Solubilization and reconstitution of the oat root vacuole H sup + /Ca sup 2+ exchanger

Journal Article · Thu Feb 01 04:00:00 EST 1990 · Plant Physiology; (USA)

DOI:https://doi.org/10.1104/pp.92.2.340· OSTI ID:5947805

Schumaker, K S; Sze, H ^[1]

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1. Univ. of Maryland, College Park (USA)

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H{sup +}/Ca{sup 2+} antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient ({Delta}pH) dependent). The first step toward purification and identification of the H{sup +}/Ca{sup 2+} antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H{sup +}/Ca{sup 2+} antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A {Delta}pH (acid inside) was generated in the proteoliposomes with an NH{sub 4}Cl gradient (NH{sub 4}{sup +}{sub in} {much gt} NH{sub 4}{sup +}{sub out}) as determined by methylamine uptake. Fundamental properties of {Delta}pH dependent calcium uptake such as the K{sub m} for calcium ({approximately}15 micromolar) and the sensitivity to inhibitors such as N,N{prime}-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H{sup +}/Ca{sup 2+} antiporter has been reconstituted in active form.

• OSTI ID: 5947805
• Journal Information: Plant Physiology; (USA), Journal Name: Plant Physiology; (USA) Vol. 92:2; ISSN 0032-0889; ISSN PLPHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METALS
CALCIUM
CATALASE
CATALYTIC EFFECTS
CEREALS
ELEMENTS
ENZYMES
ESTERS
GRASS
LILIOPSIDA
LIPIDS
MAGNOLIOPHYTA
MEMBRANE TRANSPORT
METALS
OATS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXIDOREDUCTASES
PEROXIDASES
PH VALUE
PHOSPHOLIPIDS
PLANTS
UPTAKE