Effect of recombinant boar beta-acrosin on sperm binding to intact zona pellucida during in vitro fertilization

In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.     

Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida

An assay to determine the binding of pig spermatozoa to the zona pellucida (ZP) of pig oocytes was developed using conditions compatible with in-vitro fertilization of pig eggs and with pig sperm penetration of zona-free hamster ova. These conditions were used to define which of the pig oocyte ZP components were involved in sperm binding by a competitive inhibition approach. Assay variables that were optimized included: the method of sperm preparation; sperm preincubation time; sperm-oocyte coincubation time; sperm concentration and temperature; and methods for the separation of free from oocyte-bound spermatozoa. Inclusion of solubilized ZP in the sperm preincubation medium inhibited sperm binding approximately 50%. Both the 55K and 90K components inhibited sperm binding although the 55K component was more effective. The two polypeptides derived from chemical deglycosylation of the 55K families did not inhibit sperm binding. Of several monoclonal antibodies to the ZP components tested, only one directed against the 55K alpha glycoprotein family inhibited sperm binding. Sperm binding to pig oocyte ZP is therefore dependent on the carbohydrate moiety of the glycoproteins and appears to involve more than a single ZP glycoprotein.     

Structural Proteins of Reoviruses

Polyacrylamide gel electrophoresis of the solubilized proteins from the three serotypes of reovirus revealed that each contained three major and four minor components. Subviral particles were prepared by brief treatment of complete virions with urea. Electron microscopy, density-gradient centrifugation, and chemical analyses of these particles indicated that their outer capsid structure had been selectively removed. They contained only two proteins, but their ribonucleic acid composition was similar to that of complete virions. The subviral particles were not infectious.     

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.     

Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes

Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.     

Detection of membrane-bound cytokinin-binding proteins in Arabidopsis thaliana cells.

In order to isolate cytokinin-binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase.     

Composition and genetic variability of heparin-sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs

The solubilized proteins of liver and brain from mice of two inbred strains (C57BL/6J and DBA/2J) and their hybrids were subfractionated by heparin Sepharose (H-S) CL-6B affinity chromatography. The H-S binding and nonbinding proteins were separated by two-dimensional electrophoresis. The protein patterns obtained were analyzed with regard to their protein composition and their genetic variability (qualitative and quantitative variants). Eighty to ninety percent of the H-S binding proteins were unique to this class of proteins. This class was rich in organ-specific proteins. Compared to the nonbinding proteins the portion of basic proteins was only slightly increased, suggesting that most of the H-S binding proteins interact specifically with heparin. The frequency of qualitative protein variants revealed that H-S binding proteins are more conservative than H-S nonbinding proteins. The quantitative genetic variability was higher in liver than in brain. Quantitative protein variants occurred more frequently than qualitative variants.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Taste receptor proteins

The chemistry of taste has eluded widespread investigation until only recently. Of the four basic taste qualities — sweet, salty, sour and bitter, only sweet and to some lesser extent — bitter — have had inroads made on the molecular level. Carbon-14 labeled sugars are preferently bound to taste bud proteins versus other non-sensory proteins. The binding of the sugars is in good agreement with their relative sweetness and graded conformational changes in protein molecules are seen, by a number of methods, upon these interactions. The protein may occur in two forms — soluble and membrane bound, and the bound form can be solubilized. Unique proteins are present in taste buds and would thus differentiate them from other non-sensory proteins. The sweet-sensitive protein activity has been studied in a number of animals, but purification and characterization in all has not proceeded to the point wherein comparisons can be made. Bitter reception has been suggested as the property of a protein from pig tongues, and an enzyme — phosphodiesterase has been found to be both activated and inhibited by bitter compounds. Lipids from gustatory tissue have been suggested as candidates for receptors for salty, sour and bitter, but not sweet stimulants.     

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

The receptor site for the bee venom mast cell degranulating peptide. Affinity labeling and evidence for a common molecular target for mast cell degranulating peptide and dendrotoxin I, a snake toxin active on K+ channels

The mast cell degranulating peptide (MCD) and dendrotoxin I (DTXI) are two toxins, one extracted from bee venom, the other one from snake venom, that are thought to act on voltage-sensitive K+ channels. Binding sites for the two toxins have been solubilized. The solubilized sites were stable and retained their high affinity for 125I-DTXI and 125I-MCD (Kd approximately equal to 100 pM). Interactions were found between MCD and DTXI binding sites in the solubilized state, establishing that the two different toxins act on the same protein complex. This conclusion was strengthened by the observations (i) that conditions of solubilization that eliminated 125I-MCD binding activity also eliminated 125I-DTX binding activity while both types of activities were preserved in the presence of K+ or Rb+ and (ii) that binding components for the two types of toxins had similar sedimentation coefficients and copurified in partial purifications. A component of the receptor protein for 125I-MCD has been identified; it has a Mr of 77,000 +/- 2000. This polypeptide was similar to or identical in molecular weight with that which serves as a receptor for DTXI (Mr 76,000 +/- 2000).     

Two novel brain proteins, CaBP33 and CaBP37, are calcium-dependent phospholipid- and membrane-binding proteins

Two acidic Ca2(+)-binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat-sensitivity, electrophoretic mobility, and Ca2(+)-dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane-bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KCl. However, apparent Ca2(+)-independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane-bound CaBP33 and CaBP37 could be solubilized with Triton X-100, indicating that brain membranes normally contain the two proteins as intrinsic components.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Design of an Affinity Matrix for Purification of the Histamine H1 Receptor from Guinea Pig Cerebellum

H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM [125I]iodobolpyramine specific binding represented greater than 90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki = 0.5 nM), has been coupled to a Sepharose epoxy-activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose-glycine matrix was not able to adsorb these sites.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.     

Heterogeneity in the physicochemical properties of deoxycholate-solubilized benzodiazepine receptors from calf cerebral cortex

The hydrodynamic behaviour of benzodiazepine receptors solubilized by deoxycholate from calf cerebral cortex reveals two molecular forms. The Stokes radii are 46.5 A and 67.2 A, and the sedimentation coefficients are 10.9 S and 14.6 S. The calculated apparent molecular weights and frictional ratios suggest either two nearly globular proteins of ca. 200K and 400K daltons each, or two ca. 300K daltons proteins which differ significantly in their degree of asymmetry. The benzodiazepine binding site is located on ca. 51K daltons component(s) in both forms.