3'Sulfogalactolipid binding specifically inhibits Hsp70 ATPase activity in vitro.
A method for the generation of soluble glycosphingolipid derivatives that retain the receptor activity of the parent (BBRC 257:391-394, Carb Res 335:91-100) was used to investigate the consequence of 3'sulfogalactolipid (SGL) specific binding within the N-terminal ATPase-containing domain of Hsc70. Sulfogalactosyl ceramide (SGC) was deacylated, and the resulting sulfogalactosylsphingosine coupled to an alpha-adamantane or a norbornane rigid hydrophobic frame. The resulting conjugate preferentially partitioned into water, as opposed to organic solvent. In the range of 100-300 microM, these conjugates inhibited the specific binding of bovine brain Hsc70 to immobilized SGLs. A similar dose-related inhibition of bovine brain Hsc70 ATPase activity was seen between 100 and 300 microM adamantylSGC (adaSGC). Adamantyl conjugates of glycolipids not bound by Hsp70s had no effect. Kinetic analysis indicated that adaSGC was a noncompetitive inhibitor of Hsc70 ATPase activity, a special case of mixed inhibition since the K(m) values were not statistically different, 0.89 +/- 0.024 microM to 0.93 +/- 0.038 microM, but the V(max) decreased from 0.20 +/- 0.012 pmol min(-1) microg(-1) to 0.15 +/- 0.016 pmol min(-1) microg(-1). A reproducible 5 min lag was observed prior to ATPase inhibition that could be eliminated by preincubation of adaSGC with Hsc70 or by adding the cochaperone Hdj-1. The dependence of ATPase inhibition on the rate of hydrolysis indicates that adaSGC binding occurs at a specific stage of the ATPase cycle. These studies identify a new mechanism for the regulation of Hsp70 ATPase activity.
Whetstone
H
H
Research Institute, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Canada.
Lingwood
C
C
D000215:Acylation; D000218:Adamantane / Q000138:chemical synthesis; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000476:Alkanesulfonates / Q000138:chemical synthesis; D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005690:Galactose / Q000031:analogs & derivatives* / Q000737:chemistry*; D005699:Galactosylceramides / Q000737:chemistry; D006017:Glycolipids / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D009636:Norbornanes / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity
D000476:Alkanesulfonates; D004791:Enzyme Inhibitors; D005699:Galactosylceramides; D006017:Glycolipids; D018840:HSP70 Heat-Shock Proteins; D009636:Norbornanes; C078458:3-O-sulfogalactose; D000251:Adenosine Triphosphatases; D000218:Adamantane; D005690:Galactose