Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins

Abstract

The major rat heat-shock (stress) protein and its cognate were purified to electrophoretic homogeneity from livers of heat-shocked rats. Both proteins exhibited similar behavior on a variety of column chromatography matrices but were separable by preparative isoelectric focusing under nondenaturing conditions by virtue of a 0.2 pH unit difference in isoelectric point. Both purified proteins had similar physical properties, suggesting the possibility that they may have similar biological functions as well. Both proteins were homodimers under nondissociative conditions (Mr 150 000) with isoelectric points of 5.0 (cognate) and 5.2 (major stress protein). After denaturation, both proteins had an increase in isoelectric point of 0.6 pH unit, and the resulting polypeptide chains had apparent molecular weights of 73 000 (cognate) and 71 000 (major stress protein). Similarities in the electrophoretic properties of these two proteins and serum albumin, which also undergoes a large basic shift in isoelectric point due to loss of fatty acids and conformational changes accompanying denaturation, prompted us to search for lipids associated with the purified 71-kilodalton stress protein and its cognate. Thin-layer chromatography of chloroform/methanol extracts of these two proteins revealed nonesterified fatty acids bound to both proteins. Palmitic acid, stearic acid, and a small amount of myristic acid were identified by gas chromatography/mass spectroscopy. Both proteins contained approximately four molecules of fatty acid per dimer with palmitate and stearate present in a one to one molar ratio. Possible roles of the major stress protein and its cognate as fatty acid associated proteins in cellular responses to stress are discussed.

Heat shock proteins: the search for functions.

“…This identification of an interaction between HSP70 and a protein complex with ATP, leading to hydrolysis and dissociation, is reminiscent of two other similar reactions of an HSP70 in which the substrates were a lambda phage DNA replication complex (38, 80) and a nucleolar or nuclear cytoskeleton fraction, possibly a ribonucleoprotein complex (41). Members of the HSPT0 family have ATP-binding sites (8,47,74); they also bind fatty acids (27). All of this leads me to speculate that HSP70s participate in the "scaffolding" of protein complexes as well as in their dissociation, the latter driven by ATP hydrolysis.…”

Arachidonate is a potent modulator of human heat shock gene transcription.

“…An alternative explanation for arachidonate's action is that it affects HSF1 directly or influences HSF1-DNA binding indirectly through its effects on Hsc/ Hsp7O proteins (47,48). Other free fatty acids, including palmitic and stearic acid, have been shown to associate with Hsc7O (49,50), thus offering an additional link between fatty acids and their potential regulatory role during the heat shock response.…”

The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70

“…BiP (22)(23)(24), the constitutive HSC70 (6,(25)(26)(27)(28)(29)(30), the heat shock-inducible HSP70 (31), plant HSP70 (32), and DnaK (33), all show self-association properties. Nevertheless, the structural basis and molecular mechanism of such properties have remained undocumented.…”