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.     

Studies on pituitary follitropin. III. Functional role of the amino groups in subunit and receptor interactions of the ovine hormone.

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6-P and 1,5-anhydroglucitol-6-P provided substantial protection while 2-deoxyglucose-6-P, fructose-6-P, mannose-6-P, and galactose-6-P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys.185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6-P binding sites, first reported for Glc and Glc-6-P by Ellison et al. (1975, J. Biol. Chem.250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6-P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6-P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6-P and mannose-6-P were estimated to be ?0.5 mm.     

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

1) A reproducible procedure was developed for the purification of bovine follitropin. 2) The method involved ammonium sulfate precipitation, ion exchange and adsorption chromatography, concanacaline-A-Sepharose chromatography and gel filtration. 3) A specific radioligand receptor assay was used to monitor each chromatographical step. 4) The potency of highly purified bovine follitropin as measured by Steelman and Pohley bioassay was 62 times the NIH-FSH-B1 standard preparation. 5) Contaminations of bovine follitropin by other glycoprotein hormones such as thyrotropin and lutropin amounted to 3 and 0.45 per cent by weight respectively as measured by specific radioimmunoassays and radioligand receptor assays. 6) The subunits alpha and beta of bovine follitropin were obtained by incubation in acidic urea, the chains being then separated by anion exchange chromatography. The subunits were subjitted to complete characterization. The amino-terminal residue of the alpha subunit is phenylalanine while a half cystine residue was found at the aminoterminal end of the beta chain. 8) Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 0.02 and 0.1 per cent by weight respectively.     

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.     

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.     

Studies on pituitary follitropin. IV. A conformation specific radioimmunoassay for the ovine hormone

An antiserum to partially purified ovine follitropin (50 x NIH-FSH-S10) shows species specificity. It is conformation dependent and requires the proper recombination of the alpha and beta subunits for maximal reactivity. The isolated alpha subunit is essentially inactive and the hormone specific beta subunit is weakly reactive. The homologous radioimmunoassay is valuable for estimating native ovine follitropin in the presence of free subunits. It also provides a sensitive method to study association-dissociation and structure-function relationships.     

Human pituitary thyrotropin. Isolation and characterization of the hormone and its subunits.

Procedures have been described for the isolation of highly purified thyrotropin form frozen or acetone-preserved glands or from side fractions of somatotropin isolation and for the separation of its alpha and beta subunits. The products have been characterized by terminal residue analyses, amino acid composition, carbohydrate content, disc electrophoresis, ultracentrifugation, and biological activity.     

Studies on pituitary follitropin. I. An improved procedure for the isolation of highly potent ovine hormone.

An improved method of isolation of ovine pituitary follitropin has been described. The method involves extraction of frozen glands at pH 9.0 in presence of an enzyme inhibitor, metaphosphoric acid precipitation at two stages, ammonium sulfate fractionation, glycoprotein fractionation, ion exchange chromatography on SP-C50 followed by affinity chromatography on concanavalin A-Sepharose, and filtrations on Sephadex G-100. A highly active preparation with a yield of about 11 mg/kg is obtained and has an activity of about 110 × NIH-FSH-S10 standard in the human chorionic gonadotropin (HCG)-augmentation assay in rats. Its lutropic activity as estimated by specific in vitro bioassay and radioreceptor assay was about 0.005–0.01 unit (NIH-LH-S19)/mg. It is more acidic than lutropin and thyrotropin. Its amino acid composition and carbohydrate content has been analyzed. Its isoelectric point is approximately pH 4.9.     

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.     

Isolation of mRNA from bovine pituitary. The cell-free synthesis of the alpha and beta subunits of luteinizing hormone

RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured alpha and beta subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated alpha subunit protein was approximately 14,000, while that of the beta protein was estimated to be 16,000. Since the molecular weights of authentic alpha and beta subunits are 10,600 and 14,000 respectively, the cell-free products presumably represented their pre-protein forms. The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin alpha and beta mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the alpha and beta immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin alpha and beta mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin alpha and beta subunits is coordinately expressed in certain physiological situations.     

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.