scholarly journals The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations

1985 ◽  
Vol 232 (2) ◽  
pp. 471-477 ◽  
Author(s):  
K Edman ◽  
I Ericson ◽  
I M Møller
Keyword(s):  
Surface Potential ◽  
Spinacia Oleracea ◽  
Membrane Surface ◽  
Nadh Oxidation ◽  
Spinacia Oleracea L ◽  
Spinach Leaf ◽  
Negative Surface ◽  
Proportional Increase ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2′-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.

scholarly journals Site-specific serine phosphorylation of spinach leaf sucrose-phosphate synthase

1992 ◽  
Vol 283 (3) ◽  
pp. 877-882 ◽  
Author(s):  
J L A Huber ◽  
S C Huber
Keyword(s):  
Okadaic Acid ◽  
Spinacia Oleracea ◽  
Sucrose Phosphate Synthase ◽  
Serine Phosphorylation ◽  
Spinacia Oleracea L ◽  
Spinach Leaf ◽  
Sucrose Phosphate ◽  
Phosphate Synthase

We recently reported [Huber, Huber & Nielsen (1989) Arch. Biochem. Biophys. 270, 681-690] that spinach (Spinacia oleracea L.) sucrose-phosphate synthase (SPS; EC 2.4.1.14) was phosphorylated in vivo when leaves were fed [32P]Pi. In vitro the enzyme was phosphorylated and inactivated by using [gamma-32P]ATP. We now report that SPS is phosphorylated both in vivo and in vitro on serine residues. The protein is phosphorylated at multiple sites both in vivo and in vitro as indicated by two-dimensional peptide maps of the immunopurified SPS protein. After being fed with radiolabel, leaves were illuminated or given mannose (which activates the enzyme), in the presence or absence of okadaic acid. Feeding okadaic acid to leaves decreased the SPS activation state in the dark and light and in leaves fed mannose. Across all the treatments, the activation state of SPS in situ was inversely related to the labelling of two phosphopeptides (designated phosphopeptides 5 and 7). These two phosphopeptides are phosphorylated when SPS is inactivated in vitro with [gamma-32P]ATP, and thus are designated as regulatory (inhibitory) sites [Huber & Huber (1991) Biochim. Biophys. Acta 1091, 393-400]. Okadaic acid increased the total 32P-labelling of SPS and in particular increased labelling of the two regulatory sites, which explains the decline in activation state. In the presence of okadaic acid, two cryptic phosphorylation sites became labelled in vivo that were not apparent in the absence of the inhibitor. Overall, the results suggest that light/dark regulation of SPS activity occurs as a result of regulatory serine phosphorylation. Multiple sites are phosphorylated in vivo, but two sites in particular appear to regulate activity and dephosphorylation of these sites in vivo is sensitive to okadaic acid.

scholarly journals Biplot analysis of trait relations of spinach (Spinacia oleracea L.) landraces

Genetika ◽  
2016 ◽  
Vol 48 (2) ◽  
pp. 675-690
Author(s):  
Naser Sabaghnia ◽  
Mehdi Mohebodini ◽  
Mohsen Janmohammadi
Keyword(s):  
Spinacia Oleracea ◽  
Block Design ◽  
Dry Weight ◽  
Leaf Length ◽  
First Year ◽  
Vegetable Crops ◽  
Petiole Length ◽  
Spinacia Oleracea L ◽  
Leaf Yield ◽  
Spinach Leaf

Interest in growing winter spinach (Spinacia oleracea L.) in Iran is increasing due to its good nutritional potential returns relative to other vegetable crops. The objectives of this research were to investigate the interrelationships among different traits of spinach and to evaluate different Iranian spinach landraces with application of the genotype ? trait (GT) biplot methodology in visualizing research data. 81 spinach landraces were grown during 2-years according to randomized complete block design with four replications. Ranking of the genotypes based on the ideal entry revealed that genotypes G1, G20, G7, G8, G9, G27, G49 G70 and G79 were higher in the measured traits and could be good candidates for improving most of the measured traits. Ranking of traits for the leaf yield showed that petiole diameter, petiole length, leaf numbers at flowering, 1000-seed weight and root dry weight were the most discriminating traits which influence spinach leaf yield at both years. There were 9 winning genotypes and 4 which-won-where patterns at the first year while there were 8 winning genotypes and 4 which-won-where patterns at the second year. As a result, the findings from our study are as follows: (i) traits leaf numbers at flowering, leaf length, leaf width, leaf area, petiole diameter and petiole length could be as selection indices for spinach leaf yield improvement, (ii) genotypes G1, G20, G7, G8, G9, G27, G49 G70, and G79 were the most favorable and is thus recommended for commercial release or incorporating in breeding programs; (iii) the GT biplot method can be used to identify superior genotypes in other crops and in other parts of the world.

scholarly journals Influence of the surface potential on the michaelis constant of membrane-bound enzymes

FEBS Letters ◽  
1982 ◽  
Vol 139 (2) ◽  
pp. 221-224 ◽  
Author(s):  
Lech Wojtczak ◽  
Konrad S. Famulski ◽  
Maciej J. Nałȩcz ◽  
Józef Zborowski
Keyword(s):  
Surface Potential ◽  
Michaelis Constant ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

Alterations in the Activities of Rabbit Erythrocyte Membrane-Bound Enzymes Induced by Cholesterol Enrichment and Depletion Procedures

1986 ◽  
Vol 41 (3) ◽  
pp. 301-309 ◽  
Author(s):  
Elli Kamber ◽  
Liudmila Kopeikina-Tsiboukidou
Keyword(s):  
Membrane Surface ◽  
Cholesterol Content ◽  
Inhibitory Effect ◽  
Erythrocyte Membranes ◽  
Molar Ratio ◽  
Inhibition Mechanism ◽  
Intact Cells ◽  
Egg Lecithin ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

The effect of cholesterol enrichment and depletion of rabbit erythrocytes on the activities of membrane-bound enzymes, namely (Na+ ,K+)-stimulated ATPase. NAD+ ase and acetylcholinesterase was examined. The cholesterol content of erythrocyte membranes has been modified by incubation of intact cells with sonicated egg lecithin/cholesterol vesicles (cholesterol/phospholipid molar ratio approx. 2) and with egg lecithin vesicles for time intervals up to 10 hours. The cholesterol/phospholipid molar ratio (CH /PL) of untreated rabbit red blood cell membranes was 0 .92-0.94. Linear increase (up to CH/PL molar ratio 1.72-1.9) or decrease (up to CH/PL molar ratio 0.27 - 0.43) in cholesterol content of erythrocyte membranes was observed over the 10 hours of incubation with egg lecithin/cholesterol and egg lecithin liposomes respectively. Fusion of liposom es to the m em brane or their attachment to the membrane surface was not a significant factor in the alteration of CH/PL ratio. (N+,K+ )-stimulated ATPase, NAD+ ase and acetylcholinesterase activities were measured as a function of membrane cholesterol. The specific activities of all three enzymes were progressively decreased with increase in cholesterol content. Partial reversibility of the inhibitory effect of cholesterol was demonstrated by measurements on cells depleted again after cholesterol enrichment. This was confirmed by the fact that a lowering in cholesterol content evoked an analogous activation of enzymes. The possible implications of physicochemical modifications of bulk and annular lipids of membrane-bound enzymes in the inhibition mechanism are discussed.

scholarly journals Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria

1995 ◽  
Vol 310 (2) ◽  
pp. 527-531 ◽  
Author(s):  
C Knorpp ◽  
C Szigyarto ◽  
E Glaser
Keyword(s):  
Proteolytic Activity ◽  
Spinacia Oleracea ◽  
Plant Mitochondria ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Nicotiana Plumbaginifolia ◽  
Experimental Conditions ◽  
Spinach Leaf ◽  
Membrane Bound

We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.

scholarly journals Influence of Soil-application of Fish-protein Hydrolysate Liquid on Growth and Yield of Spinach (Spinacia oleracea L.)

2020 ◽  
Author(s):  
Sheetal P. Dewang ◽  
Usha Devi C.
Keyword(s):  
Crop Yields ◽  
Spinacia Oleracea ◽  
Protein Hydrolysate ◽  
Growth And Yield ◽  
Fish Protein ◽  
Soil Application ◽  
Spinacia Oleracea L ◽  
Proportional Increase ◽  
Fish Protein Hydrolysate ◽  
Higher Doses

Background: Organic inputs, especially biostimulants, are gaining immense importance in enhancing crop yields. In the present study, the effect of soil-application of fish-protein hydrolysate (FPH) on the growth and yield of spinach was evaluated. The effect of various doses on the yield and correlation of root growth with yield was attempted to establish. Methods: In the current experiment, spinach was grown in growing pots. Four different concentrations of the FPH liquid viz. 0.5 ml, 2 ml, 5 ml and 10 ml per plant were applied to each plant through soil application at the frequency of 8 days. Result: The study revealed that amongst the various tested doses of 0.5 ml, 2 ml, 5 ml and 10 ml, the highest yield was observed with a 2 ml dose. Compared to untreated control plants, about a 40% increase in the yield was observed in the treatment with a 2 ml dose. The higher yield was associated with better root development. The higher doses of 5 ml and 10 ml did not result in a proportional increase in yields. On the contrary, these higher doses resulted in an adverse effect on the growth of roots and yield. So, the present study demonstrated the utility of FPH in increasing the yield of spinach in organic farming at the experimental level.

Some Aspects of the Polyhexamethyleneguanidine Salts Effect on Cell Cultures

2014 ◽  
Vol 1 (1) ◽  
pp. 62-67 ◽  
Author(s):  
M. Mandygra ◽  
A. Lysytsia
Keyword(s):  
Proliferative Activity ◽  
Cell Monolayer ◽  
Eukaryotic Cell ◽  
Culture Methods ◽  
Cellular Processes ◽  
Negative Effect ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Anion Type ◽  
Cell Culture Methods

Aim. To investigate the effect of polyhexamethyleneguanidine (PHMG) to eukaryotic cell culture. Methods. The passaged bovine tracheal cells culture (TCC) and primary culture of chicken embryo fi broblasts (FCE) were used in the experiments. TCC and FCE monolayers were treated with aqueous solutions of PHMG chloride or succinate. The method of PHMG polycation adsorption to the cells’ plasma membrane together with microscopy were applied. Results. The dependence of PHMG effect on the eukaryotic cells on the agent concentration, duration of exposure and the anion type has been fi xed. The PHMG concentration of 10 –5 per cent (0.1 μg/ml) never causes degradation of the previously formed cell monolayer, while the higher concentrations damage it. The conditions of the PHMG chloride and succinate’s negative effect on cell proliferation and inhibition of monolayer formation were determined. The hypothesis that under certain conditions PHMG stimulates the proliferative activity of the cells has been confi rmed. Stimulation may be associated with non-specifi c stress adaptation of cells. In this case, it is due to modifi cations of the cell membrane after PHMG adsorption to it. Conclusions. PHMG polycation binds with the membrane’s phosphoglycerides fi rmly and irreversibly. A portion of the lipids are removed from participation in the normal cellular processes at that. At the same time, the synthesis of new lipids and membrane-bound enzymes is probably accelerated. The phospholip ids’ neogenesis acceleration can stimulate mitosis under certain conditions. The obtained results can be used in the biotechnologies.

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