Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

AbstractInside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [γ-32P]ATP. Proteins were separated by SDS–PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [γ-32P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins

Numerical upscaling of discrete network models

In this paper a numerical multiscale method for discrete networks is presented. The method gives an accurate coarse scale representation of the full network by solving sub-network problems. The method is used to solve problems with highly varying connectivity or random network structure, showing optimal order convergence rates with respect to the mesh size of the coarse representation. Moreover, a network model for paper-based materials is presented. The numerical multiscale method is applied to solve problems governed by the presented network model

Network model for predicting structural properties of paper

Paper simulations that resolve the entire microscopic fiber structure are typically time-consuming and require extensive resources. Several such modeling approaches have been proposed to analyze different properties in paper. However, most use non-linear and time-dependent models resulting in high computational complexity. Resolving these computational issues would increase its usefulness in industrial applications. The model proposed in this work was developed in collaboration with companies in the papermaking industry within the Innovative Simulation of Paper (ISOP) project. A linear network model is used for efficiency, where 1-D beams represent the fibers. Similar models have been proposed in the past. However, in this work, the paper models are three-dimensional, a new dynamic bonding technique is used, and more extensive simulations are evaluated. The model is used to simulate tensile stiffness, tensile strength, and bending resistance. These simulated results are compared to experimental and theoretical counterparts and produce representable results for realistic parameters. Moreover, an off-the-shelf computer accessible to a paper developer can evaluate these models structural properties efficiently

Protein-Based Biostimulants to Enhance Plant Growth-State-of-the-Art and Future Direction with Sugar Beet as an Example

Protein-based biostimulants (PBBs) are derived from the hydrolysis of protein-rich raw materials of plant and/or animal origins, usually by-products or wastes from agro-industries. The active ingredients (AIs) produced by hydrolysis have the capacity to influence physiological and metabolic processes in plants, leading to enhanced growth, nutrient and water-use efficiency, tolerance to abiotic and biotic stresses, and improved crop yield and quality. This paper reviews the state-of-the-art and future opportunities for use of PBBs, based on potential effects on the soil, crops, and sustainability (social, economic, environmental). In this case, two examples of PBBs (hydrolyzed wheat gluten and potato protein) and their effects on the early growth of three sugar beet varieties are described and discussed. Both PBBs have a significant stimulating effect on early sugar beet growth and development. The opportunity to develop PBBs into superabsorbent polymers (SAPs) is discussed. To conclude, PBBs/SAPs developed from agro-industrial wastes have the potential for sustainably supplying water and nutrients in agricultural systems and for enhancing plant growth and development over a substantial period

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca2+ with a K0.5 of about 1 μM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca2+ with a K0.5 of 3 μM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer

Purification of a rotenone-insensitive NAD(P)H dehydrogenase from the inner surface of the inner membrane of red beetroot mitochondria

The soluble fraction of disrupted red beetroot mitochondria was resolved by anion-exchange chromatography. Three NADH-oxidising activities were found, including one duroquinone reductase oxidising both NADH and NADPH. This NAD(P)H-duroquinone reductase, which we assign as the internal rotenone-insensitive NAD(P)H dehydrogenase, was further purified by affinity chromatography into a 26 kDa polypeptide

Oxidation of External NAD(P)H by Purified Mitochondria from Fresh and Aged Red Beetroots (Beta vulgaris L.)

Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10°C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes

The presence of a short redox chain in the membrane of intact potato tuber peroxisomes and the association of malate dehydrogenase with the peroxisomal membrane

Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD−-dependent; EC 1.1.1.37) latency.The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)−1 min−1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane.When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated

Phosphoproteins and protein kinase activities intrinsic to inner membranes of potato tuber mitochondria

Inside-out submitochondrial particles (IO-SMP) were isolated and purified from potato (Solanum tuberosum L. cv.) tubers. When these IO-SMP were incubated with [γ32P]ATP more then 20 proteins became labelled as a result of phosphorylation. The 32P incorporation was stimulated by the oxidizing reagent ferricyanide. Except for a 17 kDa protein which was phosphorylated only in the absence of divalent cations, the protein phosphorylation required Mg2+. The time for half-maximum 32P incorporation was 4 min for the 22 kDa phospho-F1 δ-subunit and 2 min for the 28 kDa phospho-F0 b-subunit of the proton-ATPase. The K(m) for ATP for the detected phosphoproteins was between 65 μM and 110 μM. The pH optimum for protein phosphorylation in inner membranes was between pH 6 and 8, and for the F1 δ-subunit and the F0 b-subunit the pH optima were 6.5-8 and pH 8, respectively. A 37 kDa phosphoprotein was phosphorylated on a histidine residue while the remainder of the inner membrane proteins were phosphorylated on serine or threonine residues. Two autophosphorylated putative kinases were identified: one at 16.5 kDa required divalent cations for autophosphorylation, while another at 30 kDa did not. A 110 kDa protein was labelled only with [α-32P]ATP, suggesting adenylylation