ศูนย์นวัตกรรมเทคโนโลยีหลังการเก็บเกี่ยว

Isolation of plasma membrane from Capsicum annum fruit tissue: prevention of acid phosphatase contamination

Guodong Wang, D. James Morre and Robert L. Shewfelt

Postharvest Biology and Technology Vol: 6 Issue: 1-2 Pages: 81-90.

1995

บทคัดย่อ

Isolation of highly purified plasma membrane fractions from outer pericarp tissue of mature-green bell pepper (Capsicum annum) fruit was achieved using the two-phase polymer partitioning method. This system was optimized by adjusting the partition pH and noting the effect on various cell fraction markers. The modified system [5.8% (w/w) PEG (MW 3380), 5.8% (w/w) Dextran T-500, 20 mM NaCl, 5 mM KPi, pH 7.3] dramatically decreased thylakoid and Golgi apparatus membrane contamination. Use of normal homogenization medium resulted in acid phosphatase contamination, as evidenced by 30-40% inhibition of the apparent ATPase activity by 0.1 mM molybdate. A modified homogenization medium containing 0.3 M sucrose, 250 mM Tris-HCl (pH 8.5), 25 mM EDTA, 5 mM EGTA, 2.5 mM dithiothreitol (DTT), and 1 mM PMSF, and alternatively a microsome washing step efficiently reduced acid phosphatase contamination from 37% of the total plasma membrane ATPase activity to 8 and 12%, respectively. Two-phase partitioning of senescing fruit tissue provides a tool for studying the role of the plasma membrane in degradative changes observed during postharvest storage.