The Marker Concept in Cell Fractionation

Nagahashi, Gerald

doi:10.1007/978-3-642-82587-3_4

Cited by 12 publications

(12 citation statements)

References 47 publications

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“…For analysis, duplicate random samples of healthy young leaves and stems, and actively growing roots of each cultivar were collected, placed on ice, and taken to the laboratory. Homogenates were prepared according to Nagahashi (1985) from 2 g tissue macerated in a chilled mortar containing 0.2g of polyvinylpyrrolidone (PVP), a pinch of acid washed sea sand, and 4 ml of cold 0.05 M potassium phosphate buffer containing phenol methyl sulfonylfluoride (PMS) (pH 7.1). The substrate was squeezed through two layers of cheese cloth and centrifuged at 4 ° C and 250 x g for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

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Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

1990

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Isoenzyme analysis has been demonstrated as an effective tool for definitive identification of plant cultivars, but it has not been applied to pasture bermudagrass (Cynodon spp.) cultivars in the USA. Polyac~lamide gel electrophoresis (PAGE) was used to study five isoenzyme systems in mitochondrial, microsomal, and soluble cell fractions of actively growing leaves, stems, and roots of seven vegetatively-propagated pasture bermudagrass (Cynodon spp.) cultivars used in the southern half of the USA. Peroxidase, esterase, and, with one exception, acid phosphatase successfully differentiated between the cultivars in all leaf and stem cell fractions. Fewer cultivar differences were found for amino-and endo-peptidases. Only peroxidase and acid phosphatase were resolved from root cell fractions; and only the microsomal fraction differentiated between all cultivars. Within plant parts, cultivars were distinguishable on the basis of peptidase banding in some cellular fractions, but not in others. Plant part and subcellular fraction-specific isoenzyme variations suggest the existence of multiple molecular forms of various enzymes within the same plant.

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Section: Methodsmentioning

confidence: 99%

“…Most studies used homogenate from a single plant part, usually leaves. Typically, low speed centrifugation (250 to 3000 g) has been used to precipitate nuclei, cell wall fragments, plastids, and unbroken cells (Nagahashi, 1985). The sample homogenate is a mixture of mitochondria, microbodies, microsomes, and soluble fractions.…”

Section: Introductionmentioning

confidence: 99%

Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

1990

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“…The gradient was composed of 15 to 45% (w/w) sucrose made up in 5 mM Hepes-Mes, pH 7.7 plus 1 mM dithioerythritol. After centrifuging in a Beckman SW 28 rotor at 84,000g (average) at 4*C for 16 to 18 hours, the gradient was fractionated as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

The nature of proton‐translocating ATPases in maize roots

Loper

Bräuer

et al. 1992

Journal of Plant Nutrition

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The mechanisms of the coupling between ATP hydrolysis and proton transport catalyzed by the ATPases of the tonoplast and plasma membrane of maize (Lea mays L.) roots were investigated. Proton transport by the tonoplast ATPase was found to be much more sensitive to nitrate than ATP hydrolysis, being inhibited by 80% with almost no effect on hydrolysis at 5 mM NO 3 .Mercury was also found to be a potent inhibitor of this enzyme, inhibiting transport and hydrolysis by 50% at 60 and 100 μM, respectively. The same type of pattern was seen with other divalent cations. Millimolar concentrations of Cd 2+ , Co 2+ , Cu 2+ , and Zn 2+ in the presence of Mg 2+ inhibited proton transport significantly more than hydrolysis whereas Ba 2+ and Ca 2+ had little effect. Both free and ATP-complexed species of these inhibitory cations appeared to be effective. When the influence of temperature was investigated, both the tonoplast and plasma membrane enzymes showed a similar pattern. ATP hydrolysis by both enzymes generally obeyed the Arrhenius model, increasing in rate between 10°C and 45°C. However, the rate of proton transport deviated from this pattern above 20°C, remaining constant up to approximately 30°C and decreasing to undetectable levels by 40°C. In the presence of 50 μM vanadate, ATP hydrolysis by the plasma membrane ATPase was reduced by approximately 60% versus 30% for proton transport. In addition, vanadate significantly decreased the first order rate constant, ki, indicating a lowering of proton efflux. The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide nearly abolished transport with only a 50% reduction in ATP hydrolysis. These data arc interpreted in relation to whether the link between proton transport and ATP hydrolysis is direct or indirect for each enzyme. 929

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“…Although this crude particulate fraction contained over 94% of the large organelles, substantial amounts of various endomembrane components were also sedimented at low centrifugal force (10). The crude membrane fraction pelleted between 6000 and 100 000 6 g contained 42 to 54% of the endomembrane components and also contained the highest specific activities for the PM, Golgi, and ER markers ( Table 2).…”

Section: Differential Centrifigation Of the Crude Homogenatementioning

confidence: 99%

“…To prevent rapid browning of potato leaf homogenates, leaves were homogenized with various combinations of sulfhydryls (2-mercaptoethanol, 2-mercaptobenzothiazole, and dithiothreitol (DTT) ) in the presence and absence of 2 % BSA and (or) 1 % insoluble polyvinyl pyrollidone (10). Marker enzyme activity for PM (K+-stimulated ATPase), ER (antimycin A insensitive NADH CCR), and Golgi membranes (digitonin-stimulated UDPase) were compared with the specific activities of these markers in a microsomal fraction (13 000 to 80 000 x g) prepared in the standard homogenization medium.…”

Section: Preservation Of Marker Enzyme Activitymentioning

confidence: 99%

Preservation and separation of endomembrane marker enzyme activity in potato leaf homogenates

Nagahashi¹,

Seibles²

1986

Can. J. Bot.

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To minimize rapid browning and membrane degradation of crude microsomes, leaves of Solanum tuberosum (cv. Kennebec and cv. Katahdin) were initially homogenized in the presence of various inhibitors of polyphenol oxidase, phospholipase, and protease activity. To obtain and maintain marker enzyme activities used to identify plasma membranes, Golgi membranes, and endoplasmic reticulum, it was necessary to homogenize young leaves in the presence of sulfhydryls at pH 7.8. Further separation of these membranes, as determined by distribution of total activities of marker enzymes in linear sucrose density gradients, indicated a relatively pure plasma membrane fraction (1.15 g/cm3) free from contamination by thylakoids (1.19 g/cm3) and other endomembrane components. However, the distribution of specific activities across the gradient revealed that plasma membranes isolated from green tissue may be contaminated by Golgi membranes and not necessarily by plastid membranes.

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The Marker Concept in Cell Fractionation

Cited by 12 publications

References 47 publications

Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

The nature of proton‐translocating ATPases in maize roots

Preservation and separation of endomembrane marker enzyme activity in potato leaf homogenates

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