Spinach leaf 70-kilodalton heat-shock cognate stabilizes bovine adrenal glucose-6-phosphate dehydrogenase in vitro without apparent stable binding.

Abstract

Spinach (Spinacia oleracea L.) leaf tissue 70-kilodalton heat-shock cognate was purified by ATP-agarose affinity and gel filtration. Gel filtration of the affinity-purified protein resolved it into three forms: monomer, dimer, and oligomer. In the absence of ATP, the majority of the heat-shock cognate existed as a monomeric form with lesser amounts of dimer and oligomer. Addition of 3 mM ATP to the purified protein, containing all three forms, converted the dimeric and monomeric forms to a high-molecular-weight complex. Removal of ATP from the complex by dialysis resulted in the reappearance of the dimeric and monomeric forms. Addition of ATP to the highly purified monomer had no effect on its gel-filtration migration. Neither purified monomeric or dimeric forms showed stable binding to denatured proteins; however, both forms of the purified heat-shock cognate were able to stabilize the enzymatic activity of bovine adrenal glucose-6-phosphate dehydrogenase over a 48-h period at 25 degrees C. In addition, the activity of glucose-6-phosphate dehydrogenase in the presence of purified heat-shock cognate dimer or monomer could be rapidly decreased in an ATP-dependent fashion depending on the order of the substrate addition to the reaction mixture. Circular-dichroism studies indicated that addition of ATP to the spinach 70-kDa heat-shock cognate caused a conformation change from alpha-helical to a greater beta-sheet content. How conformational character may influence the stabilizing activity of the heat-shock cognate in a mechanism which does not require stable peptide binding is discussed.

• Authors: J V Anderson, C L Guy
• Journal: Planta (Planta) Vol. 196 Issue 2 Pg. 303-10 ( 1995) ISSN: 0032-0935 [Print] Germany
• PMID: 7599528 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• HSP70 Heat-Shock Proteins
• Plant Proteins
• Adenosine Triphosphate
• Glucosephosphate Dehydrogenase

Topics

• Adenosine Triphosphate (pharmacology)
• Adrenal Glands (enzymology)
• Animals
• Cattle
• Chromatography, Affinity
• Chromatography, Gel
• Circular Dichroism
• Electrophoresis, Polyacrylamide Gel
• Enzyme Stability
• Glucosephosphate Dehydrogenase (chemistry, metabolism)
• HSP70 Heat-Shock Proteins (chemistry, isolation & purification, pharmacology)
• Plant Leaves
• Plant Proteins (chemistry, isolation & purification, pharmacology)
• Protein Conformation
• Spinacia oleracea (chemistry)