Ovine luteinizing hormone. II. Effects of castration and steroid administration on the levels of uncombined subunits within the pituitary

Pituitaries were removed from rams, wethers, and wethers that received Silastic implants containing 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2) or DHT + E2. After homogenization and centrifugation (100,000 X g), aliquots of the supernatants were subjected to analytical gel filtration on Sephadex G-100 Superfine to separate native ovine luteinizing hormone (oLH) from its uncombined subunits. Immunoreactive oLH and oLH subunits were quantified in the elution profiles to examine the effects of castration and gonadal steroid administration on the intracellular levels of uncombined oLH subunits. Pituitaries from rams contained 1.41 +/- 0.26, 0.191 +/- 0.024, and 0.0246 +/- 0.0043 micrograms oLH, oLH alpha and oLH beta per mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.29 and approximately equal to 0.04. Castration decreased the concentrations of oLH and its subunits by approximately 50%, but did not significantly alter the oLH alpha/oLH and oLH beta/oLH molar ratios. All three steroid treatments further decreased the concentrations of oLH and oLH beta. Pituitaries from DHT-implanted wethers exhibited similar oLH alpha/oLH and oLH beta/oLH molar ratios to rams and unimplanted wethers. However, in E2- or DHT + E2-implanted wethers, there was a greater reduction in the concentration of native oLH than in the uncombined subunits. Thus, both the oLH alpha/oLH and oLH beta/oLH molar ratios were significantly higher in E2- or DHT + E2-implanted wethers than in the other groups. The apparent molecular sizes of oLH or its subunits were not significantly altered by castration or steroid administration. These results suggest that DHT and E2 decrease the concentrations of uncombined oLH beta as well as native oLH in the pituitary, but do not appear to alter the apparent molecular size of either oLH or its uncombined subunits However, because the levels of uncombined subunits were not decreased to the same degree as oLH in E2-implanted wethers, estrogens may affect the process of oLH subunit combination or may result in the production of molecular forms of oLH that are easier to dissociate.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Ovine luteinizing hormone. III. Relationships between the charge heterogeneity of partially purified subunits and the native hormone

Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 X g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed: six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI greater than 9.8), and one was bound to the column (apparent pI less than or equal to 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH alpha, and oLH beta were present at concentrations of 0.907 +/- 0.127, 0.089 +/- 0.020, and 0.010 +/- 0.023 microgram/mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.19 and approximately equal to 0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH alpha and oLH beta) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH alpha in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (approximately equal to 37%) of acidic components when chromatofocused, while more than 97% of purified oLH alpha focused as basic forms having pIs greater than 8.9. When uncombined oLH beta in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI less than or equal to 7.0); purified oLH beta displayed a nearly identical pattern. These results suggest that native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity.

The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.     

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.     

Carbohydrate chains of human thyrotropin are differentially susceptible to endoglycosidase removal on combined and free polypeptide subunits

The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free alpha and beta subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. We show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K beta subunit of hTSH could be readily deglycosylated to a 14K species while the 22K alpha subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free alpha subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-alpha and hTSH-beta could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on alpha and the single oligosaccharide present on beta in hTSH appeared to be cleaved by pure endoglycosidase F. Interestingly, one of the two carbohydrate chains present on alpha was also found to be susceptible to endoglycosidase H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the expression products of recombinant human choriogonadotropin and subunits

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.     

Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms

Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.     

Properties of equine luteinizing hormone alpha subunit alone and in combination with various beta subunits

Previous studies have shown that equine luteinizing hormone (eLH) inhibits production of cyclic adenosine monophosphate (cAMP) induced by follicle-stimulating hormone (FSH) in preparations of seminiferous tubules from immature rats. It was also shown that the inhibitory effect was a function of the equine LH (eLH) alpha subunit. To explore this phenomenon further, the intrinsic FSH-like activities of eLH alpha alone and in combination with ovine (o) LH beta, ovine FSH beta, and equine FSH beta were evaluated in several assay systems. In a radioreceptor assay employing 125I-o-FSH and testis membranes from day-old calves, eLH was twice as active as oFSH, eLH alpha was 6% as active as oFSH, and other subunits showed a lack of activity (less than 1.5%). Whereas oLH was only 0.1% as active as oFSH, the hybrid eLH alpha-oLH beta was 3.0% as active. The binding activity of eLH alpha-FSH beta hybrids tended to be higher than the oFSH alpha-FSH beta hybrids. In the cAMP production assay, eLH alpha-FSH beta hybrids exhibited dampened dose-response curves when compared to the oFSH alpha-FSH beta hybrids. In a plasminogen activator assay (PAA) employing granulosa cells from intact 21-24-day-old female rats primed with diethylstilbestrol, eLH had activity comparable to that of oFSH, while eLH alpha was inactive. When eLH alpha was recombined with oFSH beta, eFSH beta, or oLH beta, the PAA stimulatory activity was not altered compared to that of the hybrids oLH alpha-oFSH beta, oFSH alpha-eFSH beta, and the recombinant oLH alpha-oLH beta, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro activation of glycoprotein hormones. Hybridization of subunits from thyrotropin, lutropin and human choriogonadotropin.

In vitro assembly of thyrotropin alpha and beta subunits led to an increase in content of alpha helix and beta sheet very similar to that found for gonadotropins. This association-dependent active folding involved the burying of three tyrosine residues tentatively assigned to Tyr alpha 41, Tyr beta 37 and Tyr beta 59 and common to all studied glycoprotein hormones. In vitro hybridizations between alpha and beta subunits of various hormones (thyrotropin, lutropin and choriogonadotropin) from different species (ovine, bovine and human) triggered the same molecular events as assembly of homologous subunits: the burying of three tyrosine residues and the increase of periodic structure of the folding. These changes are slow, time-dependent processes. Rates and yields of hybrid formation measured by sedimentation analysis and difference spectroscopy of tyrosines are identical, within experimental error, with the rates and yields measured by the recovery of the biological activity either the stimulation of chick thyroids for thyrotropin-beta hybrids or binding to porcine testis receptors for gonadotropin-beta hybrids. Whatever the origin of the alpha subunit, the thyrotropin-beta hybrids were not able to bind to testis receptors although active on chick thyroids. Rates and yields of hybrid formation essentially depended on the origin of the beta subunit. All the hybrids could be dissociated at acid pH with rates similar to those of native hormone. The extension to thyrotropin and various hybrids of the structural features of the in vitro assembly already recognized for gonadotropins strengthens the hypothesis that one deals with a basic activation process which also occurs in vivo after the synthesis of the subunits.     

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.     

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.     

Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells

The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form. In bovine pituitary an extra O-linked oligosaccharide is added to free alpha subunit, and this modification has recently been detected at an analogous position (threonine 39) on human alpha subunit secreted by choriocarcinoma cells. To assess the contribution of N-linked and O-linked oligosaccharides to the heterogeneity of human free alpha subunit, we have compared free alpha with human chorionic gonadotropin alpha secreted by explants and cultured cytotrophoblasts of human first trimester placenta. We have also examined the free and combined forms of human alpha subunit expressed in transfected C-127 mouse mammary tumor cells. Processing of the alpha subunit in placental and C-127 cells was similar. Tryptic mapping of placental-derived and transfected alpha subunits indicated that O-glycosylation at threonine 39 was not a major modification. In the presence of the oligosaccharide processing inhibitor swainsonine the difference in size between the free and combined forms of alpha was eliminated in both placental and C-127 cells, indicating that the two forms of alpha differed in their N-linked oligosaccharides. Furthermore, the oligosaccharides of free alpha subunits from placental and transfected cells were resistant to endoglycosidase H, but the combined forms of alpha were partially sensitive to the enzyme. Thus, in human first trimester placenta and mouse C-127 cells, combination of alpha with human chorionic gonadotropin beta alters the processing of N-linked oligosaccharides on alpha subunit.     

Further characterization of the ovine lutropin alpha and beta subunits prepared by the salt precipitation method.

The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity.     

The voltage-sensitive sodium channel from rabbit skeletal muscle. Chemical characterization of subunits

The alpha and beta subunits of the rabbit skeletal muscle sodium channel have been separated and isolated preparatively under denaturing conditions. In this sodium channel, the beta subunit is not linked covalently to the alpha subunit. The isolated subunits have been subjected to amino acid and carbohydrate analysis. Both subunits are heavily glycosylated (alpha = 26.5%, beta = 29.7% carbohydrate by weight) with N-acetylneuraminic acid and N-acetylhexosamines representing the predominant monosaccharides in each. Enzymatic deglycosylation with neuraminidase and endoglycosidase F yielded single core peptides of approximately 209 kDa for the alpha subunit and 26.5 kDa for the beta subunit. Based on the known carbohydrate composition, the molecular masses for the glycosylated subunits are, therefore, 285 and 37.5 kDa for alpha and beta, respectively. Using the isolated subunits, we calibrated our protein-labeling system on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined the subunit stoichiometry for the rabbit skeletal muscle channel; in the native preparation, the molar ratio of alpha:beta is 1 : 1.     

The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.     

Effect of conformation of hCG-beta on generation of hCG-specific antibody

Most antisera generated to isolated highly purified beta subunits of human glycoprotein hormones are not sufficiently sensitive to detect physiologic blood levels of the native hormone. In the dissociated state, beta subunits assume a conformation different from that in the native hormone. Since antisera to alpha subunits have essentially no cross-reactivity between species, highly purified hCG-beta was combined with bTSH-alpha. That hybrid served as immunogen to assess whether sensitive, specific hCG antisera would more likely result than using hCG-beta alone. Of five animals immunized, three developed sufficiently sensitive and specific antisera. The results of these studies strongly suggests that human glycoprotein beta subunits combined with non-human alpha subunit are more likely to yield specific, sensitive antisera than when either isolated beta subunit or the native human glycoprotein hormone, containing common alpha determinants, serves as immunogen.