Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts

Perlin, David S.; Spanswick, Roger M.

doi:10.1104/pp.65.6.1053

Cited by 57 publications

(37 citation statements)

References 21 publications

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“…A comparison ofthe pH curves of the activity in the Golgi-rich and PM-rich fractions provided the first hint of an ATPase activity associated with the Golgi. The KCI-stimulated activity of the PM-rich fraction showed an optimum at pH 6.0 to 6.5, in agreement with the value generally reported for the K+, Mg2e-ATPase of the PM (2,12,20,32,44).…”

supporting

confidence: 78%

Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Chanson¹,

McNaughton²,

Taiz³

1984

Plant Physiol.

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Membranes of corn (Zea may:, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient ce action the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase ws selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelies. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCI, MgeATPase of the PM-rich fraction from a step gradient was 6.0 to 65, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To The present work was undertaken in an effort to clarify the status of ATPases on Golgi membranes by careful comparison of ATPase activity and the distribution of various membrane markers on isopycnic and rate-zonal sucrose and dextran gradients. The corn coleoptile was selected because it is a well characterized system which allows reasonably good separation of membrane markers on gradients.Membrane ATPases have been implicated in the transport of ions in plant cells (34). On the basis of electrophysiological and biochemical evidence, the primary transport mechanism on the plasma membrane appears to be an electrogenic H+-ATPase which pumps protons to the cell exterior (41). There is also strong evidence that the tonoplast is the site of a second H4-ATPase which pumps protons into the vacuole (9,15,26,45).Ion transport in the Golgi has received very little attention, since its primary function is to secrete macromolecules across the plasma membrane. Inasmuch as many of these secreted macromolecules are charged, e.g. pectins and glycoproteins, charge balancing and pH adjustment may be required for normal processing of secreted macromolecules

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supporting

confidence: 78%

Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Chanson¹,

McNaughton²,

Taiz³

1984

Plant Physiol.

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“…Different membrane types from corn tissues can be separated, to some extent, by centrifugation on continuous sucrose gradients (14,19). Characteristic densities have been assigned to membranes from ER, golgi, plasma membrane, and mitochondria (21).…”

mentioning

confidence: 99%

Localization of a Proton-Translocating ATPase on Sucrose Gradients

DuPont

Bennett²,

Spanswick³

1982

Plant Physiol.

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lonophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase In a previous paper (5) we described a proton-translocating ATPase in membranes from corn roots. It was stimulated by C1-and by ionophores, did not appear to be of mitochondrial origin, as it was not inhibited by oligomycin, and differed from the previously described plasma membrane ATPase of corn roots in that it was unaffected by monovalent cations, and was not inhibited by vanadate. Sze (23,24) suggested that similar vesicles obtained from tobacco callus originated from the plasma membrane and that the ionophore-stimulated ATPase, ATP-dependent methylamine uptake, and ATP-dependent SCN-uptake are the function of a proton translocating ATPase located on the plasma membrane. In this paper we describe preliminary attempts to determine the origin of the vesicles. We present evidence that the proton-translocating ATPase of corn root microsomal membranes is not of plasma membrane origin, and present evidence that the enzyme is likely to be of tonoplast origin. Similar findings have recently been reported for corn coleoptiles (8,9,15,17 Preparation of Vesicles. Sealed microsomal vesicles were prepared by a modification of the method of Sze (23). Corn seeds were germinated between damp paper towels in plastic trays. Root tips (approximately 15 mm in length) of primary and secondary roots of 4-to 5-day-old seedlings were excised into aerated 0.1 mM CaCl2 at room temperature. Roots were ground with a chilled mortar and pestle. The homogenization buffer consisted of 0.25 M sorbitol, 2 mm EGTA, 0.1% f8-mercaptoethanol, 0.1% BSA, and 25 mm Bis-Tris-propane adjusted to pH 7.5 with solid Mes. Approximately 10 ml of buffer/g fresh weight of root was used. The homogenate was centrifuged at 10,000g for 10 min, the mitochondrial pellet discarded, the supernatant centrifuged again at 10,000g for 10 min, the pellet discarded, and the supernatant centrifuged at 80,000g for 30 min to prepare the microsomal pellet. The microsomal pellet was suspended in suspension buffer consisting of 0.25 M sorbitol and 2.5 mm Bis-Tris-propane/Mes (pH 7.2), underlain with 8 ml of 10%o dextran T-70 in the same buffer, and centrifuged at 80,000g for 1 h. The cloudy interface (approximately 50%o of the protein applied to the gradient) was collected from the dextran cushion, and vesicles were stored on ice for 1 to 6 h before use. Vesicles showed little loss of activity for up to 10 h, and activity was preserved for several days if vesicles were frozen i...

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“…Simultaneously, motor cells of the flexor, characterized by poor activity of the proton pump, respond to the secretion of H^ ions, involving a negative gradient of the membrane potential, by increasing the passive transport and uptake of K"^ to motor cells of this region. Numerous reports suggest that H^-ATPases are located in the plasmalemma of plant cells (Hodges & Leonard, 1974;Perlin & Spanswick, 1980;O'Neill & Spanswick, 1984), and that both the secretion of H"*^ and the appearance of the membrane potential gradient conditioning the transport of ions depend on their activity (Poole, 1978;Bentrup, 1980). These processes and changes in the permeability of membranes are thought to account for reactions leading to cyclic changes of turgor in motor cells of the pulvinus.…”

Section: Discussionmentioning

confidence: 99%

THE EFFECT OF ABSCISIC ACID AND FUSICOCCIN ON MALIC ACID CONCENTRATION IN PULVINI OF PHASEOLUS COCCINEUS L.

Białçzyk

Lechowski

1987

New Phytologist

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SUMMARYFusicoccin and abscisic acid can modify the movements of some nyctinastic plants. This paper reports the effects of these two compounds on the concentrations of malic acid in pulvini of Phaseolus coccineus L. in the transition period of two phases of the circadian rhythm. The concentrations of malic acid in the two parts of the pulvinus, extensor and flexor, depend upon the phase of leaf movements. Abscisic acid and fusicoccin significantly affected the content of malic acid in leaves compared with the control. In the two phases of the diurnal cycle investigated, abscisic acid markedly reduced the production of malic acid in the fiexor and extensor regions of the pulvinus, and treatment with fusicoccin produced small but distinct increases in the concentration of malic acid in the same regions. The results are discussed with particular reference to the effect of abscisic acid and fusicoccin on the activity of the H+ pump associated with the control of turgor in cells.

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Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts

Cited by 57 publications

References 21 publications

Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Localization of a Proton-Translocating ATPase on Sucrose Gradients

THE EFFECT OF ABSCISIC ACID AND FUSICOCCIN ON MALIC ACID CONCENTRATION IN PULVINI OF PHASEOLUS COCCINEUS L.

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Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts

Cited by 57 publications

References 21 publications

Evidence for a KCl-Stimulated, Mg2+-ATPase on the Golgi of Corn Coleoptiles

Evidence for a KCl-Stimulated, Mg2+-ATPase on the Golgi of Corn Coleoptiles

Localization of a Proton-Translocating ATPase on Sucrose Gradients

THE EFFECT OF ABSCISIC ACID AND FUSICOCCIN ON MALIC ACID CONCENTRATION IN PULVINI OF PHASEOLUS COCCINEUS L.

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Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles