Increased sensitivity to injected 5'-guanylylimidodiphosphate of the sodium efflux in barnacle muscle fibres preexposed to aldosterone

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposed to aldosterone. 2. The response to injected Gpp(NH)p is not only greater in size than in unexposed fibres but also sustained. 3. Injection of MgCl2 following peak stimulation causes a partial reversal of the response. 4. Injection of ATPNa2 (and 5'-App(NH)p) leads to a sustained stimulatory response which is not significantly greater than that seen in unexposed fibres. 5. MgCl2 injection causes complete reversal of this response. 6. The response of preexposed fibres to injected CaCl2 in varying concentration and to injected cholera toxin is not significantly different from that seen in unexposed fibres. 7. This is also true of Gpp(NH)p when it is injected after peak stimulation by cholera toxin. 8. Prior application of verapamil (10(-4)M) drastically reduces the response to injected Gpp(NH)p. 9. The residual response is sustained but markedly reduced by injected Mg2+, Fe or Zn. 10. Injection of PKI following Gpp(NH)p reduces the response, provided PKI is also injected before Gpp(NH)p. By contrast, injection of R11 subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine, when applied externally (5 X 10(-5)M), cause almost complete reversal of the response. 12. The suggestion is made that the response to injected Gpp(NH)p is mainly due to activation of Ca2+-channels resulting in activation of the calmodulin/Ca-dependent form of adenylate cyclase and that the primary site of aldosterone action is at the level of the calmodulin form of adenylate cyclase.     

Influence of insulin on sodium efflux in barnacle muscle fibers

Summary The efflux of radiosodium in single muscle fibers from the barnacleBalanus nubilus was irresponsive to internal or external application of insulin. However, this was not the case with fibers isolated from a barnacle specimen pre-exposed overnight to a large dose of insulin. External application of insulin to pre-exposed fibers caused a decrease in the rate of decline of the radiosodium efflux and stopped the decline in the fractional rate constant for Na efflux. Such kinetics were interpreted as indicating that insulin acts either by releasing sequestered Na or abolishing the process of sequestration. Internal application of saline slowed the rate of decline but failed to completely abolish the mechanism of sequestration. Only in the presence of insulin was the fractional loss of Na each second constant. Internal application of insulin caused a prompt step-up in the rate of Na efflux, followed by a reduced efflux rate constant. This meant that injected insulin caused the release of sequestered Na, leading to partial saturation of the efflux. The response of the Na efflux to injected denatured insulin, though resembling that to native insulin was much smaller in size. Internal application of lysozyme produced a transitory step-up in the rate of Na efflux but failed to produce the kinetics observed with native or denatured insulin. Overnight exposure of the barnacle to a dose of denatured insulin failed to render the fiber sensitive to external and internal application of denatured or native insulinin vitro. Experiments with ouabain-poisoned fibers showed that external or internal application of native insulin caused stimulation of the remaining Na efflux. They also showed that a 10-fold increase in the concentration of ouabain failed to further reduce the ouabaininsensitive Na efflux. Microinjection of GTP into ouabain-poisoned fibers pre-exposed to insulin resulted in a striking rise in the remaining Na efflux. The magnitude of this effect was considerably greater than that in unexposed fibers. The response which was dose-dependent could be blunted by prior injection of CaCl₂. Similarly, the response to CaCl₂ injection could be blunted by prior injection of GTP. The evidence brought forward is compatible with the view that insulin acts by abolishing the mechanism of internal Na sequestration and by increasing the activity of the guanylate cyclase system.     

Action of different nucleotides on the last step of aldosterone synthesis

The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined.     

Protection by GTP from the effects of aluminum on the sodium efflux in barnacle muscle fibers

The idea that guanine nucleotides act as chelators of Al3+ and that Al interrupts the mechanism by which GTP or Gpp(NH)p stimulates the Na efflux in single muscle fibers from the barnacle Balanus nubilus has been tested. As a rule, injection of GTP or Gpp(NH)p into unpoisoned and ouabain-poisoned fibers produces a rise in the 22Na efflux that is usually transitory in nature. Fibers preinjected with GTP show a fall in the Na efflux following the injection of AlCl3 in an equimolar concentration. If, however, the concentration of Al for injection is halved, then GTP is found to be fully protective. Fibers preinjected with AlCl3 show little or no response to the injection of GTP. This is also the case with ouabain-poisoned fibers. Ouabain-poisoned fibers preinjected with GTP also show little or no response to the injection of AlCl3. The stimulatory response to the injection of AlCl3 into fibers preinjected with 0.5 M GTP is dose-dependent. A graded response is also found when 0.5 M AlCl3 is injected into fibers preinjected with GTP in varying concentrations. Gpp(NH)p is fully protective against the inhibitory effect of Al injection in unpoisoned fibers. Further, Gpp(NH)p abolishes the biphasic effect of Al injection on the ouabain-insensitive Na efflux. To strengthen the argument that GTP acts as a chelator of Al, a solution mixture of 0.5 M GTP/0.5 M AlCl3 (pH 1-2) was injected into unpoisoned fibers. This is found to lead to a smaller fall in the resting Na efflux than that obtained by injecting AlCl3 alone or injecting AlCl3 after GTP. It is thus quite clear that the barnacle muscle fiber is a useful preparation for studies of this type.     

Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.     

Adaptive respiratory significance of organophosphates (ATP & GTP) in air-breathing fishes

The ATP and GTP contents of erythrocytes of Protopterus annectens, Polypterus senegalus and Clarias lazera were assessed in relation to maturation and hypoxia. The steady state normal concentrations of ATP were: 446, 316 and 475 µM/100 ml and those of GTP were 286, 93 and 85 µM/100 ml in Protopterus, Polypterus and Clarias, respectively. The level of ATP decreased and that of GTP increased with increments in body growth, especially during development of the pulmonary organs. The concentration of GTP also increased progressively with hypoxia (O₂: 7.4 to 3.6 mg/l); the level ATP remained almost unaltered. The ratio of GTP increased markedly with increased hypoxia particularly in the adaptive respiratory range of aquatic oxygen concentrations (6.5–4.8 mg/l) when the fish were forced to depart from branchial respiration and became reliant on air-breathing.     

Rapid responses to aldosterone in human distal colon.

Aldosterone at normal physiological levels induces rapid increases in intracellular calcium and pH in human distal colon. The end target of these rapid signaling responses are basolateral K+ channels. Using spectrofluorescence microscopy and Ussing chamber techniques, we have shown that aldosterone activates basolateral Na/H exchange via a protein kinase C and calcium-dependent signaling pathway. The resultant intracellular alkalinization up-regulates an adenosine triphosphate (ATP)-dependent K+ channel (K(ATP)) and inhibits a Ca2+ -dependent K+ channel (K(Ca)). In Ussing chamber experiments, we have shown that the K(ATP) channel is required to drive sodium absorption, whereas the K(Ca) channel is necessary for both cyclic adenosine monophosphate and calcium-dependent chloride secretion. The rapid effects of aldosterone on intracellular calcium, pH, protein kinase C and K(ATP), K(Ca) channels are insensitive to cycloheximide, actinomycin D, and spironalactone, indicating a nongenomic mechanism of action. We propose that the physiological role for the rapid nongenomic effect of aldosterone is to prime pluripotential epithelia for absorption by simultaneously up-regulating K(ATP) channels to drive absorption through surface cells and down-regulating the secretory capacity by inhibiting K(Ca) channels involved in secretion through crypt cells.     

Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.     

Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.     

Light-induced changes in GTP and ATP in frog rod photoreceptors. Comparison with recovery of dark current and light sensitivity during dark adaptation

Light decreases GTP and ATP levels in purified suspensions of physiologically active frog rod outer segments still attached to their inner segment ellipsoids (OS-IS). (a) The GTP decrease is slower in OS-IS (t1/2 = 40 s) than in isolated outer segments (t1/2 = 7 s), which suggests there is more effective buffering in OS-IS. (b) The GTP decrease becomes detectable only at intensities greater than those required to saturate the photoresponse. As the intensity of a continuous light is increased over 4 log units, GTP levels decrease linearly with log intensity by as much as 60%. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity. (c) At levels of illumination bleaching greater than 0.003% of the rhodopsin, a decrease in ATP levels becomes detectable. (d) Following a flash, GTP levels fall and then rise with a recovery time dependent on the intensity of the flash. (e) After both 0.2 and 2% flash bleaches, the recovery of GTP levels parallels the recovery of light sensitivity, which is slower than the recovery of the dark current. This raises the possibility of a link between GTP levels and light sensitivity.     

The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP

Effect of GTP on adenylate cyclase of liver plasma membrane was examined using ATP which was extensively purified by DEAE-cellulose column chromatography. In the incubation containing 2mM purified ATP as substrate, GTP enhanced basal and glucagon- or fluoride-stimulated activities. When the unpurified ATP at 2mM was used, all the activities were high and the stimulatory effect of GTP was not detected. The substance(s) which was recovered from a small but significant peak on DEAE-cellulose column was equivalent to 10–100μM GTP in stimulating adenylate cyclase. These results indicate that, if highly purified ATP is used as substrate, GTP can enhance adenylate cyclase activity in the presence of millimolar concentration of ATP and that GTP enhances not only the glucagon-stimulated adenylate cyclase but also the basal as well as fluoride-stimulated adenylate cyclase activities.     

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.     

The role of ATP in the cytostructure of the hepatocytes

We have previously described the preparation of hepatocytes from which the plasma membrane was removed by digitonin treatment. Such "nude" cells were found to be very stable in sucrose media containing above 50 mM NaCl or KCl, but they disintegrate near instantly in salt-free media, liberating nuclei, mitochondria, and other organelles. We show here that disintegration occurs at a physiologic pH and in the presence of oxygen. Disintegration was blocked by rotenone, oligomycin, KCN, and carboxyatractyloside, establishing that oxidative phosphorylation and ATP generation is essential for disintegration to occur. The addition of ATP, GTP, ITP, or ADP (but not AMP) in the presence of the inhibitors, induced breakdown. Taxol, an inhibitor of tubulin depolymerization and phalloidin, a drug that stabilizes actin fibers, prevented disintegration in salt-free media. The effect of these drugs was counteracted by the addition of ATP. Our results show that two conditions are essential to induce the disintegration of the nude cell: media of low ionic strength, and ATP generation. The ATP effect is likely to be of physiological significance, suggesting role of ATP generation in affecting polymerization of cytoskeletal elements.     

Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools

Intracellular ATP depletion is a hallmarkevent in ischemic injury. It has been extensively characterized inmodels of chemical anoxia in vitro. In contrast, the fate of GTP duringischemia remains unknown. We used LLC-PK proximal tubular cells tomeasure GTP and ATP changes during anoxia. In 45 min, antimycin Adecreased ATP and GTP to 8% and 2% of controls, respectively.Ischemia in vivo resulted in comparable reductions in GTP and ATP.After 2 h of recovery, GTP levels in LLC-PK cells increased to65% while ATP increased to 29%. We also investigated steady-statemodels of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26%of control. Selective GTP depletion resulted in significant apoptosis.Selective ATP depletion caused mostly necrosis. These models of ATP orGTP depletion can prove useful in dissecting the relative contributionof the two nucleotides to the ischemic phenotype.

     

Protein kinase activity of bovine retina rod outer segments

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.