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D Puett

Publications and source records attributed to D Puett.

At least 73 records · Page 4Linked to original sources
PubMed1993

Mutagenesis of the 'determinant loop' region of human choriogonadotropin beta.

The hormone-specific beta subunits of the four human glycoprotein hormones are homologous, and mapping studies are underway in many laboratories to delineate the amino acid residues responsible for receptor binding and activation. Results on the human choriogonadotropin beta (hCG beta) subunit, obtained using synthetic peptides, chemically modified derivatives, and mutant forms prepared via site-directed mutagenesis, have suggested that amino acid residues enclosed by the purported disulfide loop between Cys-93 and Cys-100 may contribute to receptor binding and perhaps specificity. Indeed, the 93-100 amino acid sequence is referred to as a determinant loop. We have used site-directed mutagenesis to prepare single amino acid residue replacements at positions not previously investigated in full length beta subunits; these include Arg-95, Ser-96, Thr-97, and Thr-98. In addition, Leu-92 was studied in an effort to determine whether changes immediately adjacent to the determinant loop alter receptor binding. The wild-type and mutant cDNAs for hCG beta were subcloned into a Prsv expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The concentrations of total expressed hCG beta in heterodimer form with the bovine alpha subunit were determined by radioimmunoassays. The mutant gonadotropins were assayed in vitro using a competitive binding assay with [125I]hCG and progesterone production, both in the transformed murine Leydig cell line, MA-10. Mutant beta subunits containing the replacements Lys-92, Ser-95, Asp-96, and Tyr-97 exhibited normal alpha subunit binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells.

The effects of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen to various cell types, on proliferation and differentiated functions of the murine Leydig tumor cell line MA-10 were investigated. Kinetic binding experiments at room temperature showed that [125I] ET-1 bound to MA-10 cells and reached equilibrium in 2 h. The data from competitive binding experiments yielded an apparent single class of high affinity binding sites characterized by a Kd and maximum binding capacity of 1 nM and 59 fmol/10(6) cells, respectively. For steroidogenic assays, cells were incubated with ET-1 (1 pM to 1 microM) and with epidermal growth factor (10 ng/ml) for 4 h at 37 C, and the progesterone levels in the medium were measured by RIA. Like epidermal growth factor, ET-1 caused about a 6-fold increase in progesterone production. ET-1 also enhanced the transient expression of the protooncogenes c-jun and c-myc by 3- and 2-fold, respectively. For proliferation studies, ET-1 (1 pM to 1 microM) was added to quiescent MA-10 cells for 24 h, and cell counts were performed; no increase in cell number was observed. The results of this study demonstrate that MA-10 cells possess high affinity binding sites for ET-1 and that ET-1 stimulates progesterone production and protooncogene expression, but not mitosis in this cell line.

Animals↗
PubMed1993

Lysine residues 2 and 104 of the human chorionic gonadotrophin beta subunit influence receptor binding.

Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common alpha subunit and a hormone-specific beta subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian beta subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-beta. Using site-directed mutagenesis, each of these two lysines in hCG-beta was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine alpha subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine alpha subunit bound to hCG-beta (Glu2) and to hCG-beta (Glu104), as well as with the control, i.e. the bovine alpha subunit bound to the hCG-beta wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant beta subunit exhibited bioactivity, but their potencies were lower than that of the bovine alpha subunit bound to the hCG-beta wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-beta participate, either directly or indirectly, in receptor binding.

Amino Acid Sequence↗
PubMed1992

The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin.

The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A single amino acid residue replacement in the beta subunit of human chorionic gonadotrophin results in the loss of biological activity.

The heterodimer, human chorionic gonadotrophin (hCG), contains an alpha subunit that is common to the glycoprotein hormones and a hormone-specific beta subunit. A comparison of all known beta amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-beta) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-beta Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine alpha subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-beta cDNAs. The Arg99 beta mutant associated with the alpha subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.

Amino Acid Sequence↗
PubMed1991

Contributions of arginines-43 and -94 of human choriogonadotropin beta to receptor binding and activation as determined by oligonucleotide-based mutagenesis.

Members of the glycoprotein hormone family contain a common alpha subunit and a hormone-specific beta subunit. Human choriogonadotropin (hCG) beta is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG beta, we have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin beta amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact beta chains suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin beta, Leu-43 and Asp-94. The wild-type and mutant beta chains were expressed in CHO cells containing a stably integrated gene for bovine alpha, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard 125I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG beta wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine alpha, but the resultant heterodimer exhibited only low activity. Replacement of each arginine with lysine yielded heterodimers that were at least as potent as bovine alpha-hCG beta wild type, but the Lys-43-containing beta chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG beta wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.

Amino Acid Sequence↗
PubMed1991

Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity.

The four human glycoprotein hormones are heterodimers that contain a common alpha subunit and a hormone-specific beta subunit. Within this hormone family, 23 amino acid sequences from 11 mammalian species are available. There are 19 invariant amino acid residues in the beta subunits, 12 of which are Cys that form six disulfide bonds. Of the remaining seven conserved amino acid residues, we have investigated the role of an Asp which occurs at position 99 in human choriogonadotropin beta (hCG beta). Site-directed mutagenesis was used to replace hCG beta Asp99 with three residues, Glu, Asn, and Arg, and to prepare an inversion double mutant protein, Arg94----Asp and Asp99----Arg. The cDNAs were placed in a eukaryotic expression vector, and the plasmids were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Radioimmunoassays demonstrated that the mutant forms of hCG beta were capable of subunit assembly to the same extent as hCG beta wild type. The heterologous heterodimers were assayed in vitro using transformed mouse Leydig cells (MA-10) by competitive inhibition of 125I-hCG binding and stimulation of progesterone production. The gonadotropins containing Glu and Asn were active, although the potency was less than that associated with the hCG beta wild type-containing gonadotropin. In contrast, the Arg99-containing mutant protein and the inversion mutant protein Asp94/Arg99 were devoid of activity. Thus, in hCG beta Asp99 can be substituted with certain residues without total loss of function, although replacement with a positively charged residue leads to an inactive heterodimer. The primary role of Asp99 in hCG beta seems to involve, either directly or indirectly, receptor recognition.

Amino Acid Sequence↗
PubMed1991

Effect of 4-hydroxyandrostenedione on murine Leydig tumor cell steroidogenesis.

The murine Leydig cell tumor (M5480A) possesses high levels of estrogen receptor and is known to produce estrogens. In these studies we examined the effects of the potent aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) on Leydig tumor cell steroidogenesis both in vitro and in vivo. The addition of 4-OHA to Leydig tumor cells in primary culture resulted in a dose- and a time-dependent decrease in media progesterone levels. The observed decrease was most likely due to impaired synthesis of progesterone, inasmuch as no alteration in progesterone metabolism was seen when progesterone levels were diminishing. However, 4-OHA inhibited progesterone conversion to testosterone following 1 h of incubation, but this effect disappeared coincident with 4-OHA metabolism. Analysis of pregnenolone production revealed a biphasic dose-dependent effect of 4-OHA. At low doses (0.01-0.1 microM), 4-OHA was found to decrease pregnenolone concentrations, while at higher doses (1-10 microM) pregnenolone levels were elevated. Therefore, the actions of 4-OHA on Leydig cell steroidogenesis in vitro appear to be multifocal. Other experiments were performed to evaluate the effects of 4-OHA on tumor-bearing male mice in vivo. In these studies, the predominant effects of 4-OHA were to act as an aromatase inhibitor and to inhibit progesterone production. Thus, while 4-OHA is a potent aromatase inhibitor, we have found that this compound may alter steroidogenesis in Leydig tumor cells at several sites prior to aromatization.

Androstenedione↗
PubMed1991

Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity.

The choriogonadotropin beta subunit is unique in the human glycoprotein hormone family in containing a carboxyl-terminal extension, with four sites of O-glycosylation, that is not present in the other beta subunits. We have used site-directed mutagenesis to define boundaries on the extent to which truncations can be made at the COOH terminus without abolishing subunit assembly and biological activity. Two COOH-terminal deletion mutant chains of human choriogonadotropin beta, des(93-145) and des(101-145), were prepared and expressed in Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The heterologous gonadotropin, bovine alpha-human choriogonadotropin des(101-145) beta, formed a heterodimer and, when assayed with transformed murine Leydig cells in vitro, competed with the binding of standard human choriogonadotropin and stimulated both cAMP and progesterone production, albeit with a reduced potency relative to bovine alpha-human choriogonadotropin beta wild type. In contrast, human choriogonadotropin des(93-145) beta, expressed under identical conditions in the presence of bovine alpha, failed to form heterodimer and thus exhibited no competitive binding and was without effect on cAMP and progesterone levels. Consequently, removal of the putative determinant loop region of the beta subunit (residues 93-100), which is believed to be important in determining receptor specificity, abolishes association with alpha. Hence, in addition to its possible role as a receptor determinant, this region of the molecule appears to be critical for proper folding or subunit interaction. The truncated form of human choriogonadotropin beta lacking residues 101-145 is the shortest form of the subunit yet described that retains biological activity. Moreover, these results demonstrate that the proposed disulfide between Cys-26 and Cys-110 is not required for subunit assembly or for receptor binding and subsequent intracellular signaling.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Epidermal growth factor modulates intracellular arachidonic acid levels in MA-10 cultured Leydig tumor cells.

Epidermal growth factor (EGF) acts on various cell types, including the mouse Leydig tumor cell line MA-10, where it has been shown to stimulate steroidogenesis, apparently in a cAMP-independent manner. In the process of examining other possible signaling pathways for EGF in these cells, we found rapid changes in the intracellular concentration of arachidonic acid (AA) following addition of EGF. For example, a significant increase in AA was detected 1 min after incubating the cells with EGF, with the maximal effect observed at an EGF concentration of 10 ng/ml. In addition, exogenous AA increased steroidogenesis, and the steroidogenesis enhanced by AA and EGF was reduced by lipoxygenase inhibitors, suggesting a possible role of an AA metabolite(s) in promoting steroidogenesis. Consistent with this hypothesis is our observation that several exogenous lipoxygenase metabolites were capable of enhancing progesterone production. The EGF-stimulated steroidogenesis was also inhibited by two phospholipase A2 inhibitors, again confirming a probable role of AA or a metabolite in this process. Therefore, AA appears to be an important intracellular mediator responsible, at least in part, for some of the acute metabolic effects mediated by EGF in MA-10 cells.

Animals↗
PubMed1991

Comparison of steroidogenic potencies of homologous and heterologous gonadotropins in rat and mouse Leydig cells.

The in vitro steroidogenic potencies have been determined in rat and mouse Leydig cells for two homologous human gonadotropins, lutropin (hLH) and choriogonadotropin (hCG), and two heterologous gonadotropins, hCG beta wild-type and the product of an hCG beta clone containing a premature termination codon at position 122, each associated with a co-expressed bovine alpha subunit. hCG was found to be more potent than hLH in rat, but not mouse Leydig cells, and the heterologous gonadotropin containing the truncated hCG beta subunit was equipotent to that with hCG beta wild-type in both rat and mouse Leydig cells. Persistent steroidogenesis was determined by measuring testosterone production following pre-incubation with each of the above four gonadotropins and with ovine LH, subsequent washing of the cells, and re-incubation in the absence and presence of additional hormone. Interesting differences were found with the five gonadotropins in rat and mouse Leydig cells. The testosterone response to all gonadotropins in rat Leydig cells was essentially the same whether or not additional hormone was added after the initial cell incubation and washing. In contrast, only hLH and hCG yielded identical responses in mouse Leydig cells in the presence and absence of additional hormone, testosterone production being invariably lower with ovine LH and the two expressed heterologous gonadotropins unless they were present in the second incubation. In summary, the hCG beta C-terminal sequence from residues 122-145 makes no discernible contribution to the in vitro potency in rat or mouse Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase.

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.

Animals↗
PubMed1991

Ovarian contents of immunoreactive beta-endorphin and alpha-N-acetylated opioid peptides in rats.

beta-Endorphin was measured by radioimmunoassay in homogenates of ovaries from immature Sprague-Dawley rats (21-29 days of age) and found to be present at levels of about 0.6-0.7 ng/ovary. After administration of PMSG there was approximately a 4-fold increase (2-3 ng/ovary) in total ovarian immunoreactive (ir) beta-endorphin 48 h after injection. Analysis of follicular fluid from similarly treated rats indicated about the same amount of ovarian ir-beta-endorphin (2-3 ng/ovary) as in ovarian homogenates, suggesting that most of the ir-beta-endorphin is localized in follicular fluid of PMSG-primed immature rats. Immature rats were made pseudopregnant by administration of hCG 48 h after PMSG, and at 24 h after injection of hCG there was a slight, but significant and reproducible, increase in the ovarian content of ir-beta-endorphin. The serum concentration of ir-beta-endorphin was in the range of 1-3 ng/ml and was unaffected by PMSG and PMSG/hCG; likewise, the pituitary content of ir-beta-endorphin did not change following administration of gonadotrophins to immature rats. In mature cyclic animals, levels of 2-4 ng ir-beta-endorphin/ovary were found, comparable to those in the ovaries of PMSG-primed immature rats, and there were only small changes during the oestrous cycle. In addition to ir-beta-endorphin, we also obtained evidence for the presence of alpha-N-acetylated opioid peptides (endorphins or enkephalins) in the ovaries of PMSG-primed immature and mature rats. The physiological role of the opioid peptides in reproductive tissue is unknown, but they are presumably acting in an autocrine or paracrine fashion.

Acetylation↗
PubMed1990

Fluorescent adducts of wheat calmodulin implicate the amino-terminal region in the activation of skeletal muscle myosin light chain kinase.

Considerable attention is being directed toward defining a binding site in the central region of calmodulin that forms a high affinity interaction with certain enzymes and amphiphilic peptides. However, other regions of calmodulin are also known to be involved in the activation of enzymes such as myosin light chain kinase, regions which may not be directly involved in the binding of small peptides, e.g. mastoparan X. We investigated the properties of wheat calmodulin fluorescent derivatives, which were modified chemically in the first calcium binding site at Cys-27, in the activation of rabbit fast skeletal muscle myosin light chain kinase. Unmodified wheat calmodulin stimulated myosin light chain kinase to a greater maximal velocity than wheat calmodulin that was modified at Cys-27 by any of four fluorescent compounds, IAANS (2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid), 5-[2'-[[iodoacetyl]amino]ethyl]aminonaphthalene]-1-sulfonic acid, 5-iodoacetamidofluorescein, and 7-diethylamino-3-[4'-maleimidylphenyl]-4-methylcoumarin; the midpoints for activation of myosin light chain kinase were not significantly different for unmodified wheat calmodulin and three of the four wheat calmodulin derivatives. Myosin light chain kinase, but not mastoparan X, enhanced the fluorescence emission intensity of wheat calmodulin-IAANS. Mastoparan X reversed, in a dose-dependent manner, the changes in fluorescence intensity of a preformed complex of myosin light chain kinase and wheat calmodulin-IANNS. Thus, we propose that the region vicinal to Cys-27 participates in the activation but not the high affinity association of myosin light chain kinase. Lastly, a comparison of mammalian and plant calmodulin showed that the Vmax for the stimulation of myosin light chain kinase was 1.6-fold greater for bovine than wheat calmodulin. The difference between the two calmodulins was more pronounced at lower Ca2+ because less Ca2+ was needed to saturate the kinase rate when stimulated by bovine calmodulin.

Animals↗
PubMed1990

Divergence between ovarian aromatase activity, estrogen, and androgen levels in the cycling rat.

Serum estrogen levels vary in a cyclic fashion during the rat estrous cycle, reaching peak values on proestrus and then rapidly declining. Daily measurements of ovarian aromatase activity appear to follow a similar pattern, suggesting a potential regulatory role for the enzyme. To examine this hypothesis, mature rats were killed at various times, and ovarian microsomes were assayed for aromatase activity, while androstenedione, testosterone, estradiol, and estrone levels were measured in ovarian cytosol by RIA. The ovarian aromatase activity reached a peak about 9 h after the onset of the light period on the day of proestrus, and then began to decrease gradually to 75% lower levels by the morning of estrus. Similarly, the estradiol levels also peaked at about 9 h, but then dropped sharply; at 18 h the estradiol levels were reduced 90-95%, while aromatase activity was still close to its peak value. In contrast, the androstenedione and testosterone levels remained high for 3-4 h after the estradiol levels began to decrease. Estrone levels varied considerably less than those of estradiol, reaching lower peak values, but then decreasing only 50-75%. The dissociation between product levels and enzyme activity cannot be explained by a change in the Km of aromatase, which was about 20 nM for androstenedione throughout this period. Therefore, it appears that the decrease in estradiol levels from the peak of the surge may in part be a consequence of a shift in the ratio of the estrogen products synthesized by the ovary, rather than a decrease in the ovarian capacity for estrogen production, although other factors appear to be involved as well.

Androgens↗
PubMed1989

An enzymatically active cross-linked complex of calmodulin and rabbit skeletal muscle myosin light chain kinase.

The interaction between bovine testes calmodulin and rabbit fast skeletal muscle myosin light chain kinase was investigated with the zero-length cross-linking reagent N,N'-dicyclohexylcarbodiimide. A cross-linked product of 110 kDa was produced only in the presence of Ca2+. The reaction mixture was separated on diethylaminoethyl cellulose, and a fraction containing the cross-linked complex of calmodulin and myosin light chain kinase was found to have an elevated kinase activity in the absence of Ca2+, which constituted approximately 50% of the maximally stimulated kinase activity of control, and additional kinase activity in the presence of Ca2+, which constituted the remaining 50% of control activity. Calmodulin added exogenously to the cross-linked complex had no effect on the measured Ca2+ dependence or the maximal extent of kinase activity, which is consistent with the cross-linking of calmodulin in close proximity to a regulatory region of myosin light chain kinase. Moreover, the results are consistent with a mechanism whereby the association of calmodulin is sufficient to stimulate kinase activity and the binding of Ca2+ to bound calmodulin increases catalytic efficiency.

Animals↗