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Biomedical subjects

D Puett

Publications and source records attributed to D Puett.

At least 127 records · Page 7Linked to original sources
PubMed1983

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes.

The mitochondrial and microsomal cytochrome P-450 contents of C57B1/6 mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 +/- 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 micrograms/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N',N'-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 +/- 3.5 and 28.9 +/- 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 +/- 1.4 and 19.6 +/- 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.

Animals↗
PubMed1983

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity.

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.

Adenylyl Cyclases↗
PubMed1982

Demonstration of distinct forms of testicular adenylate cyclases associated with germinal and Leydig cell fractions.

Decapsulated testes from adult rats were digested with collagenase, and the fraction enriched in germinal and Leydig cells was applied to a 0-4% continuous metrizamide gradient and centrifuged. This leads to separation of a germinal cell fraction and two putative Leydig cell populations that bind human choriogonadotropin, but only one of which responds to the gonadotropin with marked increase in testosterone production. Adenylate cyclase activity was present in these three fractions, and Mn2+ was more effective than Mg2+ as a divalent cation. The adenylate cyclase activity associated with the germinal cell fraction was just marginally stimulated by fluoride and by the non-hydrolyzable GTP analog 5'-guanylimidodiphosphate, while that associated with the Leydig cell populations was stimulated to a greater degree depending upon the type of divalent cation. Only the Leydig cell populations exhibited marked human choriogonadotropin-sensitive stimulation of adenylate cyclase activity in the presence of 5'-guanylimidodiphosphate above that observed with the GTP analog alone. These results suggest the presence of distinct adenylate cyclases in adult rat testis and indicate that both populations of Leydig cells are capable of producing cyclic AMP in response to gonadotropins such as human choriogonadotropin.

Adenylyl Cyclases↗
PubMed1982

Epidermal growth factor and cyclic AMP stimulation of distinct protein kinase activities in Leydig cell tumor membranes.

Epidermal growth factor (EGF) and cyclic AMP were found to stimulate distinct protein kinase activities in plasma membranes prepared from the M5480P murine Leydig cell tumor. EGF stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 180,000, while cyclic AMP stimulated the phosphorylation of a minor component of molecular weight 220,000. The two types of kinases could also be distinguished on the basis of differential susceptibility to conditions of membrane preparation. These results suggest that EGF stimulates a cyclic AMP-independent protein kinase in murine Leydig cell tumors at the level of the plasma membrane.

Animals↗
PubMed1982

Testicular membranes with improved stability of the gonadotropin receptor.

A plasma membrane fraction has been prepared from rat testis using an aqueous double-phase polymer system containing dextran, poly(ethylene glycol) 6000 and Zn2+. The membrane-associated gonadotropin receptor for lutropin and human choriogonadotropin can be markedly stabilized by a thawing-washing step of frozen membranes which prolongs the apparent half-life of the unoccupied membrane-associated receptors from less than 1 h at 37 degrees C to greater than 5h. Also, no degradation of 125I-labeled human choriogonadotropin was detected following incubation with the membrane fraction. The equilibrium binding was characterized by an apparent association constant of 1.6 x 10(10) M-1 and a receptor content of 33 fmol/mg protein. Binding kinetics yielded as association rate constant of 1.0 x 10(8) M-1 x min-1, while the dissociation rate constant for human choriogonadotropin was too low to be accurately determined under the conditions used. In contrast, ovine lutropin could be reversibly bound to the membranes leaving the previously occupied receptors available for binding by 125I-labeled human choriogonadotropin.

Animals↗
PubMed1982

Conformational and molecular weight studies of tetanus toxin and its major peptides.

Two forms of tetanus toxin have been purified from Clostridium tetani cultures. These forms, obtained from filtrate and cellular extracts, were characterized by analytical ultracentrifugation using both conventional and meniscus-depletion sedimentation equilibrium. The molecular weight of filtrate toxin was found to be 128,000 +/- 3,000, while the extract toxin, which tended to self-associate, appeared somewhat larger, 140,000 +/- 5,000. The heavy and light chains were prepared from filtrate toxin, and their molecular weights were estimated to be 87,000 and 48,000, respectively, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The circular dichroic spectra of the extract and filtrate toxins are quite similar between 200-300 nm indicating that no major conformational difference exists between the two. The toxins contain both alpha-helicity and beta-structure. Interestingly, the isolated chains contain appreciable helicity (e.g., the sum of the chain helicities is over 80% of that found in filtrate toxin), but they appear to have relatively low contents of beta-structure. The sum of the spectra of the chains in both the near- and far-ultraviolet does not yield that found for filtrate toxin, although the similarity is far more striking than the difference. The prominent 293.5 nm negative circular dichroic band of tetanus toxin can be assigned to tryptophanyl residues almost exclusively in the heavy chain. The similarity in the magnitude of this band in the separated chain and toxin suggests that the microenvironments of the contributing tryptophans change very little when toxin is dissociated into its constituent chains.

Circular Dichroism↗
PubMed1982

Partial purification and characterization of a renotropic fraction from ovine pituitaries.

It has been previously established that hypophysectomy leads to renal atrophy in rats and that a crude pituitary-derived fraction is effective in restoring kidney weight to the level expected for intact animals of the same body weight. This paper reports that considerable purification of the crude renotropic fraction from ovine pituitaries has been achieved and that the purified fraction is capable of restoring kidney weights of hypophysectomized castrated rats to normal values. For example, after five daily subcutaneous injections (135 micrograms/day) there were significant increases in dry kidney weight and total renal protein and DNA. The pituitary-derived fraction was devoid of somatotropin, contained only trace amounts of corticotropin, gamma-lipotropin, vasopressin, and prolactin, and had only low levels of thyrotropin and follitropin. Daily injections of prolactin, thyrotropin, and follitropin in doses of 20 micrograms each failed to stimulate renal growth in hypophysectomized rats. Thus, it seems highly unlikely that these factors are responsible for the observed renal hyperplasia after treatment with the pituitary fraction. The purified renotropic fraction had an isoelectric pH between 8 and 9. On polyacrylamide gel electrophoresis in the presence of detergent and a reducing agent, the renotropic fraction exhibited two major bands and one minor band with mobilities that corresponded to those of a standard lutropin preparation. The renotropic fraction exhibited considerable crossreactivity with an antiserum directed against the lutropin alpha subunit, suggesting the presence of the common glycoprotein hormone subunit. Moreover, the purified fraction stimulated steroid production by Leydig tumor cells in vitro. It is noteworthy, however, that standard ovine lutropin at 135 micrograms/day failed to exhibit renotropic activity in hypophysectomized castrated rats, although effects were noted at twice that dose. It appears that the renotropic activity represents a pituitary substance that can be separated from lutropin only with difficulty.

Animals↗
PubMed1982

Circular dichroic and immunological properties of human choriogonadotropin- beta carboxyl terminal peptides.

Circular dichroic spectra have been obtained in aqueous solution and in trifluoro-ethanol for several synthetic (non-glycosylated) human choriogonadotropin carboxyl terminal peptides of the beta-subunit ranging in size from 10 residues to 40 residues. These was no evidence for formation of alpha-helicity or beta-structure, but the spectra in 90% (v/v) trifluoroethanol were consistent with the occurrence of beta-turns. The Chou-fasman predictive rules also suggest a high probability of beta-turns in these peptides which could result in the occurrence of repeating kinks. Disulfide-linked dimers were also investigated by circular dichroism, and there was evidence of stabilization of particular skewness of the disulfide dihedral angle depending upon the location of the disulfide bond. The single phenylalanyl residue at position 115 in the beta-subunit also contributed to the circular dichroic spectra above 250 nm. Antibodies raised to a peptide consisting of residues 111-145 have been shown to contain two immunological determinants, but the sum of antibodies raised to separate determinant sequences do not equal those raised to the full length peptide. These data could reflect the existence of a conformation-related determinant on the 111-145 peptide or stearic hindrance of immunoglobulin binding of two antibodies to the same peptide.

Chorionic Gonadotropin↗
PubMed1982

Alpha-subunit conformation in glycoprotein hormones and recombinants as assessed by specific antisera.

RIAs specific for the alpha-subunit of glycoprotein hormones were used to monitor the subunit conformation in ovine FSH and TSH and in recombinants of ovine LH alpha with (ovine and porcine) LH beta and hCG beta. Differences in log-logit slopes of the RIA dilution curves were interpreted to indicate changes, presumably conformational, in the local environment of the antigenic determinants of the alpha-subunit. In all but one case, free ovine LH alpha yielded a slope that was distinct from those of the hormones and recombinants. FSH, TSH, and the homologous LH recombinant all exhibited different slopes, and the recombinant ovine LH alpha-hCG beta was characterized by a slope that was distinct from the identical slopes of the recombinants ovine LH alpha-ovine LH beta and ovine LH alpha-porcine LH beta. These results suggest that a conformational change occurs in the alpha-subunit upon association with a beta-subunit and that different beta-subunits may induce distinct conformations in a common alpha-subunit.

Animals↗
PubMed1981

Changes in the circular dichroic spectrum of colchicine associated with its binding to tubulin.

Circular dichroism has been used to study the interaction of colchicine with the tubulin alpha beta dimer at 26 degrees C. Tubulin purified from bovine brain microtubule protein exhibits negligible circular dichroism at wavelengths above 310 nm. Free colchicine exhibits a negative circular dichroic band at 340 nm characterized by an extremum in molar ellipticity [theta] of -3.35 (+/- 0.27) x 10(4) deg.cm2/dmol. This negative band either vanished or was greatly reduced, i.e., [theta] = 0 (+/- 0.57) x 10(4) deg.cm2/dmol, when colchicine was bound to tubulin. This was demonstrated by circular dichroic studies on stable [3H]colchicine-tubulin complexes which were separated from all unbound colchicine by means of gel filtration. Also, tubulin was titrated with colchicine, and at low colchicine concentrations, the observed ellipticity at 340 nm could be extrapolated to zero at 0 M colchicine; saturation occurred at a molar ratio of colchicine to tubulin of about 1:1. The association constant characterizing the drug-protein interaction was estimated to be about 0.9 micro M-1. As controls, three other acidic proteins were studied at a molar ratio of colchicine to protein of 2:1, and their presence had no effect on the circular dichroic properties of colchicine. These results are consistent with the idea that a conformational change in colchicine accompanies its binding to tubulin. The spectrum of the complex between 250 and 300 nm was quite similar to that expected from simple additivity of the spectra of drug and protein except between 255 and 265 nm. The technique described herein should be applicable to other protein-drug systems.

Animals↗
PubMed1981

Characterization of the desensitized state of Leydig tumor cells.

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

8-Bromo Cyclic Adenosine Monophosphate↗