scholarly journals Phospholipid association with the bovine cardiac mitochondrial adenosine triphosphatase

1985 ◽  
Vol 225 (3) ◽  
pp. 597-608 ◽  
Author(s):  
R E Brown ◽  
R I Montgomery ◽  
P I Spach ◽  
C C Cunningham
Keyword(s):  
Phosphatidic Acid ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Sodium Cholate ◽  
Mitochondrial Atpase ◽  
Cardiac Mitochondria ◽  
Submitochondrial Particles ◽  
Relative Enrichment ◽  
Fold Stimulation ◽  
Stimulation Of

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.

scholarly journals Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression

1976 ◽  
Vol 160 (2) ◽  
pp. 335-342 ◽  
Author(s):  
D Lloyd ◽  
S W Edwards
Keyword(s):  
Atpase Activity ◽  
Schizosaccharomyces Pombe ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Early Stage ◽  
Gel Filtration ◽  
Growth Cycle ◽  
Mitochondrial Atpase ◽  
Submitochondrial Particles ◽  
Fourfold Increase

1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN′-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN′-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.

scholarly journals Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates

1980 ◽  
Vol 188 (1) ◽  
pp. 141-144 ◽  
Author(s):  
J Vanderleyden ◽  
C Peeters ◽  
H Verachtert ◽  
H Bertrand
Keyword(s):  
Neurospora Crassa ◽  
Oxidase Activity ◽  
Alternative Oxidase ◽  
Purine Nucleoside ◽  
Submitochondrial Particles ◽  
Fold Stimulation ◽  
Succinate Oxidase ◽  
Stimulation Of

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5′-monophosphates. The purine nucleoside 5′-monophosphates are more effective than the pyrimidine homologues. 5′-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825–835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309–316].

PHOSPHORYLATION OF DIGLYCERIDES BY RAT BRAIN

1962 ◽  
Vol 40 (2) ◽  
pp. 247-259 ◽  
Author(s):  
K. P. Strickland
Keyword(s):  
Phosphatidic Acid ◽  
Rat Brain ◽  
Inorganic Phosphate ◽  
Specific Activity ◽  
Alkaline Treatment ◽  
Water Soluble ◽  
Two Dimensional ◽  
Relative Specific Activity ◽  
Hydrolysis Products ◽  
Stimulation Of

The addition of D-α,β-dimyristin was observed to stimulate by three to six times the labelling of phospholipids from radioactive inorganic phosphate (Pi32) by glycolysing homogenates and respiring mitochondria of rat brain. The increase in labelling was confined to the glycerophosphate (GP) isolated by two-dimensional chromatography from the water-soluble hydrolysis products obtained on weak alkaline treatment of the labelled phospholipids. The GP formed under these conditions is presumed to be derived mainly from phosphatidic acid formed by the phosphorylation of the diglyceride. A similar effect was observed for D-α,β-dipalmitin, D-α,β-diolein, and natural diglycerides prepared from either egg lecithin or spinal cord lecithin, but not for D-α-β-distearin. L-α,β-Diolein was much less effective than the D-isomer, suggesting a stereospecificity on the part of the enzymic phosphorylation of diglyceride. Experiments on the effects of the omission of Mg++ and the addition of glycolytic inhibitors on the stimulation of the labelling from Pi32 caused by D-α,β-dimyristin and D-α,β-diolein in the anaerobic homogenate system suggested that the increased phosphorylation caused by added diglycerides was closely coupled to active glycolysis. A comparison of the relative specific activity of the lipid P, following incubation of Pi32 and ATP32 in the anaerobic homogenate system inhibited by fluoride with and without D-α,β-diolein added, showed that the phosphate of the newly formed phosphatidic acid was derived from ATP, suggesting the presence of a D-α,β-diglyceride kinase.

scholarly journals A method for determining the adenosine triphosphatase content of energy-transducing membranes. reaction of 4-chloro-7-nitrobenzofurazan with the adenosine triphosphatase of bovine heart submitochondrial particles

1976 ◽  
Vol 159 (2) ◽  
pp. 347-353 ◽  
Author(s):  
S J Ferguson ◽  
W J Lloyd ◽  
G K Radda
Keyword(s):  
Mitochondrial Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Single Amino Acid ◽  
Mitochondrial Atpase ◽  
Inner Mitochondrial Membrane ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Single Amino Acid Residue ◽  
Membrane Bound

1. Modification of a single amino acid residue by introduction of the nitrobenzofurazan group inactivates mitochondrial ATPase (adenosine triphosphatase) when membrane-bound in submitochondrial particles. The similarity between the reactions of both membrane-bound and isolated ATPase with 4-chloro-7-nitrobenzofurazan indicates that the single essential tryosine residue identified in the isolated enzyme [Ferguson, Loyd, Lyons & Radda (1975) Eur. J. Biochem. 54, 117-126] Is also a feature of the membrane-bound ATPase. 2. A procedure is presented for estimating the ATPase content of the inner mitochondrial membrane. It is based on the specificity of the incorporation of the nitrobenzofurazan group, and the ready removal of this group by compounds that contain a thiol group. This method indicates that 8.5% of the membrane protein is ATPase. The procedure should be applicable to the titration of the energy-transducing ATPases of bacterial plasma membranes and of the thylakoid membranes of chloroplasts. 3. Combination of the data obtained on the ATPase content of the bovine heart inner mitochondrial membrane with a titration of the cytochrome bc1 complex with antimycin indicates that these two components of the membrane are present in approximately equal amounts.

scholarly journals Mixed anhydrides of nucleotides and mesitylenecarboxylic acid as new specific inhibitors of mitochondrial adenosien triphosphatase

1979 ◽  
Vol 178 (2) ◽  
pp. 339-343 ◽  
Author(s):  
I A Kozlov ◽  
M V Shalamberidze ◽  
I Y Novikova ◽  
N I Sokolova ◽  
Z A Shabarova
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Covalent Binding ◽  
Mitochondrial Atpase ◽  
Mixed Anhydride ◽  
Submitochondrial Particles ◽  
Nucleoside Triphosphates ◽  
Mixed Anhydrides ◽  
Nucleotide Residue ◽  
Specific Inhibitors

Mixed anhydrides of nucleoside triphosphates and mesitylenecarboxylic acid inhibit soluble mitochondrial ATPase (adenosine triphosphatase), but do not inhibit ATPase of submitochondrial particles. Inhibition of soluble mitochondrial ATPase by the mixed anhydride of epsilon-ATP and mesitylenecarboxylic acid is followed by the covalent binding of one nucleotide residue to a molecule of the protein. It is suggested that this covalent binding occurs in the catalytic site of the mitochondrial ATPase. The mixed anhydride of ADP and mesitylenecarboxylic acid inhibits the ATPase activity of submitochondrial particles and has no effect on the activity of soluble mitochondrial ATPase. After separation of the submitochondrial particles from the mixed anhydride of ADP and mesitylenecarboxylic acid, their ATPase activity is restored to its original value (half-time of reactivation 3–4 min). Incubation of submitochondrial particles or soluble mitochondrial ATPase with the mixed anhydride of ADP and mesitylenecarboxylic acid results in AMP formation.

scholarly journals Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform

1979 ◽  
Vol 178 (2) ◽  
pp. 289-297 ◽  
Author(s):  
D. D. Tyler ◽  
Pauline R. Webb
Keyword(s):  
Extraction Method ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Hydrolytic Activity ◽  
Chloroform Extract ◽  
Soluble Enzyme ◽  
Submitochondrial Particles ◽  
Reactive Material ◽  
Bicarbonate Ions ◽  
Purification And Properties

1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60–70μmol/min per mg of protein by (NH4)2SO4 fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their Km values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1–2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533–537] is similar to other preparations described previously and that this method is superior in simplicity and speed.

PHOSPHORYLATION OF DIGLYCERIDES BY RAT BRAIN

1962 ◽  
Vol 40 (1) ◽  
pp. 247-259 ◽  
Author(s):  
K. P. Strickland
Keyword(s):  
Phosphatidic Acid ◽  
Rat Brain ◽  
Inorganic Phosphate ◽  
Specific Activity ◽  
Alkaline Treatment ◽  
Water Soluble ◽  
Two Dimensional ◽  
Relative Specific Activity ◽  
Hydrolysis Products ◽  
Stimulation Of

The addition of D-α,β-dimyristin was observed to stimulate by three to six times the labelling of phospholipids from radioactive inorganic phosphate (Pi32) by glycolysing homogenates and respiring mitochondria of rat brain. The increase in labelling was confined to the glycerophosphate (GP) isolated by two-dimensional chromatography from the water-soluble hydrolysis products obtained on weak alkaline treatment of the labelled phospholipids. The GP formed under these conditions is presumed to be derived mainly from phosphatidic acid formed by the phosphorylation of the diglyceride. A similar effect was observed for D-α,β-dipalmitin, D-α,β-diolein, and natural diglycerides prepared from either egg lecithin or spinal cord lecithin, but not for D-α-β-distearin. L-α,β-Diolein was much less effective than the D-isomer, suggesting a stereospecificity on the part of the enzymic phosphorylation of diglyceride. Experiments on the effects of the omission of Mg++ and the addition of glycolytic inhibitors on the stimulation of the labelling from Pi32 caused by D-α,β-dimyristin and D-α,β-diolein in the anaerobic homogenate system suggested that the increased phosphorylation caused by added diglycerides was closely coupled to active glycolysis. A comparison of the relative specific activity of the lipid P, following incubation of Pi32 and ATP32 in the anaerobic homogenate system inhibited by fluoride with and without D-α,β-diolein added, showed that the phosphate of the newly formed phosphatidic acid was derived from ATP, suggesting the presence of a D-α,β-diglyceride kinase.

scholarly journals Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

1969 ◽  
Vol 113 (5) ◽  
pp. 829-836 ◽  
Author(s):  
K. Ahmed ◽  
H G Williams-Ashman
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Reaction Medium ◽  
Ventral Prostate ◽  
Experimental Conditions ◽  
Enzyme Preparations ◽  
Rat Ventral Prostate ◽  
Stimulation Of

A Mg2++Na++K+-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na++K+-stimulated ATPase activity was found to be: Na+, 115mm; K+, 7–10mm; Mg2+, 3mm; ATP, 3mm; tris buffer, pH7·4 at 38°, 20mm. The average ΔPi (Mg2++Na++K+ minus Mg2++Na+) was 9μmoles/mg. of protein/hr., representing a 30% increase over the Mg2++Na+-stimulated ATPase activity. At high concentrations, K+ was inhibitory to the enzyme activity. Half-maximal inhibition of Na++K+-stimulated ATPase activity was elicited by ouabain at 0·1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of Pi release by Na++K+ was observed only with ATP as substrate. The apparent Km for ATP for Na++K+-stimulated activity was about 0·3×10−3m. Ca2+ inhibited only the Na++K+-stimulated ATPase activity. Mg2+ could be replaced by Ca2+ but then no Na++K+ stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) in vitro at 0·1–10μm under a variety of experimental conditions did not significantly increase the Na++K+-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na++K+-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.

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