Calcium-dependent lipolytic acyl-hydrolase activity in purified plant mitochondria

Ageing of isolated potato mitochondria induced by CaCl2 resulted in rapid enzymatic hydrolysis of the membrane phospholipids with the liberation of free fatty acids. The enzyme responsible for this effect was identified as a membrane bound lipolytic acyl-hydrolase which was unmasked by CaCl2. The presence of this lipolytic acyl-hydrolase induced severe functional impairments in the mitochondrial oxidative and phosphorylative properties.     

The effects of protein synthesis inhibitors on oxidative phosphorylation by plant mitochondria

Inhibitors can be successfully used if they are specific for only one process. Published data suggest that some inhibitors of protein synthesis may also inhibit respiration or oxidative phosphorylation. The effect of a range of protein synthesis inhibitors on respiration and phosphorylation has been studied, using tightly coupled mitochondria from several plant species including turnips (Brassica napus).Puromycin, actinomycin D, lincomycin, mitomycin C and d-serine did not uncouple or inhibit respiration. Cycloheximide caused a partial inhibition (maximum 22% at 3 mM) of malate but not succinate-driven respiration. Chloramphenicol was a potent inhibitor of electron transport, but not of phosphorylation. The activity of the isomers of chloramphenicol varied in the order l-threo >d-threo >l-erythro >d-erythro. From evidence presented it is concluded that chloramphenicol has three sites of action, the flavoprotein level being most sensitive, the second site of variable sensitivity lies between cytochromes b and c and the third site at the cytochrome a level is only slightly affected by the inhibitor.     

Endogenous protein phosphorylation in Escherichia coli extracts

The protein kinase activity ofEscherichia coli was analyzed through its ability to phosphorylate endogenous proteins at the expense of adenosine triphosphate in cellular extracts. The nature of the amino acids phosphorylated in these proteins was determined.     

Oxalacetate control of Krebs cycle oxidations in purified plant mitochondria

Plant mitochondria survive separation on sucrose gradients and subsequent dilution to iso-osmolar conditions. Oxalacetate penetrates these remarkably uniform and intact preparations, and inhibits all Krebs cycle oxidations. With the exception of succinate these inhibitions are caused by oxidation of a common pool of NADH, reduced by dehydrogenases, during conversion of added oxalacetate to malate.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Endogenous protein phosphorylation in adhesive plaques of substrate-attached fibroblasts

Endogenous protein kinases and acceptor proteins in the focal adhesion plaques of monolayer cells were studied using [γ-³²P]ATP as exogenous substrate. Two major phosphoproteins, p135 and pp105, besides some minor phosphoproteins, were phosphorylated in the substrate-attached material after dislodgement of cells. Evidence was obtained, that suspension cells leave the same components on the wall of the culture vessel, resulting from cell collisions with the wall and not from passive adsorption of secreted components or disintegrated cellular material.     

Simultaneous measurement of oxidative phosphorylation and adenylate kinase in plant mitochondria

An assay system capable of simultaneously measuring ATP, ADP, and AMP concentrations was used for the measurement of oxidative phosphorylation and adenylate kinase (5′-ATP:5′-AMP phosphotransferase) activities in mitochondria which were isolated from etiolated corn, soybean, or cucumber seedlings. Data obtained by this system was correlated with colorimetric P_i uptake and spectrophotometric NADH oxidation measurements. Adenylate kinase was active in both phosphorylating and nonphosphorylating mitochondria. Studies using NaCN, 2,4-dinitrophenol, atractyloside, and 2′-AMP as inhibitors indicated that exogenously supplied [¹⁴C]AMP was converted to [¹⁴C]ADP either by NADH-linked phosphorylation or by translocation and transphosphorylation from intramitochondrial nucleotides.     

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Summary Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mitochondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [¹⁴]leucine and [³⁵]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.     

Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria.

Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.     

Defining the protein complex proteome of plant mitochondria

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.     

Redox signaling and protein phosphorylation in mitochondria: progress and prospects

As we learn more about the factors that govern cardiac mitochondrial bioenergetics, fission and fusion, as well as the triggers of apoptotic and necrotic cell death, there is growing appreciation that these dynamic processes are finely-tuned by equally dynamic post-translational modification of proteins in and around the mitochondrion. In this minireview, we discuss the evidence that S-nitrosylation, glutathionylation and phosphorylation of mitochondrial proteins have important bioenergetic consequences. A full accounting of these targets, and the functional impact of their modifications, will be necessary to determine the extent to which these processes underlie ischemia/reperfusion injury, cardioprotection by pre/post-conditioning, and the pathogenesis of heart failure.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Endogenous modulators of glucocorticoid receptor function also regulate purified protein kinase C

Modulator-1 and -2, proposed to be novel ether-linked aminophosphoglycerides, were originally identified as regulators of glucocorticoid receptor function (Bodine, P. V., and Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554). We now demonstrate that these modulators are also potent new stimulators of protein kinase C activity in vitro. These endogenous biomolecules regulate purified protein kinase C activity in a biphasic and dose-dependent pattern, as determined by histone phosphorylation. Modulators, at concentrations within their apparent cellular range, stimulate protein kinase C-catalyzed histone phosphorylation 2-4-fold when added separately, or 10-12-fold when added together. This enhancement of kinase activity apparently is specific for protein kinase C, since neither protein kinase M, nor cAMP-dependent protein kinase A are stimulated by the modulators. The stimulation of purified protein kinase C occurs only when the enzyme has been initially activated by calcium, phosphatidylserine, and diacylglycerol, indicating that the modulators do not simply substitute for one of the enzyme cofactors. In addition, the modulators appear to interact directly with protein kinase C, perhaps with the regulatory domain of the enzyme, since these biomolecules inhibit the binding of phorbol ester to purified protein kinase C. Finally, time-course studies of protein kinase C-catalyzed histone phosphorylation indicate that the velocity of the enzyme reaction is increased by the modulators. Taken together, these results suggest that the modulators are a new class of regulators of protein kinase C.     

Modulation of protein phosphorylation by a factor purified from adipocytes.

1. A factor which modulates the activity of cyclic AMP-dependent protein kinase copurifies from rat adipocytes with an inhibitor of adenylate cyclase. Purification and stability studies suggest that both effects reside in a single factor previously referred to as a feedback regulator. 2. The magnitude and direction of the feedback regulator effect on cyclic AMP-dependent protein kinase activity was dependent on the concentration of feedback regulator and the concentration and type of protein substrate. Using histone type IIA as substrate, feedback regulator was inhibitory at low histone concentrations and stimulatory at high concentrations. Preincubation of protein kinase with feedback regulator resulted in inhibition at all histone concentrations. With some protein substrates, e.g. histone f2b and casein, inhibition was observed at all histone concentrations. 3. The stimulation of histone type IIA phosphorylation resulted from an increased V with no effect on either the apparent Ka for cyclic AMP or the Km for ATP. Time course studies suggest that feedback regulator increased the rate of phosphorylation without increasing the total number of phosphorylation sites. Increased histone phosphorylation was observed regardless of whether the cyclic AMP-dependent protein kinase was peak I or peak II (off Deae-cellulose), isolated from bovine or rabbit skeletal muscle or rat heart. A small stimulation was observed using cyclic GMP-dependent protein kinase. 4. These results indicate that feedback regulator can inhibit or stimulate protein kinase, an effect which is probably substrate directed, and depends on the reaction conditions. Whether feedback regulator modulated protein phosphorylation in vivo in addition to its inhibition of adenylate cyclase is unknown. However, stimulation of protein kinase activity in the presence of cyclic AMP is a valuable and rapid assay for monitoring feedback regulator fractions during purification procedures.     

pH-jump-induced ADP phosphorylation in mitochondria

Mitochondria, uncoupled by aging or by freeze-thaw treatment, are able to synthesize ATP from ADP and P_i after a fast increase (but not decrease) in the external pH. The maximal ATP yield (approx. 2.5 ATP molecules / electron-transport chain per pH jump) can be obtained under the following conditions: (1) the pH change during the jump must exceed 0.7 pH units; (2) in the course of this change, the pH of the mitochondrial suspension must cross the pH 8.1–8.3 value. This pH-jump-induced ATP synthesis is completely inhibited by oligomycin.     

Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.