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At least 19 recordsLinked to original sources
PubMed2006

Thyroid hormone receptor expression in the obligatory paedomorphic salamander Necturus maculosus.

Amphibian metamorphosis is under the strict control of thyroid hormones (TH). These hormones induce metamorphosis by controlling gene expression through binding to thyroid hormone receptors (TRs). Necturus maculosus is considered to be an obligatory paedomorphic Amphibian since metamorphosis never occurs spontaneously and cannot be induced by pharmacological means. Since metamorphosis depends on the acquisition of response of tadpole tissues to thyroid hormone, we aimed to determine TR gene expression patterns in Necturus maculosus as well as the expression of two TH-related genes: Cytosolic Thyroid Hormone-Binding Protein (CTHBP)-M2-pyruvate kinase, a gene encoding a cytosolic TH binding protein and stromelysin 3, a direct TH target gene in Xenopus laevis. Tissue samples were obtained from specimens of Necturus maculosus. We performed in situ hybridization using non-cross-hybridizing RNA probes obtained from the cloned Necturus TRalpha and TRbeta genes. We found clear expression of Necturus TRalpha gene in several tissues including the central nervous system, epithelial cells of digestive and urinary organs, as well as myocardium and skeletal muscle. TRbeta was also expressed in the brain. In other tissues, hybridization signals were too low to draw reliable conclusions about their precise distribution. In addition, we observed that the expression of CTHBP and ST3 is largely distinct from that of TRs. The fact that we observed a clear expression of TRalpha and TRbeta which are evolutionary conserved, suggests that Necturus tissues express TRs. Our results thus indicate that, in contrast to previously held hypotheses, Necturus tissues are TH responsive.

Animals↗
PubMedDate not supplied

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus.

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.

Animals↗
PubMed1993

Effects of a Shaker K+ channel peptide and trypsin on a K+ channel in Necturus enterocytes.

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Animals↗
PubMed1989

A specific androgen-binding protein (ABP) in Necturus testis and its zonal distribution.

The urodele amphibian Necturus maculosus has a zoned testis, which is advantageous for separating Leydig cells from germinal elements and for studying stage-dependent biochemical changes. Using [3H]testosterone (T) in a standard binding assay and dextran-coated charcoal (DCC) or Sephadex LH-20 to separate free and bound steroids, we identified an androgen-binding protein (ABP) in Necturus testis cytosols. This protein was of high affinity (Kd = 10(-9) M) and was saturable (Bmax = 10(-9) M) and specific for androgen (T; 5 alpha-dihydrotestosterone, DHT) but could be distinguished from the androgen receptor of Necturus testis by its relative abundance (300-550 fmol/mg protein), short half-time of dissociation (3 min at 22 degrees C), inability to adhere to DNA-cellulose, and absence from nuclear extracts. Additionally, when analyzed on sucrose gradients, the ABP of Necturus testis sedimented at 6-7 S in both low or high ionic strength buffers. In that estradiol (E2) is a poor competitor for T-binding, this protein resembles a sex steroid-binding protein previously identified in urodele serum but differs from the ABP and testosterone-estradiol-binding globulin (TEBG) of rodents, humans, goldfish, and sharks. It is differentially distributed within the testis, with the highest levels in immature lobular regions composed of Sertoli cells and germ cells in premeiotic stages and lower levels in regions composed primarily of Leydig cells. The cellular source and function of this protein in Necturus testis remain to be determined.

Androgen-Binding Protein↗
PubMed1988

Na/H exchange in antral cells isolated from Necturus, rabbit, and guinea pig.

Mechanisms of intracellular pH (pHi) regulation were investigated using the pH-sensitive dye BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to measure pHi in isolated antral cells from Necturus, rabbit, and guinea pig. The BCECF fluorescence was calibrated in terms of pHi by using a nigericin-high potassium concentration method to equilibrate pHi and pHo. Average pHi for the cells of Necturus and rabbit was approximately 7.1 in a HEPES-buffered, HCO3-free Ringer's solution. Average pHi in Necturus cells in CO2/HCO3-buffered Ringer's solution was approximately 7.0. An ammonium-loading technique was used to acidify the cell to measure intrinsic buffering capacity (beta i) and to monitor pHi recovery. Average beta i = 28 mM per pH unit in Necturus and 42 mM per pH unit in rabbit cells in HEPES-buffered Ringer's solution. Recovery from an acid load was sodium-dependent, completely inhibited by 100 microM amiloride, and unaffected by changes in potassium concentration between 6 and 25 mM. The results provide evidence in support of the presence of an amiloride-sensitive Na/H exchanger in antral cells from rabbit, guinea pig, and Necturus.

Animals↗
PubMed1979

Excretion of phenol red by the Necturus kidney.

Phenol red (phenolsulfonphthalein, PSP) is thought to be secreted by proximal kidney tubules in all vertebrates. The present study examined PSP transport by the kidney of the salamander, Necturus maculosus. In Necturus kidneys perfused with oxygenated Ringer solution, the PSP/creatinine clearance ratio was unity. Perfusion with 1 mM octanoate converted net p-aminohippurate (PAH) reabsorption to net secretion, but had no effect on PSP. In seven urethan-anesthetized Necturi, the PSP/inulin clearance ratio averaged 0.85 +/- 0.21 (SD), not significantly different from unity. Thin slices from Necturus kidneys incubated in vitro for 2 h failed to accumulate PSP; slice-to-medium (S/M) concentration ratios averaged 0.8 +/- 0.2 (n = 6). With frog kidney slices, (S/M)PSP was 9.6 +/- 1.4 (n = 6). Necturus kidney slices accumulated PAH ((S/M)PAH = 4.1 +/- 0.7) (n = 6), but uptake was not inhibited by 1 mM PSP. We conclude that Necturus kidney tubules transport PAH, but do not transport PSP. These results are consistent with the hypothesis that the organic acid secretory system in most animals involves several carriers.

Aminohippuric Acids↗
PubMed2002

Electrophysiology of Necturus taste cells.

Taste buds are sensory end organs that detect chemical substances occurring in foodstuffs and relay the relative information to the brain. The mechanisms by which the chemical stimuli are converted into biological signals represent a central issue in taste research. Our understanding of how taste buds accomplish this operation relies on the detailed knowledge of the biological properties of taste bud cells-the taste cells-and of the functional processes occurring in these cells during chemostimulation. The amphibian Necturus maculosus (mudpuppy) has proven to be a very useful model for studying basic cellular processes of vertebrate taste reception, some of which are still awaiting to be explored in mammals. The main advantages offered by Necturus are the large size of its taste cells and the relative accessibility of its taste buds, which can therefore be handled easily for experimental manipulations. In this review, I summarize the functional properties of Necturus taste cells studied with electrophysiological techniques (intracellular recordings and patch-clamp recordings). My focus is on ion channels in taste cells and on their role in signal transduction, as well as on the functional relationships among the cells inside Necturus taste buds. This information has revealed to be well suited to outline some of the general physiological processes occurring during taste reception in vertebrates, including mammals, and may represent a useful framework for understanding how taste buds work.

Action Potentials↗
PubMed1961

Oxidative and hydrolytic enzymes in the nephron of Necturus maculosus. Histochemical. biochemical. and electron microscopical studies.

The distribution of oxidative and hydrolytic enzyme activities along the nephron of Necturus maculosus Rafinesque was studied histochemically. The proximal tubule possessed all the demonstrable enzyme activities associated with the hexose-monophosphate shunt and glycolysis, but lacked detectable succinic dehydrogenase and cytochrome oxidase activities. Krebs cycle enzymes other than succinic dehydrogenase were easily detectable. The distal tubule, on the other hand, possessed no detectable hexose-monophosphate shunt enzyme activities, but all demonstrable glycolytic and Krebs cycle enzymes and cytochrome oxidase were present in high activity. These data indicate that the proximal tubule of Necturus probably cannot depend, as can the distal tubule, on the Krebs cycle and cytochrome system to provide energy for its transport processes, an inference supported, in general, by available physiological evidence. The question of the importance of the hexose shunt to proximal tubular function arises. Evidence is presented that the proximal tubular blood supply is primarily venous in nature, a hypothesis which would correlate well with its anaerobic metabolic pattern. In addition, the absence of cytochrome oxidase and succinic dehydrogenase from the proximal tubular cells implies either that they possess very few mitochondria, or that their mitochondria have a very unusual enzymatic pattern. Electron microscopical observations and data obtained from the measurement of the enzyme activities of homogenates of Necturus kidney are presented which indicate that the second hypothesis is more probably correct.

Animals↗
PubMed1991

Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds.

Little information is currently available about which neurotransmitters are involved in signal processing in the peripheral sensory organs of taste, taste buds. Synaptic contacts between taste cells and sensory axons have long been known to exist, but what substances are active at these synapses is not known. Our objective in this study was to test for the presence of the neurotransmitter candidates, GABA, glutamate, serotonin, and histamine in taste buds of Necturus maculosus. Light microscopic immunocytochemical techniques were used to investigate the location of these substances in taste buds and surrounding epithelium. GABA and glutamate were detected in nerve fibers that innervate the taste buds, and, to a substantially lesser extent, in fine, varicose axons that penetrated the surrounding nontaste epithelium. Serotonin immunostaining was strong in basal cells in frog taste discs but was only faintly detected in Necturus taste buds. Histamine was not detected at all in taste buds. We conclude that amino acid neurotransmission may be involved in taste mechanisms and that monoamines may also play a role in chemosensory transduction in the taste bud. On the basis of our inability to detect histamine with immunocytochemical techniques, we conclude that this substance is unlikely to be a major neurotransmitter in Necturus taste buds.

Animals↗
PubMed1995

Calcium is not involved in the cAMP-mediated stimulation of Cl- conductance in the apical membrane of Necturus gallbladder epithelium.

The permeability properties of the forskolin-stimulated Cl- conductance in the apical membrane of Necturus gallbladder epithelium and the possible participation of intracellular Ca2+ in its stimulation have been investigated. The anion selectivity sequence as derived from biionic potential measurements (SCN- > I- approximately NO3- > Br- > Cl- >> ISE-) differed from the sequence derived from measurements of apical membrane resistance (NO3- approximately Br- approximately Cl- > SCN- > I- approximately ISE-). Accordingly, the conductance was inhibited by SCN- and I- which, from the potential measurements, appeared to be more permeable than Cl-. This finding agrees with observations of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel reported recently. However, none of the commonly used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduced this conductance in Necturus gallbladder. In contrast to the situation in most other epithelia, elevation of intracellular Ca2+ concentration ([Ca2+]i) by ionomycin stimulated only K+ conductance and not that of Cl- in the apical cell membrane. Chelation of intracellular Ca2+ did not prevent the stimulation of Cl- conductance by forskolin. This indicates that [Ca2+]i does not have even a permissive role in the cyclic adenosine monophosphate-(cAMP)-mediated stimulation process, as would have been expected if exocytosis was involved. Further evidence against the involvement of exocytosis in the stimulation process came from the observation that the stimulation was not associated with an increase in apical membrane capacitance and was not suppressed by disruption of the cytoskeleton by preincubation of the tissue with cytochalasin D. The data indicate that Necturus gallbladder epithelium contains homologues of the CFTR Cl- channel which reside permanently in the apical cell membrane and which can be stimulated by a cAMP-dependent phosphorylation process without involvement of cell Ca2+ or exocytosis.

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Animals↗
PubMed1981

Noise analysis of the K+ current through the apical membrane of Necturus gallbladder.

Current noise power spectra of the voltage-channel (V = 0) Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f alpha noise component, with alpha being about 2. Substitution of all Na+ by K+ on either the serosal or mucosal side increased the relaxation as well as the 1/f alpha noise component considerably. In Necturus gallbladder both noise components are reduced by addition of 10 mM, 2,4,6-triaminopyrimidine (TAP) or 5 mM BA2+ to the mucosal side, as well as by acidification of the mucosal solution to pH 5 and lower. Five mM of tetraethylammonium (TEA+) added to the mucosal solution, abolished K+ relaxation noise and decreased the 1/f alpha noise component. Applying a Cs+ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb+ was substituted for all Na+ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K+ channels in the apical membrane of the Necturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K+ greater than Rb+ greater than Cs+. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.

Animals↗
PubMed1981

Noise analysis of the K+ current through the apical membrane of Necturus gallbladder.

Current noise power spectra of the voltage-clamped (V = 0) Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f alpha noise component, with alpha being about 2. Substitution of all Na+ by K+ on either the serosal or mucosal side increased the relaxation as well as the 1/f alpha noise component considerably. In Necturus gallbladder both noise components are reduced by addition of 10mM 2,4,6-triaminopyrimidine (TAP) or 5 mM of tetraethylammonium (TEA+) added to ification of the mucosal solution to pH 5 and lower. Five mM of tetraethylammonium (TEA+) added to the mucosal solution, abolished K+ relaxation noise and decreased the 1/f alpha noise component. Applying a Cs+ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb+ was substituted for all Na+ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K+ channels in the apical membrane of the Necturus gallbladder. Furthermore it can be concluded that these channels have a permeability sequence K+ greater than Rb+ greater than Cs+. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.

Animals↗
PubMed1983

KCl cotransport: a mechanism for basolateral chloride exit in Necturus gallbladder.

K+ and Cl--selective double-barreled microelectrodes were used to study the effect of changes in external K+ concentration on intracellular Cl- activity (aiCl) in epithelial cells of Necturus gallbladder. Decreasing the K+ concentration simultaneously in both bathing solutions produced a decrease in aiCl. Steady-state values of aiCl were related to the values of the chemical potential gradient for K+ (delta microK) across either the apical or the basolateral cell membrane. A similar dependence between aiCl and delta microK appeared when the K+ concentration was changed in the serosal solution only. This indicates that aiCl depends on delta microK across the basolateral membrane. aiCl was virtually independent of the membrane potential. This supports the idea that both the mucosal and the basolateral membranes of Necturus gallbladder cells have very low passive permeabilities to Cl-. These results indicate that the exit of Cl- from Necturus gallbladder cells is driven by delta microK across the basolateral membrane, and suggest that KCl electroneutral coupled mechanism in this membrane plays an important role in transcellular Cl- transport.

Animals↗
PubMed1997

Ion, fluid and charge transport across Necturus intestinal epithelium in response to alanine.

Fluxes of Na, Cl and volume were followed across Necturus small intestine under zero voltage clamp. 20 mM L-alanine doubles the net Na and fluid transfer. Although there is a ouabain-sensitive Na pump present in Necturus a major fraction of the net Na flux can be measured for an hour after application of 10(-3) M ouabain. Collected fluid transferred by the epithelium is quasi-isotonic over a range of luminal osmolarities from 100 to 250 milliosmolar in alanine saline. The net Na fluxes account for the Na found in this transported fluid. Fluid transfer also shows a large ouabain-insensitive fraction after the addition of alanine. Compartmental analysis of 22Na-loaded epithelium was used to separate cellular and paracellular fluxes. The estimated Na concentration in the cell derived from its Na content is 9-10 mM, in agreement with that determined with microelectrodes. The Na efflux from cell to serosa is stimulated by alanine, but this increase accounts for only a quarter of the simultaneous rises in Na, fluid and current flow across the epithelium. The increase of Na efflux from the cell induced by alanine is apparently insensitive to ouabain although the cell Na content rises to circa 20 mM but no higher even after 20 hr. From the initial rate of rise of Na in the cell on treatment with ouabain the activity of the Na pump can be estimated to be approximately 92 pM/cm2 . sec, a value much smaller than the transepithelial net flux. The results are not consistent with the standard model in which Na-alanine influx stimulates the Na pump and enhances fluid transport by osmotic coupling in the lateral interspace system. A scheme is proposed based upon that for absorption in Necturus gallbladder by which alanine stimulates an active paracellular fluid transfer driven by motile elements of the junction.

Alanine↗
PubMed1980

Cytochalasin B does not stimulate sugar uptake into small intestine of necturus or chick.

3-O-Methylglucose accumulation by Necturus intestine was studied in the presence and absence of cytochalasin B. The effect of this agent on 3-O-methylglucose-induced changes in transepithelial potential difference was also investigated. Cytochalasin B (50 microM) blocked the rise in potential difference induced by 4 mM 3-O-methyglucose. 3-O-Methyglucose accumulation by Necturus intestine was measured; it was inhibited by cytochalasin B at concentrations of 50 and 100 microM. These results indicate that, in Necturus intestine, sodium-dependent sugar transport across the apical cell membrane is blocked by cytochalasin B.

Animals↗
PubMed1992

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.

Animals↗
PubMed1984

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus.

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.

Androgens↗