-protein interface.

17,21-Dimethyl-19-nor-pregn-4,9-diene-3,20-dione (promegestone) was used to characterize the mechanism of inhibition of nicotinic acetylcholine (ACh) receptors (AChR) by progestin steroids. Promegestone reversibly inhibited ACh-induced currents of Torpedo AChRs expressed in Xenopus oocytes. Between 1-30 microM promegestone produced a concentration-dependent enhancement of the equilibrium binding affinity of [3H]ACh to Torpedo AChR-rich membranes. For AChRs in the presence of agonist (desensitized state) promegestone was a more potent inhibitor of the binding of the noncompetitive antagonist [3H]phencyclidine (IC50 = 9 microM) than of [3H]histrionicotoxin (IC50 approximately 100 microM). To identify AChR domains in contact with the steroid, AChR-rich membranes equilibrated with [3H]promegestone were irradiated at 312 nm, and 3H-labeled amino acids were identified by amino-terminal sequencing of fragments isolated from subunit proteolytic digests. Within AChR alpha-subunit, 70% of 3H was covalently incorporated in a 10-kDa fragment beginning at Asn-339 and containing the M4 membrane spanning segment, and 30% was in a 20-kDa fragment beginning at Ser-173 and containing the M1-M3 segments. Fragments containing the M2 channel domains as well as the M4 segments were isolated from proteolytic digests of AChR subunits and subjected to amino-terminal sequence analysis. No evidence of [3H]promegestone incorporation was detected in any of the M2 segments. The amino acids in the M4 segments labeled by [3H]promegestone were among those previously shown to be in contact with the lipid bilayer (). These results indicate that the steroid promegestone is an AChR noncompetitive antagonist that may alter AChR function by interactions at the lipid-protein interface.

Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound -3H-diazofluorene.

The uncharged photoactivable probe 2-[3H]diazofluorene ([3H]DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcholine receptor (AChR) ion channel induced by agonists. Photoincorporation of [3H]DAF into the AChR consisted of the following two components: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface, and a specific component, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF incorporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforce the conclusion that the M4 segments are organized as transmembrane alpha-helices with a common "face" of each helix in contact with lipid. Within the M2 segments, in the absence of agonist [3H]DAF specifically labeled homologous residues betaVal-261 and deltaVal-269, with incorporation into deltaVal-269 at a 5-fold greater efficiency than into betaVal-261. This observation, coupled with the lack of detectable incorporation into alpha-M2 including the homologous alphaVal-255, indicates that within the resting channel [3H]DAF is bound with its photoreactive diazo group oriented toward deltaVal-269. In the presence of agonist, there is an approximately 90% reduction in the labeling of betaVal-261 and deltaVal-269 accompanied by specific incorporation into residues (betaLeu-257, betaAla-258, deltaSer-262, and deltaLeu-265) situated 1 or 2 turns of an alpha-helix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist-induced rearrangements of the M2 (ion channel) domain of the AChR.

p-(4-Hydroxybenzoyl)phenylalanine: a photoreactive amino acid analog amenable to radioiodination for elucidation of peptide-protein interaction. Application to substance P receptor.

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.

Speract receptors are localized on sea urchin sperm flagella using a fluorescent peptide analog.

In two species of sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus, the egg jelly-associated decapeptide, speract, binds to specific sperm surface receptors resulting in increased sperm motility and respiration rate. Previously, a peptide analog, GGG[Y2]-speract, was used to identify a 77-kDa receptor on intact sperm cells using chemical cross-linking. In this paper we describe the synthesis and characterization of a fluorescent derivative of GGG[Y2]-speract for use as a probe for the sperm receptor. Fluorescein isothiocyanate (FITC) was conjugated to the amino terminus of GGG[Y2]-speract and the resulting analog (FITC-GGG[Y2]-speract) was purified by size exclusion chromatography and reverse-phase HPLC. Competition binding studies with the fluorescent peptide and intact spermatozoa yielded IC50 values which were indistinguishable from native speract and GGG[Y2]-speract (approximately 20 nM). FITC-GGG[Y2]-speract half-maximally stimulated sperm respiration at a concentration nearly identical to that of the native peptide (EC50 approximately 50 pM). Using digitally enhanced video imaging fluorescence microscopy, FITC-GGG[Y2]-speract was used to localize the speract receptor on the flagella of intact sperm. Excess concentrations of both unlabeled speract and GGG[Y2]-speract abolished the binding of the fluorescent analog, yet unrelated peptides did not. Further, results of cross-linking experiments using 125I-GGG[Y2]-speract and purified sperm flagella and heads were consistent with the fluorescent labeling results on whole cells. The finding that the speract receptor is localized exclusively to the sperm flagella may reveal its role in the regulation of flagellar motility.

Cloning of the mRNA for the protein that crosslinks to the egg peptide speract.

An apparent receptor for the egg peptide speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was identified by covalently coupling a radiolabeled speract analogue to intact spermatozoa and was then purified by DEAE-Sepharose chromatography and preparative gel electrophoresis after solubilization with Lubrol PX. The purified, crosslinked protein was digested with Staphylococcus aureus V8 protease and a resultant peptide, purified from polyacrylamide slab gel slices, was shown to have the amino acid sequence Val-Ser-Ala-Pro-Phe-Asp-Leu-Glu-Ala-Pro-Phe-Ile-Ile-Asp-Gly-Ile. Polyclonal antiserum, generated against a synthetic peptide that corresponded to the above sequence, immunoprecipitated the radiolabeled crosslinked protein and reacted with a Mr 77,000 protein on immunoblots, demonstrating that the sequenced peptide originated from the apparent receptor. A clone containing a 2.5-kilobase insert was subsequently isolated from a sea urchin testis cDNA library that contained DNA sequences encoding an open reading frame of 532 amino acids that included the above peptide sequence. The deduced amino acid sequence suggests that the protein contains a 26-residue amino-terminal signal peptide, a large extracellular domain relatively rich in cysteine (5%) that includes a four-fold repeat of about 115 amino acids, a single membrane-spanning region, and only 12 amino acid residues extending into the cytoplasm. Analysis of total RNA from Strongylocentrotus purpuratus testis by Northern blot revealed a 2.5-kilobase RNA. Preliminary data show the presence of hybridizing RNA of the same apparent size in other sea urchin species, including Arbacia punctulata, which does not respond to speract.

Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases.

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.

Covalent coupling of a resact analogue to guanylate cyclase.

GGGYG-resact (Gly-Gly-Gly-Tyr-Gly-Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg -Leu-NH2) was synthesized and shown to possess the same respiration-stimulating activity and receptor-binding ability as resact. The incubation of intact sperm cells with radioiodinated peptide, 125I-GGGYG-resact, and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single, major radioactive band of apparent molecular weight 160,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The interaction was specific since 150 nM nonradioactive resact but not speract (200 nM) blocked formation of the radioactive band. The radioactive, cross-linked protein co-migrated with 32P-labeled guanylate cyclase and could be immunoprecipitated with a polyclonal antibody raised in rabbits against the sperm guanylate cyclase. The incubation of intact cells with NH4Cl resulted in the partial dephosphorylation of guanylate cyclase and a change in its apparent molecular weight from 160,000 to 150,000; NH4Cl also caused the same conversion in the apparent molecular weight of the cross-linked protein. These data demonstrate that an analogue of resact can be covalently coupled to guanylate cyclase with the specificity predicted for the peptide receptor.

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.

Peptides associated with eggs: mechanisms of interaction with spermatozoa.

Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), a peptide obtained from the culture medium of Strongylocentrotus purpuratus eggs, stimulates the respiration and motility of S. purpuratus spermatozoa under appropriate conditions. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2), a peptide obtained from Arbacia punctulata eggs also stimulates the metabolism and motility of A. punctulata spermatozoa, however, it fails to stimulate S. purpuratus spermatozoa. Early biochemical responses of the spermatozoa to the egg peptides include a net H+ efflux and elevations of cyclic AMP and cyclic GMP concentrations. In addition, in A. punctulata spermatozoa, a major plasma membrane protein is modified in response to resact such that its apparent molecular weight shifts from 160,000 to 150,000. If cells are incubated with 32P, the 160,000 molecular weight form of the protein becomes radiolabeled; subsequent addition of resact causes a rapid loss of 32P from the protein. The plasma membrane protein appears to be the enzyme, guanylate cyclase; coincident with the shift in apparent molecular weight, enzyme activity decreases by as much as 90%. Since speract fails to cause these responses in A. punctulata, it can be concluded that the events are receptor-mediated.

Identification and partial characterization of the receptor for speract.

Here, we report the first identification of a sperm receptor for an egg-associated peptide (speract). Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly (GGG[Y2]-speract), was chemically synthesized and shown to possess equivalent respiration-stimulating activity and the same receptor binding characteristics as speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly). The incubation of Strongylocentrotus purpuratus intact sperm cells with radioiodinated GGG[Y2]-speract and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single radiolabeled band with an estimated Mr = 77,000 (determined by Na dodecyl SO4-gel electrophoresis under reducing conditions). The covalent coupling of 125I-GGG[Y2]-speract was prevented by biologically active analogues of speract but not by inactive analogues and the 125I analogue was not detectably coupled to any protein in Arbacia punctulata spermatozoa, a species with which speract does not cross-react. Comparisons of the amount of 125I-GGG[Y2]-speract bound in the presence of variable concentrations of unlabeled GGG[Y2]-speract showed that about 1 nM unlabeled peptide reduced 125I-GGG[Y2]-speract binding by 50% when estimated as either specific binding to intact cells or as the reduction in radioactivity found in the 77,000 molecular-weight band on autoradiographs after covalent coupling. The radiolabeled receptor could be solubilized with 0.5% Lubrol PX and was bound to wheat germ lectin-Sepharose. It could be subsequently eluted with N-acetylglucosamine or diacetylchitotriose but not by methyl-alpha-D-mannoside or NaCl, suggestive that the receptor is a glycoprotein.