Reversed-phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents.

Reversed-phase, high-pressure liquid chromatography has been successfully applied to the analysis of peptides and proteins by the addition of hydrophilic (for example, phosphoric acid) or hydrophobic (for example, hexanesulfonic acid) ion-pairing reagents, or both, to the mobile phase. Examples described included proteins such as insulin, glucagon, and 1-24 ACTH pentaacetate (ACTH is adrenocorticotrophic hormone).

Thermodynamic analysis of the stabilisation of pig heart mitochondrial malate dehydrogenase and maize leaf phosphoenolpyruvate carboxylase by different salts, amino acids and polyols.

As part of our investigations into the inactivation of pig heart mitochondrial malate dehydrogenase (phm-MDH) and maize leaf phosphoenolpyruvate carboxylase (ml-PEPC) in the presence of various cosolvents, the denaturation kinetics as a function of temperature have been determined based on Arrhenius plots derived from transition state theory analysis over the temperature range from 3.5 degrees C to 65 degrees C. The experimental data for phm-MDH were obtained in the presence of 1 M concentrations of various salts of monovalent and polyvalent anions, 1 M amino acids or 1 M sucrose and 6.1 M glycerol. Similarly, Arrhenius plot data were obtained for ml-PEPC in the presence of 2.5 M NaOAc and 0.8 M sodium glutamate. Distinct regimes of inactivation corresponding to high and low values of inactivation enthalpy were identified for the phm-MDH in the presence of all cosolvents and for the ml-PEPC in the presence of 2.5 M NaOAc, but not in the presence of 0.8 M sodium glutamate. A significant temperature-dependent effect dominated the inactivation of phm-MDH and ml-PEPC at elevated temperatures (e.g., > or = 45 degrees C), whilst the inactivation of these enzymes over a lower temperature range (< or = 25 degrees C) was dominated by temperature-independent phenomenon. The corresponding thermodynamic activation parameters (deltaG++, deltaH++ and deltaS++) associated with the transition state complexes involved in the inactivation of phm-MDH and ml-PEPC in the presence of the various cosolvents have been determined. The results indicate that the transition states associated with the inactivation of these two enzymes at elevated temperatures are characterised by large, positive enthalpic and entropic changes. In contrast, the inactivation process observed for phm-MDH at low temperatures in the presence of various cosolvents was marked by a large, negative entropic contribution and a small, positive enthalpic contribution. The results obtained in this study indicate that more than one mechanism of inactivation can occur with these two multimeric enzymes depending on the selected temperature range and the type of cosolvent. The relationship of these results to stabilisation models for phm-MDH and ml-PEPC in the presence of various cosolvents, as well as the application of Arrhenius plot data to extrapolate the long term solution stability of these enzymes at lower temperatures from the pseudo-first order rate constants of inactivation experimentally derived over a range of temperatures, are discussed.

Stability studies on pig heart mitochondrial malate dehydrogenase: the effect of salts and amino acids.

The effect of different salts and amino acids on the thermal stability and quaternary conformation of pig heart mitochondrial malate dehydrogenase (phm-MDH) in solution has been determined. The effectiveness of salts of anions in the stabilisation of phm-MDH followed the order: Citrate > SO(4)2- > or = Tartrate > Phosphate > F-, CH3COO- > Cl- > Br-. Anions above and including Cl- in this series were increasingly effective in stabilising phm-MDH with a rise in salt concentration from 0.05-2 M, whilst Br- was destabilising under similar conditions. The effect of potassium salts of acetate, chloride and bromide at a concentration of 1 M on the quaternary conformation of phm-MDH correlated also with the relative order of anion stabilisation above, with the anions higher in the series increasingly promoting the formation of the dimeric conformation of the enzyme. The cations of the corresponding salts had a relatively neutral (Cs+, K+, Na+, (CH3)4N+, NH4+) to a destabilising ((CH3)4N+, NH4+, Li+) effect on phm-MDH. Potassium ferrocyanide and potassium ferricyanide conferred complex, concentration dependent effects on the stability of phm-MDH, unlike the salts described above. Salts of amino acids were effective in the stabilisation of phm-MDH against temperature induced changes, following the order: NaGlutamatec = NaAspartate > NaGlycinate > lysine. HCl > arginine. HCl. The magnitudes and trends of the effects of these salts and amino acids on the stability and quaternary structure of phm-MDH were observed to correlate well with considerations based on the Hofmeister series of anions and solvophobic concepts as they apply to the influence of co-solvents at intermediate to higher concentrations. Other, more specific effects were also evident in the stabilisation and destabilisation of phm-MDH by low concentrations of the salts, as noted most particularly in the presence of potassium ferrocyanide and potassium ferricyanide.

Inhibition of activin A signalling in a mouse model of pre-eclampsia.

INTRODUCTION: Pre-eclampsia remains a major cause of maternal and fetal morbidity and mortality. Despite intensive research over the last 50 years, significant therapeutic advances have yet to be realised. We recently reported on the role of activin A in the pathophysiology of pre-eclampsia, whereby a pre-eclampsia-like disease state was induced in pregnant mice through activin A infusion. Using the same animal model, the effects of inhibiting activin A signalling on this pre-eclampsia-like disease state have now been assessed with low molecular weight compounds structurally related to activin-receptor-like kinase (ALK) inhibitors. METHODS: 23 synthetic compounds were screened for ability to reduce activin A-induced free radical production in HUVECs. Further, following administration of activin A (50 µg) via a subcutaneous mini-osmotic pump from day 10 of pregnancy, the most active inhibitor, MKP-1-140A, (1 mg/kg) was also concomitantly administered via subcutaneous injections. RESULTS: Significant reductions in activin A-induced systolic blood pressure and urine albumin:creatinine ratio were observed with inhibitor-treated animals. However, these findings were accompanied by sustained elevation of liver enzymes and albumin extravasation in the brains of pregnant mice that received MKP-1-140A. Furthermore, inhibition of activin A signalling with MKP-1-140A failed to rescue fetal growth restriction, and treatment with MKP-1-140A alone resulted in craniofacial and karyotypic abnormalities. DISCUSSION: These data indicate that whilst inhibition of activin A signalling by the low molecular weight ALK kinase inhibitor, MKP-1-140A, reduced some of the physiological manifestations of pre-eclampsia, the potential for serious maternal and fetal side effects may preclude it from clinical applications.

Microheterogeneous forms of radioiodinated bovine thyrotropin: discrimination of different receptor-active components by gel permeation chromatography.

The products of the radioiodination and subsequent receptor adsorption of bovine TSH (bTSH) radiolabeled by the lactoperoxidase method have been further investigated. After receptor adsorption, [125I]bTSH was resolved by gel permeation chromatography on Sephadex G-100 (superfine) under low ionic strength conditions into three peaks of radioactivity (tracers 2a, 2b, and 2c, respectively). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions demonstrated that each tracer component was radiolabeled on both the alpha- and beta-subunits. Analysis of the three tracers by TSH radioreceptor assay (under different radioreceptor assay conditions) showed that tracers 2b and 2c exhibited saturable rebinding to crude thyroid membranes containing functional TSH receptors. However, tracer 2c exhibited a maximum binding 2-fold greater than tracer 2b. This difference has been attributed to the abundance of an apparently low affinity binding component in tracer 2c. Rechromatography of tracers 2b and 2c on Sephadex G-100 (superfine) under high ionic strength conditions yielded tracer profiles that were coincident, demonstrating that the initial separation under low ionic strength conditions was not based on differences in molecular volume. The data indicate that radioiodination of highly purified bTSH yields multiple tracer components. Further, receptor adsorption, commonly used to purify freshly iodinated bTSH before radioreceptor assay, purifies at least two species of receptor-active [125I] bTSH.

Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography.

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

Isolation and physicochemical characterization of human follicle-stimulating hormone isoforms.

Twenty hFSH isoforms were isolated from human pituitary extracts, 15 of which were highly pure. The mild purification procedure, which used a FSH RRA to monitor FSH activity, involved an initial fractionation of pituitary extracts by gel filtration and isoelectric focusing. Six pI regions (mean pI values, 3.63, 3.88, 4.07, 4.23, 4.84, and 5.13) of human (h) FSH were obtained and further fractionated on ion exchange and gel filtration HPLC. Recoveries of FSH radioreceptor activity at each stage were greater than 80%. Fifteen isoform preparations were judged as near homogeneous by HPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibiting elution and migration behaviors consistent with the known mol wt of intact hFSH and its subunits. The remaining 5 isoform preparations contained a higher mol wt component that is probably hFSH related, as this component was detected after iodination, immunoprecipitation with hFSH antiserum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar amino acid compositions were obtained for the 20 hFSH isoforms, except for evidence of some oxidative degradation of serine, threonine, and tyrosine and decreased levels of glutamic acid, possibly due to carboxy-terminal heterogeneity of the beta-subunit. An average amino acid composition value for all isoforms was comparable to that of 2 other highly purified hFSH preparations. Using the First International Standard for pituitary hFSH (83/575) as standard, radioreceptor activities were obtained ranging from 7,800-56,300 IU/mg protein. It is concluded that a mild purification procedure for the isolation of hFSH isoforms has been developed which gives high recoveries and has enabled the isolation of 15 isoforms in high purity suitable for further physicochemical and biological characterization.

Hypoglycemic action of a novel constrained analog of human growth hormone-(6-13).

The amino-terminal region of human GH (hGH), in particular the amino acid sequence Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala[hGH-(6-13)], has been implicated as a functional region for the regulation of energy metabolism by exerting an insulin-potentiating action on insulin-sensitive tissues. Recent structural studies have revealed that the cyclization of the aspartate (Asp11) residue to form the alpha-aminosuccinimide (Asu11) ring is essential for the biological action of peptides related to this hGH fragment. The pharmacological application of these hGH-(6-13) peptides has been hindered by the vulnerability of the alpha-aminosuccinimide to hydrolytic modification leading to the loss of biological action. We have succeeded in stabilizing the structure of the Asu11-hGH-(6-13) peptide by replacing the alpha-aminosuccinimide ring with compatible and less rapidly metabolized gamma-lactam structures. In the present paper we report the bioactivity profile of an analog of hGH-(6-13) containing a gamma-lactam at residue position 11 that mimics the stereoelectronic and conformational characteristics of the alpha-aminosuccinimide ring. In vitro, the gamma-lactam11-hGH-(6-13) peptide analog increased [14C]glucose incorporation into glycogen in muscles and conversion to lipid in adipose tissues. In vivo, the gamma-lactam11-hGH-(6-13) peptide enhanced hypoglycemia during iv insulin tolerance tests. The results demonstrate that the gamma-lactam11-hGH-(6-13) peptide analog has similar biological properties to the Asu11-hGH-(6-13) peptide fragment, but with improved molecular stability and bioavailability.

Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid.

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.

Adrenocorticotropin, beta-endorphin, and beta-lipotropin in normal thyroid and lung: possible implications for ectopic hormone secretion.

The expression of the proopiomelanocortin (POMC) gene by normal lung and thyroid was examined by measurement of the content of ACTH, beta-lipotropin (beta LPH), and beta-endorphin (beta EP) in porcine lung and thyroid tissue. Acid extracts of normal porcine lung and thyroid tissue each contained appreciable amounts of immunoreactive (ir) ACTH, ir-beta LPH, and ir-beta EP. The content of ir-beta LPH in both tissues exceeded by severalfold, on a molar basis, the content of ir-ACTH and ir-beta EP, suggesting that the common precursor POMC was processed predominantly to peptides other than ir-ACTH and ir-beta EP. A porcine thyroid extract (Calcitare, porcine calcitonin, Armour) showed equivalent levels of beta EP-like immunoreactivity and bioactivity, measured by opiate radioreceptor assay; in contrast, ACTH-like bioactivity, measured by rat zona fasciculata steroidogenesis, was only 4% of ACTH-like immunoreactivity. On reversed phase high performance liquid chromatography, Calcitare showed multiple peaks of ACTH-like immunoreactivity, one of which coeluted with porcine ACTH-(1-39), and two much smaller peaks of beta EP-like immunoreactivity, of which the smaller coeluted with porcine beta EP. These data suggest that both lung and thyroid gland synthesize POMC, which in normal tissue is usually predominantly processed to species other than ACTH and beta EP. Ectopic secretion of ACTH and beta EP by lung and thyroid neoplasms may thus represent the loss of a system(s) normally responsible for processing the precursor beyond ACTH and beta EP.

Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid.

Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

Physicochemical and biological characterization of recombinant human inhibin A.

Recombinant human inhibin A was isolated from recombinant mammalian cell line culture media. Two forms of inhibin were identified with Mr of 34 and 31 Kd composed of subunits (alpha, beta) of 24 and 15 Kd and 21 and 15 Kd respectively. Both forms are bioactive in an inhibin in vitro bioassay and immunoactive with potencies comparable to or higher than purified bovine inhibin. Amino acid analyses and NH2-terminal sequences of each of the subunits are consistent with those predicted from their cDNA structures. The inhibin alpha- but not beta-subunit is glycosylated based on its binding to the lectins concanavalin A and wheat germ lectin. The difference in molecular weight of 31 and 34 Kd inhibin is attributed to variation in glycosylation of the alpha-subunit. The 31+34 Kd inhibin is heterogeneous on isoelectric focusing gels consisting of four isoforms in the pH range 6.2-7.6. Inhibition also exhibits in vivo biological activity by suppressing serum FSH but not LH in castrate male rats. These physicochemical and biological characteristics of recombinant human inhibin are similar to those described for native inhibin isolated from a variety of other species.

Regenerating neurons. Changes in protein phosphorylation.

We have been studying the phosphorylation of proteins of both normal and regenerating superior cervical ganglia of the rat. Here we report the incorporation of radioactive phosphate into proteins of ganglia homogenates incubated with 32P-labeled ATP under various conditions at day 3 after postganglionic axotomy. The proteins were analyzed by two-dimensional electrophoresis followed by autoradiography. Incubation in the presence of Ca2+ or Ca2+ plus cyclic AMP produced only about 20 spots corresponding to distinctly labeled proteins. This number was reduced to about five under EGTA plus cyclic AMP conditions, whereas the presence of EGTA alone suppressed the phosphorylation reaction almost totally. All these proteins fell within the narrow pI range of 4-6, whereby no qualitative differences between regenerating and control cases were observed. However, the growth-associated protein, variously designated GAP-43, B-50, F-1, and pp-46, had enhanced levels of phosphate incorporation in regenerating ganglia compared to controls. Injury also caused consistently higher levels of phosphorylation of proteins running in the position of alpha- and beta-tubulin. Since these three proteins are major constituents of regenerating axons, these results suggest that the changes in their phosphorylation induced by injury may be involved in the regulation of their transport.

Cloning and regulation of the rat activin betaE subunit.

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

Localization of basic fibroblast growth factor mRNA (FGF-2 mRNA) in the uterus of mated and unmated gilts.

In mated sows, the level of placental vascularization has a direct effect on fetal growth and litter birth weight. Vascularization of the endometrium and uterus under the control of various polypeptide growth factors is an important early stage in this process. Basic fibroblast growth factor (FGF-2), a polypeptide distributed throughout the mesodermal and neuroectodermal tissues of many species, is a vascular endothelial cell mitogen in vitro and has been implicated in neovascularization and wound healing in vivo. As part of our studies of the distribution of FGF-2 in uterine tissue and its role in placental development and embryo implantation, the localization and changes in the abundance of porcine FGF-2 mRNA in the uterus of mated and unmated gilts were investigated by in situ hybridization procedures. These procedures were based on the use of [alpha35S]-dATP-labeled oligonucleotide probes and a novel set of digoxigenin-labeled oligonucleotide probes generated by reverse transcriptase-polymerase chain reaction (RT-PCR) methods and anti-sense labeling strategies from the corresponding mRNA templates. With these in situ hybridization procedures, porcine FGF-2 mRNA was localized during the first 30 days of pregnancy to specific tissue areas in the porcine uterus comprising glandular and luminal epithelial cells and stromal cells of both the stratum functionalis and stratum basalis regions of the endometrium, and within the smooth muscle of myometrium and the associated blood vessels. However, no significant increase in the level of FGF-2 mRNA within these tissues was detected during these early stages of pregnancy or during the estrous cycle of unmated gilts. These distribution and abundance patterns are only partially compatible with other recent observations suggesting a possible role for changing levels of the mature polypeptide form of FGF-2 in the reproductive tract of sows during the early stages of pregnancy.

Isolation of FSH from bovine pituitary glands.

An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5.7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay. SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19.5 kDa and 15.8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with alpha- and beta-subunits, and a third minor (< 20%) sequence consistent with the alpha-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators.

Isolation and characterization of human LH isoforms.

Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (> 90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38,650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were < 0.5% and < 7.8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23.5-24.5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21.1-22.4 kDa and 18.0-20.5 kDa, probably corresponding to the alpha and beta subunits of hLH respectively. The remaining three less pure isoform preparations (70-90% pure) contained additional bands of 16 kDa and 26.3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11-14 kDa which was < 7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two alpha and two beta hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of alpha and beta subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.

Immunological activities of highly purified isoforms of human FSH correlate with in vitro bioactivities.

In a recent study, a five- to eightfold range in human FSH radioreceptor activity (RRA) was documented for highly purified isoforms of FSH when the data were expressed on an FSH protein content basis as determined by amino acid analysis. This study examined the FSH in vitro bioactivity and immunoactivity of these preparations. FSH in vitro biological activity showed a five- to eightfold range in activity with a high correlation with the RRA values (r = 0.82). A similar five- to eightfold range of values was obtained with a specific FSH radioimmunoassay and an FSH two-site immunoassay with high correlations again observed between each other, between each immunoassay and with either the in vitro bioassay or the RRA method (r = 0.77-0.995). Although there was overall a close correlation between these assays, significant differences in ratios of activities between the in vitro bioassay and other methods were observed with highly purified FSH isoform preparations from different pI regions. The high correlation between in vitro bioassay/RRA methods and immunoassay methods over a wide range of isoform specific activities suggests that these methods are detecting similar structural features on each isoform. It is thus concluded that these immunoassays are not solely measuring hormone mass based entirely on amino acid composition. This conclusion raises questions about ratio measurements of FSH, where immunoassay methods are presumed to measure total protein content, and their application in physiological situations and clinical practice.

Inhibin, activin and follistatin bind preferentially to the transformed species of alpha 2-macroglobulin.

alpha 2-Macroglobulin (alpha 2-M), a major serum glycoprotein, has been implicated as a low-affinity binding protein for inhibin and activin. In serum, alpha 2-M exists as two major species, a native form that is abundant and stable, and a transformed ('fast') species that is rapidly cleared from the circulation via alpha 2-M receptors. In this study inhibin, activin and their major binding protein follistatin were investigated for their ability to bind to the native or transformed species of purified human alpha 2-M. Using native PAGE and size exclusion chromatography, radiolabelled inhibin, activin and follistatin bound to the transformed alpha 2-M. Inhibin and follistatin did not bind significantly to native alpha 2-M, whereas activin was able to bind to the native species but with a lower capacity compared with that to transformed alpha 2-M. Under reducing conditions, binding of these hormones to alpha 2-M was abolished. These findings implicate alpha 2-M as a mechanism whereby inhibin, activin and follistatin may be removed from the circulation through alpha 2-M receptors, but also whereby activin can be maintained in the circulation through its ability to bind to native alpha 2-M.

Isolation of inhibin from ovine follicular fluid.

Two forms of inhibin with molecular weights of 65,000 and 30,000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in greater than 95% purity (1210-fold purification and 4.2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20-21 and 16 kD of which the 20-21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.