Differential influence of ATP on native spinach 70-kilodalton heat-shock cognates.
A constitutively expressed class of 70-kD heat-shock cognate (HSC70) proteins from spinach leaf tissue was purified based on their affinity for ATP-agarose. The affinity-purified spinach proteins were resolved into at least three different forms on two-dimensional gels. Under native conditions, and iN the absence of ATP, the affinity-purified proteins were separated into three molecular mass classes by gel-filtration chromatography; a monomer of 85 kD, a multimer of 280 kD, and a large molecular mass oligomer of > 650 kD. All molecular mass forms contained a major protein that migrated at 79 kD on sodium dodecyl sulfate-polyacrylamide gels. N-terminal sequencing of the 79-kD purified monomer showed the highest homology to the endoplasmic reticulum-luminal HSC70. Addition of Mg-ATP to monomeric HSC70 did not alter its migration during gel filtration. Addition of Mg-ATP to the dimer converted it to monomer and oligomeric forms, whereas the presence of ATP converted a fraction of the large molecular mass oligomeric form of HSC70 to dimeric and monomeric forms. Only the large molecular mass oligomeric HSC70 appears to autophosphorylate in vitro in the presence of [gamma-32P]-ATP. Dimers and monomers can bind ATP by a nonhydrolytic mechanism and undergo a conformational change in the presence of Mg-ATP. In this paper we discuss the effects that ATP may have on the regulation of plant HSC70.
Department of Environmental Horticulture, University of Florida, Gainesville 32611-0512.
J V
JV
Anderson
D W
DW
Haskell
C L
CL
Guy
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D014675:Vegetables / Q000235:genetics / Q000378:metabolism*
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate