Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Characterization of a soluble follitropin receptor from porcine testis.

Porcine testis receptors for follitropin (FSH) were solubilized by treatment with the non-ionic detergent Nonidet P-40 and receptor-bound and free ¹²⁵I-porcine FSH were separated by ammonium sulfate precipitation. The soluble receptor retained both its high affinity and specificity for FSH. The soluble hormone-receptor complex exhibited an equilibrium association constant of 4.7 × 10¹⁰ M^?1 at 4°C. Its hydrodynamic properties were consistent with those obtained for other solubilized peptide hormone receptors, and its molecular weight estimated to 244,000.     

Analysis of the kinetics of ovine follitropin agonist-antagonist interactions with pig ovarian membranes

The binding of 125I-labeled ovine follitropin (oFSH) and 125I-labeled deglycosylated ovine follitropin (DG-oFSH) to porcine granulosa cell membranes was studied at equilibrium and nonequilibrium binding conditions and statistically analyzed. Saturation and competition binding experiments revealed homogeneity in the population of binding sites labeled with 125I-oFSH, having a pK estimation of approximately equal to 10. 125I-DG-oFSH similarly interacts with a single uniform class of receptors of equal affinity (pK approximately equal to 10) and binding capacity as oFSH. In contrast, displacement experiments using 125I-DG-oFSH as tracer and unlabeled oFSH as competing ligand demonstrate slope factors less than unity, suggesting apparent heterogeneity of sites not observed with 125I-DG-oFSH vs. DG-oFSH competition experiments. Under these conditions, it appears that FSH binds to two sites in near equal proportion but of unequal affinities. The total specific binding capacities of these sites equal those observed in 125I-DG-oFSH/unlabeled DG-oFSH competition experiments. Analysis of oFSH association kinetics at 37 degrees C by curve-fitting methods is best explained by a biexponential rate equation describing a fast and a slow association component that are equally distributed. DG-oFSH demonstrates a disproportionately greater amount of fast vs. slow binding component. The binding half-times for each component of oFSH and DG-oFSH are similar, i.e., minutes for the fast and hours for the slow t 1/2 times. At 37, 25, and 4 degrees C, DG-oFSH exhibits greater velocity of binding to the receptor than oFSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of follitropin binding to solubilized calf testis receptor

Thermodynamic parameters of follitropin binding to solubilized testicular receptors were measured in order to assess the forces involved in the binding reaction. Reversibility of follitropin binding to solubilized receptor decreased only 20% over the temperature range 4-24 degrees C, whereas earlier studies indicated reversibility of binding to membrane-bound receptor decreased by more than 40% over the same range [Anderson, T. T., Curatolo, L. M., & Reichert, L. E., Jr. (1983) Mol. Cell. Endocrinol. 33, 37-52]. Thermodynamic analysis of follitropin binding to solubilized receptors showed that the hydrophobic effect was important in the binding reaction. The mean values, at 25 degrees C, for delta H and delta S were -31.8 kcal/mol and -66.0 cal mol-1 K-1, respectively, and delta Cp was -3.0 kcal mol-1 K-1. This is an unusually large heat capacity for protein-protein association reactions, indicating an enhanced role for the hydrophobic effect with the solubilized (compared to membrane-bound) receptor. Since glycerol was necessary to stabilize the solubilized receptor, we determined whether glycerol affected the thermodynamic parameters measured for the binding reaction. Control experiments, performed with membrane-bound receptor in the presence or absence of glycerol, indicated that delta Cp actually decreased upon addition of glycerol (-0.8 kcal mol-1 K-1 in the presence of glycerol compared to -2.3 kcal mol-1 K-1 in the absence of glycerol). Thus, the large negative delta Cp observed for the soluble receptor was a result of its removal from the membrane and was not due to the presence of glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...     

Photoaffinity labeling of the follitropin receptor

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.     

Porcine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

The properties of porcine follitropin and its subunits which have not yet been characterized are presented. The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P-1 preparation. Its contamination by lutropin and thyrotropin amounted to 1 and 0.5 percent by weight respectively, as measured by radioimmunoassay. The alpha and beta subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography. The amino acid composition of porcine follitropin alpha subunit was found to be identical to that of alpha chain of porcine lutropin and thyrotropin. These porcine alpha chains differ, nevertheless, markedly in their carbohydrate composition particularly with respect to their mannose and galactose contents. The amino-terminal residue of the follitropin alpha subunit is threonyl. The carboxy-terminal end of the alpha chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin beta chain with glutamic acid at its carboxy-terminal end. Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively.     

Purification and properties of porcine testis acylphosphatase

Acylphosphatase has been purified from porcine testis and its properties were compared with those of porcine skeletal muscle acylphosphatase. The molecular weight of the testis enzyme was found to be 10,600, similar to that of porcine skeletal muscle acylphosphatase, on sedimentation equilibrium analysis. The specific activity of the testis enzyme was 10,800 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate, i.e., higher than that of the muscle enzyme, 7,200 mumol/min/mg, under the same conditions. The pI of the testis enzyme was 8.3, i.e., lower than that of the muscle enzyme, 10.6. There were marked differences in the amino acid compositions of the two enzymes. In particular two histidine residues were present in the testis enzyme but none were present in the muscle enzyme, and no cysteine residue was present in the testis enzyme but one was present in the muscle enzyme. The carboxyl terminal amino acid of the testis enzyme seemed to be lysine, while that of the muscle enzyme is tyrosine. The peptide maps of the testis and muscle enzymes indicated considerable differences in the amino acid sequences of the two enzymes. Differences in the antigenic structures of the two enzymes were demonstrated on enzyme linked immunoassaying and double immunodiffusion. These results indicate that the porcine testis acylphosphatase is an isozyme different from the porcine skeletal muscle acylphosphatase.     

Studies on pituitary follitropin. V. Isolation of the subunits of the human hormone and reaction of the amino groups with acylating agents.

The alpha and beta subunits of human follitropin were isolated in a high state of purity. The tryptophan fluorescence of the native hormone and the isolated beta subunit are different. The N-terminus of the alpha and beta subunits was identified as valine and aspartic acid respectively. While recombination of the isolated alpha and beta subunits restores the electrophoretic mobility of the intact hormone, its receptor binding activity cannot be fully regenerated. Substitution of the human follitropin alpha by an ovine lutropin alpha subunit, to form a recombinant with the follitropin beta subunit, generates a complex with 2-3 receptor binding activity of the native human follitropin and the same activity as ovine follitropin. Acylation of the intact hormone does not disrupt the quaternary structure but leads to complete inactivation. Acylation studies with the subunits suggests the crucial role of the epsilon-amino groups of the alpha subunit in determining biological activity.     

Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.     

Isolation and characterization of the subunits of bovine follitropin.

Highly purified bovine follitropin was dissociated into its alpha- and beta-subunits after treatment with 1 M-propionic acid. The dissociated subunits were fractionated by chromatography on DEAE-cellulose and further purified by gel filtration on Sephadex G-100. The isolated alpha- and beta-subunits were biologically inactive, but their recombinants regenerated 80% of the follitropin activity. The alpha-subunit of bovine follitropin recombined with the beta-subunits of bovine lutropin and thyrotropin to regenerate 70% of lutropin and 50% of thyrotropin activities respectively. The beta-subunit of bovine follitropin recombined with the alpha-subunit of either bovine lutropin or thyrotropin to regenerate about 75% of follitropin activity. Recombinations were monitored by specific radioligand-receptor assays and polyacrylamide-gel electrophoresis. The elution volumes of the alpha- and beta-subunits of bovine follitropin after gel filtration on Sephadex G-100 were almost identical. The amino acid composition of bovine follitropin-alpha was low in histidine, arginine, isoleucine and leucine, but relatively high in lysine, threonine and glutamic acid. The bovine follitropin-beta contained one methionine residue and low amounts of histidine and phenylalanine, but relatively high in aspartic acid, threonine and glutamic acid. The N-terminal residues of the alpha- and beta-subunits of bovine follitropin were identified to be phenylalanine and glycine respectively.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Studies on pituitary follitropin. II. Isolation and characterization of the subunits of the ovine hormone.

The α and β subunits of highly potent ovine follitropin have been isolated by dissociation in 8 m urea, pH 7.5, and chromatography on DEAE-Sephadex A25. The isolated subunits display microheterogeneity on polyacrylamide gel electrophoresis and have very low activity in follitropin-specific radioreceptor and radioimmunoassays. The tryptophan fluorescence spectra of native follitropin and the isolated β subunit are different. The recombinant of follitropin α + β subunit had the same activity as the native hormone in the radioimmunoassay, but its activity in the radioreceptor and in vivo bioassay was about 65% of the intact hormone. Substitution of the follitropin α by ovine lutropin α subunit (prepared by a method not involving urea) to form the recombinant restored full activity in all the three assays investigated. The formation of recombined hormone proceeds at a rapid rate and is almost complete by 6 h. The α and β subunits of ovine follitropin differ from each other in amino acid composition. No significant differences were apparent in their carbohydrate composition. The amino acid composition of the ovine follitropin α and lutropin α subunits are very similar. The oxidized α subunit has phenylalanine at its NH₂-terminus while aspartic acid is present at this position in the oxidized β subunit.     

Specific interaction between follitropin and GM1 ganglioside incorporated into lipid membranes.

A specific interaction was demonstrated between a monosialo-ganglioside (galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide) incorporated in glycerol monoleate planar bilayer membranes and follitropin. Conductance of glycerol monoleate planar bilayer membranes containing gangliosides were measured before and after addition of follitropin in the aqueous phase. A 5 fold increase of membrane conductance was observed in presence of a specific monosialo-ganglioside. No significant effect was obtained with di- and trisialogangliosides. Membrane conductance changes were suppressed by the presence of an equimolar mixture of the saccharide residues (galactose, N-acetylgalactosamine and N-acetylneuraminic acid) present in the hydrophilic moeity of the ganglioside. The results favour the hypothesis that gangliosides may contribute to the formation of functional glycoproteins receptors.     

Purification and properties of bovine pituitary follitropin.

A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE2 binding sites in four subcellular fractions (F1-F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of 3HPGE2 to fractions F2-F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitrochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE2 binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.