In order to evaluate the importance of carbohydrate side chains on the separate subunits in the biological action of human chorionic gonadotropin (hCG), we have treated each subunit with anhydrous hydrogen fluoride (HF) to remove carbohydrate and tested the products for recombination, immunologic recognition, ovarian receptor binding, and adenylate cyclase activation. The glycoproteins were reacted with H F for 1 h at 0 OC in the presence of anisole scavenger and separated from the reagents by gel filtration. The H F treatment removed 80% of total carbohydrate from, the a-subunit and 66% from the @-subunit. Treatment of hCG@ with H F for 3 h removed 85% of the carbohydrate. Chemical characterization, including compositional and sequence analysis after reduction and carboxymethylation, confirmed that the peptide backbone had remained intact and undamaged during the H F reaction. Circular dichroic spectra were unaltered. The deglycosylated hCG bound to antibody with a lowered affinity, suggesting either subtle conformational changes or involvement of the carbohydrate in the antigenic determinant. The HF-treated subunits recombined readily H u m a n chorionic gonadotropin (hCG),' like the other glycoprotein hormones (LH, FSH and TSH), contains a series of carbohydrate side chains on both the a-(common) and @-(hormone-specific) subunits. Accounting for about 30% of hCG molecular weight, they are comprised principally of branched long-chain carbohydrates attached to asparagine residues at positions 52 and 78 in a-subunit and 13 and 20 in fl-subunit. There are also four short-chain carbohydrates linked to serine residues in the C-terminal region of the flsubunit of hCG at positions 121, 127, 132, and 138.
The aquatic leech, Theromyzon rude, secretes a £ex-ible, proteinaceous cocoon that is resistant to a broad range of denaturing conditions (e.g. heat, denaturing chemicals). We have partially solubilized the Theromyzon cocoon membrane in 10% acetic acid and identi¢ed two major protein fragments. Microsequencing of both Theromyzon cocoon protein (Tcp) fragments generated an identical stretch of the amino-terminal sequence that was used to clone the corresponding gene. The predicted linear amino acid sequence of the resulting cDNA contained an unusually high cysteine content (17.8%). Sequence analysis identi¢ed six internal repeats, each comprising 12 ordered Cys residues in a V V62 amino acid repeating unit. Sequence comparisons identi¢ed homology with undescribed, Cys-rich repeats across animal phyla (i.e. Arthropod, Nematoda). ß
Evidence indicates that dietary lipids influence adrenocortical function. In the present study, weanling rats were fed isocaloric synthetic diets for 6 and 12 months that contained 10% of one of the selected fatty acids as the predominant lipid: butter fat (high saturated, low polyunsaturated fat); olive oil (monounsaturated); corn oil (polyunsaturated); omega-3 ethyl ester mixture (long-chain polyunsaturates); elevated eicosapentaenoic acid; elevated docosahexaenoic acid. Adrenocortical cells derived from individual rats were evaluated for corticosterone and aldosterone responses to adrenocorticotropic hormone (ACTH). All comparisons were to the butter fat diet. Adrenocortical cell sensitivity to ACTH was not affected by the diets. However, there were differences in basal and maximal ACTH-induced corticosteroid production. Compared to the butter fat diet, the other diets variably decreased cellular corticosteroid production. Corticosterone and aldosterone production were affected similarly. The greatest decrease was most often seen with the omega-3 mixture diet (about -67%). At 6 months, the docosahexaenoic acid-elevated diet had selective suppressive actions on adrenocortical function whereas at 12 months, both docosahexaenoic and eicosahexaenoic acid-elevated diets had similar suppressive efficacies. The data indicate that a diet rich in high saturated, low polyunsaturated fat augments adrenocortical function and increasing the representation of long-chain unsaturated fatty acids suppresses adrenocortical function.
The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.
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