An Improved Method for the Determination of Orthophosphate Suitable for Assay of Adenosine Triphosphatase Activity.

Mozersky, Samuel M.; Pettinati, Julio D; Kolman, Stanley D.

doi:10.1021/ac60241a015

Cited by 40 publications

(11 citation statements)

References 29 publications

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“…The reaction was started by the addition of the fraction to be assayed, and stopped after 15 min by the addition of I ml of icecold quench solution (1.8 M NaClO4, 0.12 M glycine, and 0.3 M HCl). Inorganic phosphate was measured on a portion of the quenched mixture by the Mozersky et aL method (9), which was modified as described previously (8). A unit of ATPase activity is defined as the amount of enzyme required to liberate I pmol of phosphate in I min, under the above assay conditions.…”

Section: Methodsmentioning

confidence: 99%

Oligomycin-sensitive ATPase of Submitochondrial Particles from Corn

Grubmeyer¹,

Spencer²

1978

Plant Physiol.

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Research into the mechanisms of oxidative and photosynthetic phosphorylation has shown that the energy-transducing ATPases (ATP: phosphohydrolase EC 3.6.1.3) from a wide variety of organisms are extremely similar. Well studied preparations from chloroplasts, from yeast and mammalian mitochondria (I 1), from bacteria (1), and from pea mitochondria (4,8) show similarities in many catalytic and structural properties. In particular, the mitochondrial ATPases in their membrane-bound state are noted for their sensitivity to the antibiotic oligomycin (10, 11). This sensitivity, conferred upon the Fi-ATPase' by integral membrane components, is lost when the Fi-ATPase is solubilized (10). The soluble enzyme is noted for extreme cold lability (12) and a high mol wt (380,000 daltons) (10).Recently, workers from two laboratories have reported that the ATPase activity of sonicated corn mitochondria is not inhibited by oligomycin (5, 15). In addition, the solubilized form of the corn ATPase was shown to be stable in the cold, and of low mol wt (40,000-6,000 daltons) as demonstrated by gel filtration (15). The workers suggested that mitochondria of corn, and of the other monocotyledons tested, may possess a unique energy-transducing ATPase system (15).In this paper we report on the results of experiments designed to test this hypothesis. Submitochondrial particles, which are low in contaminating soluble enzymes, were prepared from sonicated corn mitochondria and were shown to be inhibited by oligomycin in the normal fashion; the remaining soluble fraction contained an ATPase that resembled F1-ATPase. several times in running water. The seeds were then allowed to germinate in the dark for 3 days in Vermiculite in a growth chamber at 27 C. The etiolated shoots (100-200 g) were harvested and mitochondria isolated according to a differential centrifugation procedure that was described previously (14). A greenish layer around the brown mitochondrial pellet was removed by suction. The mitochondria were resuspended in 0.25 M sucrose and repelleted at 20,000g for 8 min. The washed mitochondrial pellet was suspended in 20 to 30 ml of "SMP buffer" (0.25 M sucrose and 50 mm TES brought to pH 7 at 25 C with Tris). The mitochondria were then sonicated at 5 C for two 1-min bursts at full power on an Artek Sonic Dismembranator. Unbroken mitochondria were removed by centrifugation at 20,000g for 8 mi. The translucent supernatant layer (whole sonicate) was then centrifuged at 90,000g for 60 min at 4 C. The pellet (SMP) was resuspended in 2 ml of SMP buffer preparatory to assay. The supernatant layer (soluble fraction) was assayed directly.Trypsin treatments were carried out in SMP buffer by the addition of 0.1 g of trypsin for each 1 ,ug of protein. After 15 min at 30 C, 1.6 jig of trypsin inhibitor for each 1 pug trypsin protein was added to stop the reaction.ATPase activity was assayed at 30 C by the method described previously (4, 8). The assay medium was as described in Table I. The reaction was started by the addition of the fraction to b...

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Section: Methodsmentioning

confidence: 99%

Oligomycin-sensitive ATPase of Submitochondrial Particles from Corn

Grubmeyer¹,

Spencer²

1978

Plant Physiol.

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“…(1954). Water-extractable P was determined by shaking 20 g soil in 100 ml H2O for 24 h. The suspension was filtered and the inorganic P concentration of the extract determined using isobutanol-benzene extraction (Mozersky et al 1966). Totai P (inorganic * organic) concentration of the water extract was determined by evaporating a 1O-ml aliquot followed by ignition of the residue at 500 C for 4 h. Total P concentration in soil was determined by wet digestion and the vanadomolybdophosphoric yellow color method (Jackson 1958). Organic P concentration in soils was determined by an ignition method (Olsen and Dean 1965) and by an extraction method (Mehta et al 1954 occurred as a function of time (Table 3).…”

Section: Canadian Journal Of Soil Sciencementioning

confidence: 99%

Organic and Inorganic P Content, Movement and Mineralization of P in Soil Beneath a Feedlot

Campbell¹,

Racz²

1975

Can. J. Soil. Sci.

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CenrrrnLL, L. B. lr.ro Rlcz, G. J. 1975. Organic and inorganic P content, movement and mineralization of P in soil beneath a feedlot. Can. J. Soil Greater amounts of 0.5 M NaHCO3 and water-exftactable P were found in soil beneath a cattle feedlot located on an alkaline sandy soil than in soil in an adjacent non-manured field. The 0.5 M NaHCOs-extractable P contents of the feedlot soil samples were greater than for the adjacent field to a depth of cm, suggesting that P from the manure had moved to this depth. Water extracted very little P from all field samples and the feedlot samples obtained below 120 cm. Concentration of total P in the feedlot soil was usually greater than in the corresponding field soil. The field soil contained more organic P than the feedlot soil at depths of 0-90 cm.Organic P concentrations at the 0 to l5-cm depths were 268 and 56 ppm for the field and feedlot sites, respectively. The organic C:N:P ratios for the 0 to l5-cm feedlot and field samples were 214

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“…where Tc is the center temperature, p is the bulk density, c is the specific heat, and K is the thermal conductivity of the cylinder material B and a are constants depending on initial conditions and the thermal constants respectively 1 Present address, Wigan Mining and Technical College, England. 2 Present address, Leeds University, England.…”

Section: Electrical Analogs Heat Flow Equationsmentioning

confidence: 99%