Time Course Of Phosphorylation Research Articles

The rate and extent of phosphorylation of the two light-harvesting chlorophyll a/ b binding protein complex (LHC-II) polypeptides in isolated spinach thylakoids

The level of phosphorylation of the 24 kDa and the 25 kDa light-harvesting chlorophyll a/ b binding protein complex (LHC) II polypeptides in isolated spinach thylakoids has been determined by quantitative SDS-polyacrylamide gel electrophoresis. The time-course of phosphorylation, after correction for the molar abundance of these two polypeptides, shows that (a) the rate of phosphorylation of the 24 kDa polypeptide is at least 3-fold faster compared with the 25 kDa polypeptide, (b) the final extent of phosphorylation for both the polypeptides is very similar, and (c) the final extent of phosphorylation is typically between 0.15 and 0.25 mol phosphate per mol polypeptide. The low extent of phosphorylation is not simply a consequence of inactivation of the kinase and / or LHC II substrate or ATP depletion. These observations suggest that there are at least three different sub-populations of LHC II in isolated thylakoids: (i) phosphorylated ‘mobile’, (ii) phosphorylated ‘PS II-coupled’ and (iii) non-phosphorylated. Furthermore, the reported differences in the specific activity of phosphorylation for the ‘mobile’ and the ‘PS II-coupled’ LHC II sub-populations (Kyle, D.J. et al. (1984) Biochim. Biophys. Acta 765, 89–96) are no longer observed following correction for the non-phosphorylated LHC-II population.

Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulin-dependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under conditions (0 degree C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of phosphorylation of threonine in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the beta subunit. The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2+/calmodulin-independence of Ca2+/calmodulin-dependent protein kinase II.

Phosphorylation of the α-subunit of (Na + + K +)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses ( M r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M r 96 000 protein precipitated at 120 000 × g while most of the M r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M r 96 000 protein was phosphorylated by ATP in the presence of Na + and Mg 2+. It was dephosphorylated by K +. This proves that the M r 96 000 dalton protein is the α-subunit of the (Na + + K +)-ATPase.

Myosin light chain phosphorylation during phasic contractions of tracheal smooth muscle.

Rapid, coordinated contractions of tracheal smooth muscle were elicited by either direct electrical depolarization of muscle cells or treatment with tetraethylammonium which produced spontaneous phasic contractile activity. Both types of contraction were blocked by the calcium channel antagonist verapamil, indicating that these contractions are supported primarily by calcium of extracellular origin. With direct electrical stimulation, force was biphasic and phosphate content of the phosphorylatable light chain (P-light chain) of myosin increased rapidly (approximately 2.5 s) from 0.1 to 0.4 mol phosphate/mol P-light chain, then decreased to levels above resting values. Phosphorylation increased more rapidly than force. Under conditions of spontaneous activity, phasic contractions occurred above a level of basal tone significantly greater than resting force, and minimum values of phosphorylation measured at the base of contraction were significantly greater than those observed in the resting muscle. Phosphorylation oscillated with force (from 0.2 to 0.4 mol phosphate/mol P-light chain) and peak values occurred during the rising phase of contraction. Time courses of phosphorylation and force showed evidence of a prolonged state of activation of myosin following dephosphorylation. These results suggest that phosphorylation and dephosphorylation of myosin P-light chain are sufficiently rapid to participate in regulation of contractility during phasic mechanical activity.

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin.

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.

Phosphorylated proteins from anuran intestinal microvilli membranes—I. Relations with alkaline phosphatase

1. 1. The degree of phosphorylation of intestinal microvilli membrane proteins in an adult amphibian, Rana esculenta, was investigated under various experimental conditions. 2. 2. The microvilli protein phosphorylation rate rapidly increases during the first 4 min of incubation in a medium containing [γ- 32P]ATP. This increase is slower afterwards. 3. 3. Cyclic nucleotides (cyclic AMP, cyclic GMP) and sorbitol do not modify the microvilli protein phosphorylation rate. 4. 4. On the contrary, this phosphorylation rate significantly decreases in the presence of l-lysine, when its concentration in the incubation medium is greater than 25 mM. The time course of phosphorylation confirms the inhibitory effects of l-lysine (100 mM). 5. 5. The microvilli membrane proteins were distinguished by polyacrylamide gel electrophoresis. In heated samples, electrophoresis followed by an radioautograph systematically reveals the existence of a very phosphorylated protein with a mol. wt of 86 kDa. 6. 6. The phosphorylation of this protein is partially inhibited by l-lysine (100 mM). 7. 7. The very phosphorylated protein could be the monomer of alkaline phosphatase. The dimer (170 kDa) is visualized on electrophoretograms by its catalytic activity. 8. 8. In mammals, several authors have established a correlation between phosphorylation of the microvilli membrane proteins and the intensity of intestinal calcium absorption. Such a control is presently being investigated in adult Rana esculenta.

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains.

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response.

When platelets were stimulated with ADP to cause shape change without aggregation or secretion, myosin 20,000-Da light chain phosphorylation was rapid and appeared to precede slightly the shape change response. While the shape of the platelets remained spheroidal, myosin phosphorylation was transient and after 2-5 min returned to the same level as that of unstimulated cells. Phosphorylation of the 47,000-Da platelet protein was minimal under these conditions. The phosphorylation time course was not altered by the addition of indomethacin or allowing the cells to aggregate. The dose-response curve of myosin phosphorylation very closely paralleled that of shape change with a midpoint at 0.7 microM ADP. ATP, a competitive antagonist of ADP, inhibited both shape change and myosin phosphorylation with the same concentration of ATP causing 50% inhibition of each response. Similarly, when platelets were stimulated with either 15-hydroxy-9,11-azo-prostadienoic acid or collagen, myosin phosphorylation slightly preceded shape change. These results suggest that myosin phosphorylation is required for the initial change in platelet shape but is not necessary for maintenance of the spherical shape.

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN

AbstractWe have continued to characterize the blue light‐regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol.92, 179–185). By incubating pea stem sections in 32P‐phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30‐fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence‐response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse‐chase experiments on unirradiated, blue light pulse‐irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light‐induced change to persist for several minutes at 30°C. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light‐induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V‐8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.

The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state.

The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction.

Solubilization of EGF receptor with Triton X-100 alters stimulation of tyrosine residue phosphorylation by EGF and dimethyl sulfoxide.

In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation.

Effect of magnesium on the calcium-dependent transient kinetics of sarcoplasmic reticulum ATPase, studied by stopped flow fluorescence and phosphorylation.

At pH 7 and 20 degrees C and in the absence of potassium and magnesium, the intrinsic fluorescence rise after addition of calcium to a calcium-deprived enzyme was monoexponential. On the other hand, when the calcium-deprived enzyme was preincubated with magnesium, this fluorescence rise was clearly biphasic at high calcium concentrations. For a constant magnesium concentration (higher than millimolar), the rate constant of the slow phase and the amplitude of the fast phase rose for the same range of calcium concentrations, between pCa 5 and 4. At pH 6, fluorescence signals were monophasic even with 20 mM Mg2+. We also found that at pH 7, phosphorylation of the enzyme after simultaneous addition of calcium, magnesium, and ATP was faster when sarcoplasmic reticulum was originally calcium-deprived in the presence of magnesium. At pH 6, on the contrary, preincubation with magnesium did not influence the phosphorylation time course. It is concluded that (a) magnesium produces an enzyme conformation which is able to react with calcium and ATP as required by the kinetics of the ATPase reaction; (b) the magnesium-enzyme complex presents one readily accessible site of relatively low affinity (Kd congruent to 25 microM) for calcium; (c) occupancy of this site by calcium triggers a relatively slow (4-6 s-1) conformational change unmasking a second high affinity site; (d) interaction between and occupancy of these two calcium sites occur with positive cooperativity and yield enzyme activation; and (e) the kinetic difference between the low time constant of the second component of fluorescence rise following addition of calcium (in the absence of ATP) and the relatively fast phosphorylation obtained in the same conditions (but in the presence of ATP) is attributed to an Mg2+-dependent accelerating effect of ATP on enzyme isomerization.

Endogenous protein phosphorylation by resting and activated human neutrophils

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.

Endogenous Protein Phosphorylation by Resting and Activated Human Neutrophils

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen-dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl-phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.

Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin.

The time courses of phosphorylation of the Mr 20 000 light chains by purified myosin light chain kinase plus calmodulin were determined. In confirmation of an earlier report [Persechini, A., & Hartshorne, D. J. (1981) Science (Washington, D.C.) 213, 1383-1385], a steady-state kinetic analysis indicates that the phosphorylation occurs in an ordered manner; i.e., at a phosphorylation level of 0.5 mol of 32P incorporated per mol of bound Mr 20 000 light chain, each myosin molecule would have one phosphorylated head. The kinetic parameters obtained for the phosphorylation of the more reactive myosin head are similar to those determined by using isolated light chains. It is suggested that the ordered, or sequential, phosphorylation, and the different reactivities of the two Mr 20 000 light chains, is the result of preexisting asymmetry of the myosin molecule. Similar patterns of myosin phosphorylation are obtained in both the absence and presence of skeletal muscle actin.

Role of Ca2+ and myosin light chain phosphorylation in regulation of smooth muscle

The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of 32P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.

Control of adenosine 3′,5′-monophosphate level and protein phosphorylation by depolarizing agents in Coprinus macrorhizus

The treatment of mycelial cells with membrane-active antibiotics, uncouplers of oxidative phosphorylation and KCl leads to a transient increase in adenosine 3′,5′-monophosphate (cyclic AMP) levels in Coprinus macrorhizus. The maximal values and duration of increase in the cyclic AMP level depended on the kind and amount of these drugs. The treatment with thse drugs simultaneously resulted in a rapid increase in the phosphorylation of three cellular proteins. The levels and time course of phosphorylation of these proteins were paralleled with the increase of cyclic AMP level in response to the drugs used. Thus, the treatment of these drugs causes the transient increase of cyclic AMP level and cyclic AMP stimulates the phosphorylation of particular proteins by activating protein kinases.

Phosphorylation of Plasma Membrane Proteins Dependent on Adenosine 3′,5′‐Monophosphatein Rat‐Glial C6 Cells

The presence of protein kinase(s) dependent on adenosine 3′,5′‐monophosphate (cyclic AMP) and their endogenous substrates were demonstrated in a glial plasma‐membrane preparation. Three proteins, C, D and E, are the major substrates for a cyclic‐AMP‐regulated phosphorylation system in the plasma membrane of C6 cells, a glial tumor cell line. Plasma‐membrane fractions were isolated by a modification of the ZnCl2 method of Warren et al. and were endogenously phosphorylated in vitro in the presence of [γ‐32P]ATP. Phosphorylated proteins were separated by electrophoresis in 7, 9 and 12% discontinuous gradient polyacrylamide gels. Phosphorylated protein bands were visualized by autoradiography. The time course of phosphorylation and dephosphorylation of protein bands C, D and E was regulated by cyclic AMP. The approximate molecular weights of these proteins are 51000, 47000 and 38000, respectively. Quantitative microdensitometry of the autoradiograms showed that the endogenous phosphorylation of the three proteins was affected by the concentration of cyclic AMP, cyclic GMP, ATP and divalent cations. The concentration of cyclic AMP required for optimal stimulation of the endogenous phosphorylation of the three proteins was in the range of 0.1 to 1 μM. Cyclic GMP gave maximal stimulation above 10 μM. Mg2+ was a more effective metal ion than either Mn2+ or Co2+. The optimal concentration of those divalent cations was in the range of 10–25 mM. When Mg2+ was replaced by Ca2+, Cu2+ or Zn2+, little or no phosphorylation was observed. However, two proteins, A and B, were specifically phosphorylated in the presence of Cu2+ and Zn2+ respectively, but not in the presence of Mg2+, Mn2+ or Ca2+. We propose that such cyclic‐AMP‐dependent phosphorylation of plasma‐membrane proteins may mediate cyclic AMP action in regulating membrane functions, such as amino acid transport in C6 cells.

The ATPase inhibitor protein in oxidative phosphorylation The rate-limiting factor to phosphorylation in submitochondrial particles

1. 1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an ‘on-line’ digital computer. 2. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate (‘lag’); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (ATP hydrolysis) after all the oxygen had been consumed. 3. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor protein inhibits both ATP synthesis and hydrolysis by the ATPase. 4. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. 5. The rate constant for inhibitor release was dependent on ‘energisation’ of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.