An amphitropic cAMP-binding protein in yeast mitochondria. 2. Phospholipid nature of the membrane anchor

We describe the first example of a mitochondrial protein with a covalently attached phosphatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cerevisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified cAMP-binding protein through a phospholipase activity.     

An amphitropic cAMP-binding protein in yeast mitochondria. 1. Synergistic control of the intramitochondrial location by calcium and phospholipid

A cAMP-binding protein is found to be integrated into the inner mitochondrial membrane of the yeast Saccharomyces cerevisiae under normal conditions. It resists solubilization by high salt and chaotropic agents. The protein is, however, converted to a soluble form which then resides in the intermembrane space, when isolated mitochondria are incubated with low concentrations of calcium. Phospholipids or diacylglycerol (or analogues) dramatically increases the efficiency of receptor release from the inner membrane, whereas these compounds alone are ineffective. Also, cAMP does not effect or enhance liberation from the membrane of the cAMP-binding protein. Photoaffinity labeling with 8-N3-[32P]cAMP followed by mitochondrial subfractionation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis does not reveal differences in the apparent molecular weight between the membrane-bound and the soluble form of the cAMP receptor. The two forms differ, however, in their partitioning behavior in Triton X-114 as well as in their protease resistance, indicating that the release from the membrane is accompanied by a change in lipophilicity and conformation of the receptor protein. Evidence is presented that a change of the intramitochondrial location of the yeast cAMP-binding protein also occurs in vivo and leads to the activation of a mitochondrial cAMP-dependent protein kinase. The cAMP-binding protein is the first example of a mitochondrial protein with amphitropic character; i.e., it has the property to occur in two different locations, as a membrane-embedded and a soluble form.     

Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine

Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, Mr = 60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50 mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 microM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+-dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 microM and 2.5 microM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.     

Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes.

Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.     

Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.     

Cyclic AMP- and Ca2(+)-dependent protein kinases in Plasmodium falciparum

The cyclic AMP- and Ca2(+)-dependent protein kinase activities of Plasmodium falciparum were partially characterized after purification of parasites from host erythrocytes by N2 cavitation and Percoll gradient centrifugation. Proteins of molecular weights 80, 54, 51, and 31.5 kDa were phosphorylated in a cAMP-dependent manner in cytosolic extracts of isolated P. falciparum. Cytosolic extracts also contained cAMP-dependent histone II-A kinase activity with an average Vmax of 131.1 pmol/32P/min/mg protein and a Km for cAMP of 85nM. Upon photoaffinity labeling with [32P]-8-N3-cAMP, a 53-kDa protein was specifically labeled in parasite cytosol. A metabolically labeled protein of the same molecular weight was identified by cAMP-agarose affinity chromatography. The 53-kDa protein cochromatographed with cAMP-dependent histone II-A kinase activity on DEAE-cellulose, suggesting that it is the regulatory subunit of the kinase. Ca2(+)-dependent phosphorylation of proteins of molecular weights 195, 158, 51, 47.5, and 15 kDa was demonstrated in a membrane fraction from parasites free of the erythrocyte membrane. This activity was not stimulated by either calmodulin or phospholipid plus diacylglycerol and was absent from the membranes of uninfected erythrocytes. Of several exogenous substrates tested, none were found to be a substrate for this Ca2(+)-dependent kinase. Both cAMP- and Ca2(+)-dependent kinases phosphorylated serine and threonine residues.     

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation

Interaction of Cu(II) and Gly-His-Lys, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-n.m.r. and e.p.r. spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The results indicate that the n.m.r. line-broadening is due to the presence of minor species in rapid exchange and not due to the major species in solution, which has a large tau M. It is concluded that the technique of 13C- and 1H-n.m.r. line broadening, caused by paramagnetic Cu(II) ion, should be undertaken with caution, since the method may not be useful for obtaining structural information on the major species. The e.p.r. spectra over a wide pH range are almost entirely due to similarly co-ordinating species. Starting at pH 5.5, the narrowest absorption near 340 mT shows superhyperfine structure, which comes out sharply in the pH region 6.0-9.6. The spectra in this pH range showed the seven lines of nitrogen superhyperfine splitting, indicating clearly the co-ordination of three nitrogen atoms to Cu(II). The e.p.r. parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

Free fatty acids activate a vigorous Ca(2+):2H(+) antiport activity in yeast mitochondria.

The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.     

ATP-dependent Ca2+ uptake and Ca2+-dependent protein phosphorylation in basolateral liver plasma membranes

ATP-dependent Ca2+ uptake was measured in vesicles of rat liver cell basolateral plasma membranes. Nucleotide-dependent uptake was specific for ATP and observed at pH 7.0 and 7.4/7.5 but not at pH 8.0. ATP-dependent Ca2+ transport was only observed in the presence of Mg2+. Kinetic analysis of ATP-dependent transport revealed an apparent Km in the submicromolar region. Addition of calmodulin and trifluoperazine had no effect on ATP-dependent uptake. A Ca2+-dependent, phosphorylated intermediate with the apparent molecular weight of 135,000 could be demonstrated in the basolateral plasma membranes. Phosphorylated intermediates with apparent molecular weights of 200,000 and 110,000 were demonstrated in microsomes and appeared to contaminate 'basolateral' membrane protein phosphorylation. The results suggest that a 135,000 molecular weight protein is a Ca2+-ATPase and the enzymatic expression of the liver cell basolateral membrane Ca2+ pump.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Increased phospholipase a(2) activity with phosphorylation of peroxiredoxin 6 requires a conformational change in the protein

We have shown previously and confirmed in this study that the phospholipase A(2) (PLA(2)) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluates the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, strengthened ANS binding, and a lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ΔG°(D) was 3.3 ± 0.3 kcal mol(-1) for Prdx6 and 1.7 ± 0.7 kcal mol(-1) for pPrdx6, suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (50:25:15:10 DPPC:egg PC:cholesterol:PG molar ratio) were evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with a higher affinity (K(d) = 5.6 ± 1.2 μM) than Prdx6 (K(d) = 24.9 ± 4.5 μM). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (ΔH = -31.49 ± 0.22 kcal mol(-1)). Correlating our conformational studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA(2) activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA(2) enzymatic activity.     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.     

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.     

Involvement of Ca2+ release and activation of phospholipase A2 in mitochondrial dysfunction during anoxia

During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.