Dissociation and recombination of the subunits of human chorionic gonadotropin

Swaminathan, N.; Bahl, Om P.

doi:10.1016/0006-291x(70)91026-0

Cited by 177 publications

(40 citation statements)

References 6 publications

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“…One glycoprotein secretory product, HCG, has been localized to the syncytium and is known to contain galactose (47). However, placental secretion of this glycoprotein hormone is highest in first trimester and lowest in term placentas.…”

Section: Nelson ~T XL Glycoprotein Synthesis In Cellular and Syncytimentioning

confidence: 99%

Cytological events involved in glycoprotein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]galactose incorporation.

Nelson

Enders

King

1978

The Journal of Cell Biology

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Electron microscope autoradiography was used to study glycoprotein synthesis in cellular trophoblast (cytotrophoblast) and syncytial trophoblast of term human placental villi incubated in vitro with D-[1-aH]galactose ([SH]gal). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15 [aH]gal incorporated into cytotrophoblast during the pulse incubation remained intracellular through the duration of the experiment. There was little autoradiographic evidence for secretion of tritiated macromolecules. Cytotrophoblast incubated for the longest time period studied (4 h +) showed a substantial concentration of tritiated macromolecules in the Golgi complex and in the ground plasm but not in the rough endoplasmic reticulum.KEY WORDS placenta glycoproteins electron microscopy trophoblast 9 autoradiography Tritiated monosaccharides have been used in a number of studies to label glycoproteins whose synthesis and secretion are to be followed by light and/or electron microscope autoradiography. These studies have demonstrated that some glycoproteins that are synthesized remain within the cell (5,6,15,37,38,43) while others are added either to the surface coat (3,4,6,23,28,29,41) or to the basal lamina (12, 13). In other cases, the glycoprotein synthesized is destined for secre- 418

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Section: Nelson ~T XL Glycoprotein Synthesis In Cellular and Syncytimentioning

confidence: 99%

Cytological events involved in glycoprotein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]galactose incorporation.

Nelson

Enders

King

1978

The Journal of Cell Biology

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“…In the case of ␣␤-dimerization, the situation is complicated by the presence of cystine knots (57) and the "seat belt" structure that is formed by a 21-amino acid arm of the ␤-subunit that "embraces" the ␣-subunit (58,59). Native hCG can be readily dissociated into its subunits by reduction and alkylation (60), but the usual mode of subunit preparation is by dissociation at low pH in the presence of urea (60,61). Prevention of disulfide bridge formation in vivo does not interfere with heterodimer formation, as demonstrated above.…”

Section: Pulse-chase Kinetics Of the Hcg Subunits In The Unperturbedmentioning

confidence: 99%

Disulfide Bond Formation Is Not Required for Human Chorionic Gonadotropin Subunit Association

Singh¹,

Merz

2000

Journal of Biological Chemistry

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To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER) in vivo. In the presence of 5 mM DTT during pulse-chase experiments all of the ␤-subunit precursors observed in unperturbed cells (p␤ 0 , p␤ 1 , p␤ 2 , and ␤ * ) collapsed into the p␤ 0 form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the p␤ 0 precursor from 8.8 to 65.2 min. Under reoxidation conditions, the ␤-subunit precursors folded back from p␤ 0 in less than 5 min. In unperturbed JEG-3 cells, the ␣-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-␣) forms. The attachment of the second N-linked glycan residue of the ␣-subunit was accelerated in the presence of DTT, and consequently pre-␣-subunit was missing from the DTT-treated cultures. The formation of ␣␤-dimers appeared to be at least partially independent of the oxidation state in the ER. The ␣␤-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mM DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place.Secretory and membrane glycoproteins of eukaryotic cells are co-translationally translocated into the lumen of the endoplasmic reticulum (ER) 1 from where they travel to Golgi complex on the secretory pathway and to other destinations. Recently, it became evident that the transport of proteins out of the ER is limited by a unique "quality control" system that involves recognition and retention of misfolded or misassembled proteins. If further attempts of acquiring the correct folding fail, these proteins may be directed into a degradation pathway (1-4). In the case of oligomeric proteins, the correct formation of disulfide bonds plays an important role in the assembly of secretory and membrane proteins (5-7), which in turn determines stability, intracellular transport, maturation, and function.The disulfide bonds are generated through oxidation in the ER. The ER lumen is unique among the various compartments in the eukaryotic cells because it provides an oxidizing environment for the disulfide bond formation with the help of the protein disulfide isomerase that promotes the disulfide bond formation (8,9). Recently, it was demonstrated that the cotranslational disulfide bond formation, folding, and oligomerization of proteins within the ER can be reversibly inhibited by the addition of the disulfide bridge disrupting agent dithiothreitol (DTT) to living cells (10 -12). Interestingly, upon the removal of DTT, the disulfide bond formation, folding via normal ER folding intermediates, and oligomerization seems to take place (10 -17). Moreover, DTT does not inhibit the transport within t...

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“…Fraction 2 with the highest biological activity displayed no outstanding absorption features. The differences in the spectra of the a and/3 subunits of HCG were to be expected, as it is known that they have different molecular weights, amino acid and carbohydrate contents [7,8]. The significance of the two Cotton effects of the native HCG, the IEF fractions and the HCG subunits is not yet clear.…”

Section: Hcgmentioning

confidence: 99%

“…Swaminathan and Bahl [7] and Morgan and Canfield [8] separated two subunits, ct and ~, from a highly purified HCG, with different molecular weights, amino acid and sugar composition.…”

Section: Introductionmentioning

confidence: 99%