Disassembly and assembly of glycoprotein hormones.
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Animals↗
Biomedical subjects
T F Parsons
Publications and source records attributed to T F Parsons.
Free alpha-like material from bovine pituitaries. Removal of its O-linked oligosaccharide permits combination with lutropin-beta.
Further characterization of the free alpha subunit immunoreactive material, not combined with beta subunit in extracts of bovine pituitaries, shows that the only significant modifications, relative to alpha subunits themselves, are the oligosaccharide O-linked to threonine-43, and heterogeneity of the carboxyl terminus. Removal of the O-linked carbohydrate with a mixture of glycosidases from Streptococcus pneumoniae results in an alpha-like material capable of combining with lutropin beta subunit and, thus, the presence of the oligosaccharide is responsible for the inability of the free alpha-like material to combine with beta subunits. Amino acid compositions of tryptic peptides spanning the entire sequence indicate no change in amino acid sequence of the free alpha-like material as compared to lutropin alpha. Further, based on the similar behavior reverse phase high performance liquid chromatography of the tryptic peptides as compared to their lutropin alpha counterparts, it is concluded that no additional post-translational modifications are present. The N-linked oligosaccharides of the free alpha-like material most likely contain terminal O-sulfated N-acetylhexosamines (as do the asparagine-linked carbohydrates from the pituitary hormones) as indicated by the presence of 3 mol of sulfate/mol of free alpha-like material and the resistance of these oligosaccharides to enzymatic deglycosylation. The O-linked oligosaccharide does not contain sulfated residues.
Amino Acid Sequence↗
Rapid and easy separation of the subunits of bovine and human glycoprotein hormones by use of high performance liquid chromatography.
A single method of reverse phase high performance liquid chromatography is used to separate the subunits of human and bovine glycoprotein hormones. This rapid and easy method is applicable for the separation and detection of subunits from as little as 10 micrograms hormone or the isolation of subunits from as much as 100 mg hormone. Separation is achieved by chromatography on a Vydac 218TP1010 column with a linear (60-min) gradient of 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide to a solvent containing 50% acetonitrile and 50% 0.1 M sodium phosphate, pH 6.8, plus 1 mM sodium azide. Although in some cases the interaction between the hydrophobic support and the hormone is sufficient for dissociation, preincubation of the hormone with guanidine hydrochloride ensures optimum dissociation and improves resolution of the subunits. The subunits isolated by high performance liquid chromatography are functional in that they will reassociate with their counterpart subunits.
Animals↗
Purification of an alternate form of the alpha subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide.
Extracts of bovine anterior pituitary glands contain significant amounts of material with immunological properties similar to those of the common, alpha, subunit isolated from the pituitary glycoprotein hormones. Purification of this "free alpha-like" material and analysis show it to contain an additional site of glycosylation not present in the alpha subunit isolated from intact glycoprotein hormones. This additional oligosaccharide is O-linked to a threonine residue corresponding to threonine-43 of bovine lutropin-alpha. Carbohydrate analysis shows 1.7 mol of sialic acid, 0.8 mol of galactose and 0.9 mol of galactosamine/mol of oligosaccharide. A similar structure for the free alpha-like material as compared to bovine lutropin-alpha is evident from equal potency in an anti-lutropin-alpha radioimmunoassay, a similar amino acid composition and similar but not identical peptide maps. The free alpha-like material is distinct from lutropin-alpha in that the free alpha-material contains sialic acid and galactose, has a slightly higher apparent molecular weight, an increased negative charge, and will not reassociate with native lutropin-beta. Peptide maps of the tryptic peptides of the free alpha-like material show additional differences (other than the O-linked oligosaccharide) when compared to peptide maps of lutropin-alpha; thus additional modifications are probably present.
Amino Acids↗
Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance.
Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.
Animals↗
Enzymatic deglycosylation of the subunits of chorionic gonadotropin. Effects on formation of tertiary structure and biological activity.
Both the O- and N-linked oligosaccharide moieties of the subunits of the placental glycoprotein hormone, human choriogonadotropin (hCG), are removed by treatment with a mixture of glycosidases produced by Streptococcus (Diplococcus) pneumoniae. The resulting deglycosylated subunits recombine with their native counterparts in good yield, and the reassociated hormones bind to gonadotropin receptors equally as well as the untreated hormone. Stimulation of steroidogenesis by the deglycosylated alpha-native beta recombinant, however, was markedly less than the stimulation by unmodified hCG both in terms of relative potency (0.10-0.15) and the maximal amount of steroid (40-50%) produced. The native alpha-deglycosylated beta recombinant produced a maximum level of steroid production of 80-90% that of control hCG although its relative potency had decreased approximately 4-fold. The data are in accord with results by others in which either hCG or lutropin was partially deglycosylated by treatment with anhydrous hydrofluoric acid. In addition, the effects of deglycosylation on the ability of each subunit to refold after reduction of their disulfide bonds was studied. Of particular interest is that, after deglycosylation, the beta subunit can correctly refold to a significant degree, in contrast to several unsuccessful attempts to demonstrate correct refolding of the unmodified beta subunit of either lutropin or hCG. Alpha subunit, as measured by a conformation sensitive radioimmunoassay, refolds with equal facility both before and after deglycosylation.
Animals↗
Glycoprotein hormones: structure and function.
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Amino Acid Sequence↗
Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa.
The ADPglucose pyrophosphorylases from Rhodopseudomonas sphaeroides and Rhodopseudomonas gelatinosa are activated by fructose-6-phosphate, pyruvate and fructose-1,6 biophosphate-P2. The effects of the activators are to increase significantly the Vmax of ADPglucose synthesis and to lower the S0.5 values (concentration of substrates giving 50% maximal velocity) for ATP and MgCl2. The R. sphaeroides enzyme is inhibited by Pi while the R. gelatinosa enzyme is inhibited by AMP as well as by Pi. The interaction between inhibitor and activator is complex. At very low concentrations of activator the enzyme is more sensitized to inhibition. However, at higher concentrations of activator there is a decrease in the sensitivity of the enzyme towards inhibition. The findings are discussed with respect to glycogen synthesis in these microorganisms and may be related to findings that indicate that Rhodopseudomonads have the ability to degrade sugars via the Entner-Duodoroff or Embden-Meyerhoff pathways.
Adenosine Diphosphate Glucose↗
Oligosaccharide moieties of glycoprotein hormones: bovine lutropin resists enzymatic deglycosylation because of terminal O-sulfated N-acetylhexosamines.
The oligosaccharides of the bovine pituitary gonadotropin lutropin are N-linked to asparagine residues. These carbohydrates are unusual in that, although they contain the mannose, N-acetylglucosamine, and fucose typical of N-linked oligosaccharides, they also contain one residue of N-acetylgalactosamine but insignificant amounts of sialic acid or galactose. These oligosaccharides exhibit complete resistance to several exoglycosidases. This is in contrast to the ready release of peripheral sugars from human chorionic gonadotropin, a placenta hormone which has oligosaccharides of the complex type with terminal sialic acid and galactose residues. Stability of the lutropin hexosamines to periodate oxidation and reduction (Smith degradation) together with other data show that one residue of N-acetylgalactosamine and one of N-acetylglucosamine are peripheral to two periodate-sensitive mannose residues. The insensitivity to periodate of these two terminal amino sugars is found to result from a sulfate group covalently linked to each; sulfation of these hexosamines is also the most probable reason for the resistance to enzymatic deglycosylation. The alpha subunits of bovine thyrotropin and human pituitary lutropin also contain sulfate, in contrast to human chorionic gonadotropin. The results indicate that sulfating enzymes are present in the pituitary and that sulfation of peripheral sialic acids in the placental gonadotropin. The data lead to a partial structure for the oligosaccharides of bovine LH as follows: (formula see text).
Acetylgalactosamine↗
Biologically active covalently cross-linked glycoprotein hormones and the effects of modification of the COOH-terminal region of their alpha subunits.
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Animals↗
Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.
The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.
Amino Acid Sequence↗
Glycoprotein hormones: similar molecules with different functions.
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Amino Acid Sequence↗
Purification and receptor binding properties of complexes between lutropin and monovalent antibodies against its alpha subunit.
A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its alpha subunit, which is common to the glycoprotein hormones, has been purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex is heterogenous with respect to molecular size; 70--80% of the hormone is complexed with either two or three Fab fragments. The LH-Fab alpha complexes retain only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permits direct measurement of competition between receptor and the bovine complex because the alpha portion of the human hormone does not cross react significantly with antibodies directed against bovine alpha subunits.) Complex formation does not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-beta immunoassay which distinguishes free beta subunit from intact LH. Complexing of LH with Fab-alpha fragments also causes little or no change in the affinity of the hormone's beta subunit for anti-LH-beta antibodies indicating that significant changes in beta subunit conformation did not occur. The data show that at least two well-separated antigenic regions on the alpha subunit are exposed to the surface in the intact hormone. They are also in agreement with the proposal that the loss of binding activity to receptor is due to steric effects rather than to changes in conformation or dissociation, and that there may be sites on the alpha subunit which interact directly with the receptor.
Animals↗
Biosynthesis of bacterial glycogen. Isolation and characterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site of Escherichia coli B ADP-glucose synthase.
[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of the allosteric effector, 1,6-hexanediol-P2, protects against pyridoxylation of the allosteric activator site and allows modification of the ADP-glucose-protected site. The activator site CNBr [3H]pyridoxyl-P peptide was purified to homogeneity in the presence of urea by Sephadex G-50 and CM-cellulose chromatography. The peptide consists of 59 residues, with a molecular weight of 6750. The NH2-terminal of the peptide has a 16-residue sequence overlap with the previously determined NH2-terminal sequence of the native enzyme. The activator site pyridoxyl-P lysine is identified as residue 38 of the native enzyme's NH2 terminus. The ADP-glucose-protected site CNBr [3H]pyridoxyl peptide was purified to homogeneity by Sephadex G-50 and DEAE-cellulose chromatography. The peptide consists of 21 residues, with a molecular weight of 2460. The sequence of this peptide has been elucidated.
Adenosine Diphosphate Glucose↗
Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.
[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.
Adenosine Diphosphate Glucose↗
Biosynthesis of bacterial glycogen. Activator-induced oligomerization of a mutant Escherichia coli ADP-glucose synthase.
ADP-glucose synthase (EC 2.7.7.27) was purified to homogeneity from SG5-504, Aa mutant strain of Escherichia coli B. It had a molecular weight of approximately 2 X 10(5) and four identical subunits. In the presence of its allosteric activator, fructose-1-6-P2, the mutant enzyme formed oligomers with several times the tetramer molecular weight, as revealed by sedimentation equilibrium centrifugation, polyacrylamide gel electrophoresis, and gel filtration column chromatography. Enzyme purified from a wild type strain, AC70R1, did not exhibit the activator-induced oligomerization. Catalytically, the mutant enzyme had a 2-fold higher affinity for activator and a 2- to 3-fold lower affinity for the allosteric inhibitors 5'-adenylate and KH2PO4 than the wild type. The two enzymes appeared identical in subunit molecular weight, amino acid composition, COOH-terminal amino acid, and the first 27 residues of the NH2-terminal sequence. Subtle structural differences were revealed by chemical modification of specific residues. Compared to wild type, the mutant enzyme had 1 cysteine/subunit more accessible to modification by Ellman's reagent. SG5-504 enzyme was inactivated more slowly than AC70R1 enzyme by phenylglyoxal and by trinitrobenzenesulfonate. These results reflected an altered conformation of the SG5-504 enzyme molecule. The unique ability of it to oligomerize in the presence of activator may be the result of a single amino acid substitution.
Amino Acid Sequence↗
Mechanistic implications of the pH independence of inhibition of phosphoglucose isomerase by neutral sugar phosphates.
In contrast to the strongly pH-dependent inhibition of phosphoglucose isomerase by substrate analogues with a free carboxyl group, inhibition of this enzyme by neutral sugar phosphates is essentially invariant between pH 7 and 9. Competitive inhibition constants for glucitol 6-phosphate (40 muM), arabinose 5-phosphate (50 muM), and erythritol 4-phosphate (100 muM) were found to be of the same order of magnitude as that reported previously for substrate binding constants (50 to 240 muM). The unique exception is erythrose 4-phosphate whose Ki (0.7 muM, independent of pH) reflects a tightness of binding similar to that found at pH values near or below neutrality for the transition state analogue 5-phosphorarabinonate. The pH independence of inhibition by erythrose 4-phosphate and other neutral sugar phosphates may reflect a mode and locus of binding to phosphoglucose isomerase different from that of the aldonate inhibitors.
Animals↗