Certain aspects of transformation of 21-acetate of the Reichstein substance "S" by the culture of Tieghemella orchidis]

The transformation of acetate from the Reichstein substance "S" by the culture of Tieghemella orchidis was studied upon a single and fractional addition of the substrate. The effect of streptomycin sulphate, Tween-80, ethanol (steroid solvent) and steroid solution on the respiratory activity of the culture was investigated upon a single and fractional addition of the steroid. During transformation the respiratory activity of the culture decreased--by the end of the process (18-20 hours) it fell 2-2.5-fold upon a single addition of the steroid (1 g/l) and by 20% upon a fractional addition of the steroid (2 g/l). In the latter case the respiratory activity dropped step-by-step if the concentration of the steroid was 1.4-1.6 g/l; simultaneously the transformation activity declined.     

Compartmentation of metabolic systems in the regulation of dormancy in fungus spores

Dormancy in fungus spores can be due to a variety of causes relating to structural, physiological, or biochemical functions. Based on data reported here and earlier, compartmentation of endogenous reserves or of enzymes is proposed as the mechanism controlling dormancy in spores ofMyrothecium verrucaria andTrichoderma reesei. Spores of both organisms contain a pool(s) of reserves composed of trehalose, amino acids, as well as unidentified compounds. Addition of hot water extracts of these reserves to spores results in rapid increases in respiratory activity and germination. This observation coupled with other data showing the stimulation of endogenous respiration by heat, freezing, or azide demonstrates that dormancy is due primarily, if not entirely, to sequestering or compartmentation of the endogenous reserves and not of the enzymatic systems involved in utilization of metabolites for germination. Presumably these reserves are contained within the vacuoles. Data on the interactions of treatments that stimulate endogenous respiration and on the effects of metabolism of exogenous substrates indicate that the transport pathways within the cell, from the pools or from the plasma membrane, to the loci of initial metabolism are not identical and that the total regulatory system is composed of a number of separable processes. It appears probable that examination of the spores of other fungi will show that compartmentation of reserves is not of uncommon occurrence and is not an unusual cause of dormancy.     

Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.     

Reduction of nifurtimox and nitrofurantoin to free radical metabolites by rat liver mitochondria. Evidence of an outer membrane-located nitroreductase

Nifurtimox and nitrofurantoin are reduced by intact rat liver mitochondria to nitro anion radicals whose autoxidation generates superoxide anion as detected by direct electron spin resonance spectroscopy and by spin-trapping experiments, respectively. Although nitroreduction occurred in the presence of respiratory substrates such as beta-hydroxybutyrate, malate-glutamate, succinate, or endogenous substrates, nitro anion radical formation activity was much greater on addition of exogenous reduced pyridine nucleotides. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H nitroreductase reactions unless the mitochondria were solubilized by detergent. In addition, NAD(P)H nitroreductase activity was detected in the crude mitochondrial outer membrane fraction, with a higher activity than in mitoplasts and intact mitochondria. These results provide direct evidence of a nitrofuran reductase activity associated with the mitochondrial outer membrane that is far more important than that of respiratory chain enzymes.     

Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent

Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH.     

Kinetic Modeling of cometabolic degradation of ethanethiol and phenol by Ralstonia eutropha

Cometabolism, as a complex phenomenon in microbial world, is a special mechanism for transformation of many compounds of environmental and toxicological significance. Several models have been proposed to describe the cometabolic transformations of non-growth substrates in the absence or presence of growth substrates. In this study, a model was proposed to simulate the degradation kinetics of phenol and ethanethiol (ET) by a pure culture of Ralstonia eutropha, including the effects of cell growth, endogenous cell decay, loss of transformation activity, competitive inhibition between growth and non-growth substrates, and self-inhibition of non-growth substrate. The model parameters were determined independently and were then used for evaluating the applicability of the model by comparing experimental data with model predictions. The model successfully predicted ET transformation and phenol utilization for a wide range of concentrations of ET (0 ～ 40 mg/L) and phenol (0 ～ 100 mg/L).     

Adventures in Rhodococcus - from steroids to explosives

Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis, arguably the world's most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.     

Structural Basis for Featuring of Steroid Isomerase Activity in Alpha Class Glutathione Transferases

Glutathione transferases (GSTs) are abundant enzymes catalyzing the conjugation of hydrophobic toxic substrates with glutathione. In addition to detoxication, human GST A3-3 displays prominent steroid double-bond isomerase activity; e.g. transforming Δ⁵-androstene-3-17-dione into Δ⁴-androstene-3-17-dione (AD). This chemical transformation is a crucial step in the biosynthesis of steroids, such as testosterone and progesterone. In contrast to GST A3-3, the homologous GST A2-2 does not show significant steroid isomerase activity. We have solved the 3D structures of human GSTs A2-2 and A3-3 in complex with AD. In the GST A3-3 crystal structure, AD was bound in an orientation suitable for the glutathione (GSH)-mediated catalysis to occur. In GST A2-2, however, AD was bound in a completely different orientation with its reactive double bond distant from the GSH-binding site. The structures illustrate how a few amino acid substitutions in the active site spectacularly alter the binding mode of the steroid substrate in relation to the conserved catalytic groups and an essentially fixed polypeptide chain conformation. Furthermore, AD did not bind to the GST A2-2-GSH complex. Altogether, these results provide a first-time structural insight into the steroid isomerase activity of any GST and explain the 5000-fold difference in catalytic efficiency between GSTs A2-2 and A3-3. More generally, the structures illustrate how dramatic diversification of functional properties can arise via minimal structural alterations. We suggest a novel structure-based mechanism of the steroid isomerization reaction.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

Biotransformation of HCFC-22, HCFC-142b,HCFC-123, and HFC-134a by methanotrophic mixed culture MM1

This research investigated the potential for methanotrophic biotransformation of three HCFCs — chlorodifluoromethane (HCFC-22); 1-chloro-1,1-difluoroethane (HCFC-142b); and 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123); and one HFC — 1,2,2,2-tetrafluoroethane (HFC-134a). All of these compounds were biotransformed to differing degrees by methanotrophic mixed culture MM1. Rates of transformation were obtained by monitoring disappearance of the target compounds from the headspace in batch experiments. Henry's constants were determined over a range of conditions to enable estimation of the intrinsic rates of transformation. Intrinsic rates of transformation were obtained by combining a second order rate expression with an expression describing loss of transformation activity due to either endogenous decay or product toxicity. For HCFC-123 and HFC-134a, the independently measured endogenous decay rate for mixed culture MM1 (0.594/day) was sufficient to account for the observed loss of transformation activity with time. However, the endogenous decay rate did not account for the loss of transformation activity for HCFC-22 and HCFC-142b. A model based on product toxicity provided a reasonable representation of the loss of transformation activity for these compounds. The order of reactivity was HCFC-22>HCFC-142b>HFC-134a>HCFC-123, with second order rate coefficients of 0.014, 0.0096, 0.00091, and 0.00054 l/mg-day, respectively. Transformation capacities for HCFC-22 and HCFC-142b were 2.47 and 1.11 µg substrate/mg biomass, respectively.     

Profile of sex steroids in healthy volunteers' urine during experiment in isolated object

The quantitative determination a number of endogenous steroids and their metabolites in urine of healthy volunteers by means of gas chromatography - mass spectrometry was performed. The dynamic of steroid profile of healthy individuals as well as possible ranges of several endogenous steroid parameters have been investigated. Samples were obtained during 105-days experiment with 6 volunteers in isolated on ground modules where were modeling the main life conditions which could influence the steroid profile: meal volume and composition, water consumption, motion activity, air composition and temperature, rate sleep - wakefulness and emotional tension. The parameters of urine steroid profile of healthy volunteer which were affected by life conditions in isolated object were revealed. The parameters of individual and group variability of steroid profile and its dependence from definite experiment conditions - change of salt consumption periods, autonomy of vital activity were detected.     

Dynamic flux cartography of hairy roots primary metabolism

A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Intramitochondrial cholesterol transfer

Cholesterol serves as the initial substrate for all steroid hormones synthesized in the body regardless of the steroidogenic tissue or final steroid produced. The first steroid formed in the steroidogenic pathway is pregnenolone which is formed by the excision of a six carbon unit from cholesterol by the cytochrome P450 side chain cleavage enzyme system which is located in the inner mitochondrial membrane. It has long been known that the regulated biosynthesis of steroids is controlled by a cycloheximide sensitive factor whose function is to transfer cholesterol from the outer to the inner mitochondrial membrane, thus, the identity of this factor is of great importance. A candidate for the regulatory factor is the mitochondrial protein, the steroidogenic acute regulatory (StAR) protein. Cloning and sequencing of the StAR cDNA indicated that it was a novel protein, and transient transfections with the cDNA for the StAR protein resulted in increased steroid production in the absence of stimulation. Mutations in the StAR gene cause the potentially lethal disease congenital lipoid adrenal hyperplasia, a condition in which cholesterol transfer to the cytochrome P450 side chain cleavage enzyme, P450scc, is blocked, filling the cell with cholesterol and cholesterol esters. StAR knockout mice have a phenotype which is essentially identical to the human condition. The cholesterol transferring activity of StAR has been shown to reside in the C-terminal part of the molecule and a protein sharing homology with a region in the C-terminus of StAR has been shown to display cholesterol transferring capacity. Recent evidence has indicated that StAR can act as a sterol transfer protein and it is perhaps this characteristic which allows it to mobilize cholesterol to the inner mitochondrial membrane. However, while it appears that StAR is the acute regulator of steroid biosynthesis via its cholesterol transferring activity, its mechanism of action remains unknown.     

Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.     

Verapamil directly inhibits aldosterone synthesis by adrenal mitochondria in vitro

The action of verapamil, a calcium channel blocker, on the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) was studied using duck adrenal mitochondria in the absence of regulatory factors. Results show that 10(-5) M verapamil inhibits the transformation of 18-hydroxycorticosterone into aldosterone by 52.8%. Moreover, our findings show that verapamil induces only a slight inhibition of respiratory capacity without action on respiratory control and does not displace 18-hydroxycorticosterone from cytochrome P450 11 beta which catalyses the reaction. Thus, this study does not explain the mechanism of inhibition induced by verapamil on the last step of aldosterone synthesis but it is of interest to note, for clinical use, that this inhibition is not linked to regulatory factors of aldosterone production. Since primary hyperaldosteronisms are characterized by their independence vis-á-vis regulatory factors, administration of verapamil may be particularly interesting for treatment of primary hyperaldosteronisms.     

The pheromone production of female Plodia interpunctella is inhibited by tyraminergic antagonists

Several compounds were found to suppress the calling behavior and in vitro pheromone biosynthesis of the Indian meal moth, Plodia interpunctella. The compounds were screened by means of a calling-behavior bioassay with female P. interpunctella. Five derivatives with activities in the nanomolar range were identified, in order of decreasing pheromonostatic activity: 4-hydroxybenzaldehyde semicarbazone (42) > 5-(4-methoxyphenyl)-1,3-oxazole (38) > 5-[4-(tert-butyl)phenyl]-1,3-oxazole (40) > 5-(3-methoxyphenyl)-1,3-oxazole (35) > 5-(4-cyanophenyl)-1,3-oxazole (36). These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis, as determined with isolated pheromone-gland preparations that incorporated [1-(14)C]sodium acetate in the presence of the so-called pheromone-biosynthesis-activating neuropeptide (PBAN). The non-additive effect of the inhibitor with antagonist (yohimbine) for the tyramine (TA) receptor suggests that it could be a tyraminergic antagonist. Three-dimensional (3D) computer models were built from a set of compounds. Among the common-featured models generated by the program Catalyst/HipHop, aromatic-ring (AR) and H-bond-acceptor-lipophilic (HBAl) features were considered to be essential for inhibitory activity in the calling behavior and in vitro pheromone biosynthesis. Active compounds, including yohimbine, mapped well onto all the AR and HBAl features of the hypothesis. Less-active compounds were shown to be unable to achieve an energetically favorable conformation, consistent with our 3D common-feature pharmacophore models. The present hypothesis demonstrates that calling behavior and PBAN-stimulated incorporation of radioactivity are inhibited by tyraminergic antagonists.     

Molecular mechanism of telokin-mediated disinhibition of myosin light chain phosphatase and cAMP/cGMP-induced relaxation of gastrointestinal smooth muscle

Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.