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  • 1
    ISSN: 1432-2048
    Keywords: H+-ATPase ; Cucurbita ; Plasma membrane ; Platelet-activating factor ; Phospholipid ; Protein kinase, phospholipid-activated ; Tonoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160–1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127–130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state ΔpH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H+ transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca2+. When Ca2+ was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H+-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.
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  • 2
    ISSN: 1432-2048
    Keywords: Cucurbita ; Filipin ; H+-ATPase ; Lepidium ; Plasma membrane ; Tonoplast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H+-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles.
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