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PubMed1977

Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.

Allosteric Site↗
PubMed2000

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

Allosteric Site↗
PubMed2005

Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators.

Metal ion homeostasis in prokaryotes is maintained by metal-responsive transcriptional regulatory proteins that regulate the transcription of genes encoding proteins responsible for metal detoxification, sequestration, efflux and uptake. These metalloregulatory, or metal sensor proteins, bind a wide range of specific metal ions directly; this in turn, allosterically regulates (enhances or decreases) operator/promoter binding affinity or promoter structure. Recent structural studies reveal five distinct families of metal sensor proteins. The MerR and ArsR/SmtB families regulate the expression of genes required for metal ion detoxification, efflux and sequestration; here, metal binding leads to activation (MerR) or derepression (ArsR/SmtB) of the resistance operon. In contrast, the DtxR, Fur, and NikR families regulate genes encoding proteins involved in metal ion uptake; in these cases, the metal ion functions as a co-repressor in turning off uptake genes under metal-replete conditions. Inspection of the structures of representative members from each metal sensor family reveals several common characteristics: (1) they function as homo-oligomers (either dimers or tetramers); (2) metal-binding ligands are found at subunit interfaces, with ligands derived from more than one protomer; this likely helps drive quaternary structural changes that mediate allosteric coupling between the metal and DNA binding sites; and (3) the primary determinant of metal ion selectivity within each protein family is dictated by the coordination geometry of the metal chelate, with trends consistent with expectations from fundamental inorganic chemistry. This review highlights recent efforts to elucidate the structure of metal sensing chelates and the molecular mechanisms of allosteric coupling in metal sensor proteins.

Allosteric Site↗
PubMed1996

Regulation of muscle glycogen phosphorylase activity during intense aerobic cycling with elevated FFA.

This study examined muscle glycogenolysis and the regulation of glycogen phosphorylase (Phos) activity during 15 min of cycling at 85% of maximal O2 consumption (VO2max) in control and high free fatty acid (FFA; Intralipid-heparin) conditions in 11 subjects. Muscle biopsies were sampled at rest and 1, 5, and 15 min of exercise, and glycogen Phos transformation state (%Phos alpha), substrate (Pi, glycogen), and allosteric regulator (ADP, AMP, IMP) contents were measured. Infusion of intralipid elevated plasma FFA from 0.32 +/- 0.04 mM at rest to 1.00 +/- 0.04 mM just before exercise and 1.12 +/- 0.10 mM at 14 min of exercise. In the control trial, plasma FFA were 0.36 +/- 0.04 mM at rest and unchanged at the end of exercise (0.34 +/- 0.03 mM). Seven subjects used less muscle glycogen (46.7 +/- 7.6%, mean +/- SE) during the Intralipid trial, and four did not respond. In subjects who spared glycogen, glycogen Phos transformation into the active (alpha) form was unaffected by high FFA except for a nonsignificant reduction during the initial 5 min of exercise. Total AMP and IMP contents were not significantly different during exercise between trials, but total ADP was significantly lower with Intralipid only at 15 min. The calculated free ADP, AMP, and Pi contents were lower with Intralipid but not significantly different. However, when the present results were pooled with the data from a previous study using the same protocol [Dyck et al., Am. J. Physiol. 265 (Endocrinol, Metab. 28): E852-E859, 1993], the free ADP, AMP, and Pi contents of all subjects who spared glycogen (n = 13) were significantly lower at 15 min in the Intralipid trial. The findings suggest that the elevation of plasma FFA during intense cycling spares muscle glycogen by posttransformational regulation of Phos. This may be due to blunted increases in the contents of AMP, an allosteric activator of Phos alpha, and Pi, a substrate for Phos.

Adult↗
PubMed2003

Carbon-source-dependent nitrogen regulation in Escherichia coli is mediated through glutamine-dependent GlnB signalling.

The P(II) signal transduction proteins GlnB and GlnK are uridylylated/deuridylylated in response to the intracellular glutamine level, the primary signal of the cellular nitrogen status. Furthermore, GlnB was shown to be allosterically regulated by 2-oxoglutarate, and thus GlnB was suggested to integrate signals of the cellular carbon and nitrogen status. Receptors of GlnB signal transduction in Escherichia coli are the NtrB/NtrC two-component system and GlnE, an enzyme which adenylylates/deadenylylates glutamine synthetase. In this study, the authors investigated the effect of different carbon sources on the expression of the NtrC-dependent genes glnA and glnK and on the uridylylation status of GlnB and GlnK. With glutamine as nitrogen source, high levels of glnA and glnK expression were obtained when glucose was used as carbon source, but expression was strongly decreased when the cells were grown with poor carbon sources or when cAMP was present. This response correlated with the uridylylation status of GlnB, suggesting that the carbon/cAMP effect was mediated through GlnB uridylylation, a conclusion that was confirmed by mutants of the P(II) signalling pathway. When glutamine was replaced by low concentrations of ammonium as nitrogen source, neither glnAglnK expression nor GlnB uridylylation responded to the carbon source or to cAMP. Furthermore, glutamine synthetase could be rapidly adenylylated in vivo by the external addition of glutamine; however, this occurred only when cells were grown in the presence of cAMP, not in its absence. Together, these results suggest that poor carbon sources, through cAMP signalling, favour glutamine uptake. The cellular glutamine signal is then transduced by uridylyltransferase and GlnB to modulate NtrC-dependent gene expression.

Bacterial Proteins↗
PubMed2004

Nitric oxide, S-nitrosothiols and hemoglobin: is methodology the key?

Two main hypotheses describe the role of hemoglobin in the regulation of nitric oxide (NO) bioavailability. It has been suggested that hemoglobin interacts with circulating NO, forming Fe-nitrosyl hemoglobin and then S-nitrosothiols, which deliver NO extracellularly by an allosterically regulated mechanism. Alternatively, the existence of diffusional barriers that protect NO from hemoglobin-mediated degradation has been proposed. The reliability of each model in vivo is supported by the detection of physiological hematic levels of S-nitrosohemoglobin. However, the measured concentrations of S-nitrosohemoglobin are largely divergent between the two models. Moreover, recent reports suggest that circulating levels of S-nitrosohemoglobin in human blood could be significantly lower than assessed previously. We suggest that solving the methodological controversies that make the field of NO research a 'minefield', even for skilled analysts, is fundamental to understanding the role of S-nitrosothiols in the vasculature.

Hemoglobins↗
PubMed2004

Streptococcus pneumoniae isoprenoid biosynthesis is downregulated by diphosphomevalonate: an antimicrobial target.

The toll that Streptococcus pneumoniae exacts on the welfare of humanity is enormous. This organism claims the lives of approximately 3700 people daily, the majority of whom are children below the age of 5, and the situation could worsen due to the increasing incidence of pernicious, multiple-antibiotic-resistant strains. Here we report the discovery and characterization of a new allosteric site, shown to be absent in humans, that can be used to switch off an essential pathway in S. pneumoniae, the mevalonate pathway. Diphosphomevalonate (DPM), an intermediate in the pathway, binds with high affinity (K(d) = 530 nM) to mevalonate kinase, the first enzyme in the pathway, and inactivates it. Steady-state and equilibrium binding measurements reveal that DPM binding is noncompetitive versus substrates. DPM binds at an allosteric site, and inhibition cannot be overcome by an increasing substrate concentration. The DPM-binding site is a promising target for the development of new antimicrobial agents.

Allosteric Regulation↗
PubMed1988

Regulation of binding of phosphofructokinase to myofibrils in the red and white muscle of the barred sand bass, Paralabrax nebulifer (Serranidae).

The binding of phosphofructokinase (PFK) to myofibrils from the white muscle of the fish Paralabrax nebulifer (Girard, 1854) is sensitive to factors known to be allosteric regulators of PFK activity. PFK in Triton-X-100-extracted muscle remains bound to myofibrils at pH 7.0 and is fully solubilized by increasing the pH to 8.0. The curve describing the pH-dependence of PFK binding to myofibrils is similar in its steepness to pH versus activity curves of PFK at low temperature. Nucleotides are also potent modulators, preventing the association of PFK with myofibrils at concentrations between 20 and 60 mumol l-1 of ATP, ADP, MgATP or GTP, listed in order of effectiveness. PFKs in the red and white muscle extracts differ in their pH-dependence of binding to myofibrils, and their kinetic and regulatory properties (response to citrate, pH and fructose-2,6-bisphosphate). Reversible binding of PFK to myofibrils may be important in the control of glycolysis, especially in the highly glycolytic white muscle fibres.

Animals↗
PubMed2002

The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase.

A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial target for the apicomplexan parasite Eimeria tenella [Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald, R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley, T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol. Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in their regulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sites and has distinctive activation properties, including a very large stimulation by cGMP ( approximately 1000-fold) with significant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we found that 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides a maximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP can synergize with the analogue to provide full activation. The results suggest that partial activation is a consequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directed mutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirms that this site is an important functional participant in the allosteric regulation of the kinase and that it exhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activation of Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additional cGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present in the PKGs of higher organisms containing only two allosteric sites.

Allosteric Site↗
PubMed1999

Complementary regulation of anaesthetic activation of human (alpha6beta3gamma2L) and Drosophila (RDL) GABA receptors by a single amino acid residue.

1. The influence of a transmembrane (TM2) amino acid located at a homologous position in human beta1 (S290) and beta3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2. GABA-evoked currents mediated by human wild-type (WT) alpha6beta3gamma2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated alpha6beta3gamma2L, but not RDL, receptors. 3. GABA-evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. alpha6beta3N289Mgamma2L) were potentiated by propofol or pentobarbitone with approximately 2-fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4. Incorporation of the homologous GABAA beta1-subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous beta3-subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5. The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (alpha6beta3gamma2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.

Amino Acids↗
PubMed1999

Regulation of Ca2+ release by cAMP-dependent protein kinase. A mechanism for agonist-specific calcium signaling?

Calcium is an ubiquitous second messenger that is involved in the regulation of a number of cell functions. The mechanism by which the specificity of calcium signaling is achieved is not well understood. We suggest that calcium release from the ER can occur selectively at different spatial locations in response to different extracellular stimuli. We discuss a possible mechanism for such selectivity and present a model based on this mechanism. The suggested mechanism is based on the regulation of local Ca2+ release by cyclic AMP-dependent protein kinase (PKA) and relies upon two experimental observations: first, some G-protein coupled signaling pathways activate PLC and regulate adenylate cyclase at the same time, leading to IP3 production and altering PKA activity via changes in cAMP level; second, phosphorylation by PKA alters the properties of IP3 receptor (IP3R). In our model we consider allosteric regulation of IP3Rs by IP3 and cAMP-dependent phosphorylation. The differences in IP3Rs and PKA densities at different spatial locations within the cell allow the release of calcium selectively at each location in response to certain combination of IP3 and cAMP concentration. Specificity of agonist-response coupling is achieved if different combinations in the levels of these second messengers are specific for different extracellular stimuli.

Animals↗
PubMed2001

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Alleles↗
PubMed2003

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

Binding Sites↗
PubMed2005

Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector.

The hairpin ribozyme can catalyze the cleavage of RNA substrates by employing its conformational flexibility. To form a catalytic complex, the two domains A and B of the hairpin-ribozyme complex must interact with one another in a folding step called docking. We have constructed hairpin ribozyme variants harboring an aptamer sequence that can be allosterically induced by flavin mononucleotide (FMN). Domains A and B are separated by distinct bridge sequences that communicate the formation of the FMN-aptamer complex to domains A and B, facilitating their docking. In the presence of a short oligonucleotide that is complementary to the aptamer, catalytic activity of the ribozyme is completely abolished, due to the formation of an extended conformer that cannot perform catalysis. However, in the presence of the small molecule effector FMN, the inhibitory effect of the oligonucleotide is competitively neutralized and the ribozyme is activated 150-fold. We thus have established a new principle for the regulation of ribozyme catalysis in which two regulatory factors (an oligonucleotide and a small molecule) that switch the ribozyme's activity in opposite directions compete for the same binding site in the aptamer domain.

Allosteric Regulation↗
PubMed1998

Organization of the human glucokinase regulator gene GCKR.

Glucokinase plays an important role in regulating insulin secretion in response to changes in blood glucose levels. As a result, one form of maturity onset diabetes of the young (MODY) results from haploinsufficiency of glucokinase. In both liver and pancreatic islet, glucokinase is allosterically regulated by an inhibitory protein (glucokinase regulatory protein, GCKR). GCKR has therefore become an important gene for functional analysis in type 2 diabetes. To allow genetic assessment of any such role, we have determined the structure of the human GCKR gene. Characterization of P1 and YAC clones containing GCKR shows it to consist of 19 exons spanning 27 kb. RT-PCR, RACE, and RNase protection experiments defined a transcriptional start site for GCKR 66 bp upstream of the initiation codon, but provided no evidence for islet cell specific alternative splicing in the rat. By SSCP screening, a common polymorphic sequence variant has been defined within exon 15 of human GCKR, at nt 1400 of the cDNA. This alters amino acid residue 446 from proline, conserved in rat and Xenopus, to leucine.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A common interface on histidine-containing phosphocarrier protein for interaction with its partner proteins.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system accomplishes both the transport and phosphorylation of sugars as well as the regulation of some cellular processes. An important component of this system is the histidine-containing phosphocarrier protein, HPr, which accepts a phosphoryl group from enzyme I, transfers a phosphoryl group to IIA proteins, and is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface on HPr that interacts with this multiplicity of proteins from Escherichia coli was previously undefined, we investigated these interactions by nuclear magnetic resonance spectroscopy. The chemical shift changes of the backbone and side-chain amide (1)H and (15)N nuclei of uniformly (15)N-labeled HPr in the absence and presence of natural abundance glycogen phosphorylase, glucose-specific enzyme IIA, or the N-terminal domain of enzyme I have been determined. Mapping these chemical shift perturbations onto the three-dimensional structure of HPr permitted us to identify the binding surface(s) of HPr for interaction with these proteins. Here we show that the mapped interfaces on HPr are remarkably similar, indicating that HPr employs a similar surface in binding to its partners.

Bacterial Proteins↗
PubMed1995

Affinity labelling of the catalytic and allosteric ATP binding sites on pyruvate kinase type I from Escherichia coli.

The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.

Adenosine Triphosphate↗
PubMedDate not supplied

Routes and regulation of NADPH production in steroidogenic mitochondria.

The first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system that is dependent on NADPH. The pathways of NADPH generation in steroidogenic mitochondria include three major routes catalyzed by: 1. NADP-linked malic enzyme, 2. NADP-linked isocitrate dehydrogenase, and 3. nicotinamide nucleotide transhydrogenase. The main route may differ among cell types and across species. Generally operation of alternative routes, with different substrates is not excluded. The oxidation of NADPH by the mitochondrial P450 systems is not tightly coupled with substrate metabolism, as these systems can reduce O2 by a single electron to produce harmful superoxide radical. To minimize such futile NADPH oxidation, NADPH generation may be regulated by two types of mechanisms: 1. Feedback mechanisms that maintain the ratio of NADPH/NADP+ at a steady-state level by enhancing the rate of NADPH production to keep up with its rate of oxidation, e.g., allosteric regulation of enzymes involved in NADPH production. 2. Hormonal signals that enhance the level of NADPH production in coordination with steroidogenesis. One major hypothesis with experimental evidence is that stimulation of mitochondrial NAD(P)H synthesis is mediated by Ca++ as a second messenger of tropic factors. Tropic stimulation of cells increases the levels of Ca++ in the cytosol and then in the mitochondrial matrix, and the rise in Ca++ activates enzymes involved in NAD(P)H synthesis. These regulatory mechanisms most probably operate in concert adjusted to the steroidogenic activity of the cell.

Animals↗