Fermin Pacheco | Universidad de Guadalajara (original) (raw)

Physiologia Plantarum, 1997

Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficul... more Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficult. In the present work, plasma membranes were isolated from dry maize (Zea mays L.) embryos with an enrichment of 11-fold as estimated by glucan synthase II (GSII, EC 2.4.1.34) activity and a purity of 78 to 90% as judged by the sensitivity of ATP hydrolysis to vanadate, a specific inhibitor of the plasma membrane H+-ATPase (EC 3.6.1.35). The procedure involved a double homogenization of the dry embryos and the addition of a 1500-g supernatant to an aqueous polyethyleneglycol-dextran two-phase partitioning system; the optimal ratio of polyethyleneglycol-dextran for purification of plasma membranes from dry seeds was 6.8/6.8% (w/w). In the isolated membranes a trace of a tonoplast enzyme marker (tonoplast H+-ATPase, EC 3.6.1.3) could be detected, but there were negligible amounts of mitochondrial and rough endoplasmic reticulum markers, H+-ATPase (EC 3.6.1.34) and diacylglycerol acyltrans-ferase (EC 2.3.1.20), respectively. The technique could also be used in hydrated embryos. The entire procedure can be carried out in 5 to 6 h. The resulting preparation is stable for at least 2 months at −70°C. The membranes of dry and hydrated embryos exhibited a high level of vanadate-sensitive ATPase activity that was increased by lysophosphatidylcholine.