Purification of acidic phosphatase from mustard seedlings

Purification of acidic phosphatase from mustard seedlings

Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases. Many phosphatases are highly substrate-specific, like those enzymes involved in signal transduction. A number of phosphatases, however, cleave virtually any phosphate ester. Such unspecific enzymes function mainly in the catabolic breakdown of metabolites or nutrients.

• Since the phosphatase is active only at acidic pH values, but p-nitrophenol is colored at basic pH values, we must change the pH following the enzyme reaction. We will incubate for 30 min at pH 4.8, and then stop the reaction by adding NaOH.

  EXPERIMENTAL PROTOCOL

  Week 1 - Day 1

  1. Remove the 5 - 10 cm long seedlings from the vermiculite, wash and pat dry on paper towel. Weigh and record the weight. (use 25-50 g).

  
  

  2. Grind in a mortar with 75 ml of ice-cold water.

   

  3. Make the volume up to 150 ml and transfer the slurry into the Polytron homogenizer. Homogenize 1 min at speed 5 (3x20 sec).

  4. Filter the homogenate through 8 layers of cheese cloth into a cold beaker on ice. Remove 1 ml aliquot to an Eppendorf microfuge tube, and label as HOM and leave on ice.

  5. Transfer the extract into one 250 ml centrifuge bottle. Balance against water or, if ready, against the extract of the other group. Always keep the extract on ice.

  6. Centrifuge in the Sorvall superspeed centrifuge (B 72