A sulfotransferase from spinach leaves using adenosine-5'-phosphosulfate

Area of Science:

• Plant biochemistry
• Enzymology
• Sulfate metabolism

Background:

• Sulfotransferases play a crucial role in sulfate assimilation in plants.
• The specific sulfuryl donor for sulfotransferase activity in higher plants remained unclear.

Purpose of the Study:

• To investigate the substrate specificity of sulfotransferase from spinach (Spinacea oleracea L.) and other higher plants.
• To elucidate the role of adenosine-5 omino-phosphosulfate (APS) and 3 omino-phosphoadenosine-5 omino-phosphosulfate (PAPS) in plant sulfate reduction.

Main Methods:

• Extraction and stabilization of active sulfotransferase from spinach leaves.
• Enzymatic assays using radiolabeled APS and PAPS as sulfur donors.
• Investigating the effects of various ions (Mg2+, Ca2+, NaF), buffer components (KCl, thiol reagents), and inhibitors (phosphate, cyanide) on enzyme activity.
• Competition experiments with unlabeled APS and PAPS to determine the obligatory intermediate.

Main Results:

• Spinach sulfotransferase was extracted and stabilized using ammonium sulfate and mercaptoethanol.
• The enzyme reduced both APS and PAPS to acid-volatile radioactivity in the presence of dithioerythrol.
• Magnesium chloride was required for PAPS reduction but not APS reduction.
• Competition experiments demonstrated that APS is an obligatory intermediate in the conversion of PAPS to acid-volatile radioactivity, indicating APS specificity.
• Similar APS-dependent sulfotransferase activity was observed in five other plant species.

Conclusions:

• The sulfotransferase from spinach specifically utilizes APS as the sulfuryl donor for sulfate reduction.
• This study demonstrates the use of APS for assimilatory sulfate reduction in higher plants, extending findings from green algae.
• The findings clarify a key step in plant sulfate assimilation pathways.