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Temperature interactions of the molecular chaperone Hsc70 from the eurythermal marine goby Gillichthys mirabilis

Sean P. Place and Gretchen E. Hofmann^*

Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA

View larger version (25K): [in a new window]   Fig.1. Water temperature for the estuary inhabited by Gillichthys mirabilis. (A) Seawater temperature readings taken during the summer from 29 June to 19 September 2000 and (B) during the winter from 28 February to 3 March 1999. (C) Seawater temperature during tidal cycles in the estuary between 11 July and 13 July 2000. Temperature data were collected using programmable Stowaway data loggers (Onset Instruments Inc.).

View larger version (73K): [in a new window]   Fig.2. Analysis of sequential steps in the purification of Gillichthys mirabilis Hsc70. (A) Silver-stained gel of various stages in the purification process. For silver staining, 5µg of total protein was applied to lanes 1–4 and separated by electrophoresis on a 7.5% SDS–polyacrylamide gel. Lanes are as follows: (1) silver-stained protein molecular mass standards; (2) 10700g supernatant from G. mirabilis white muscle; (3) pooled eluant from the DEAE anion-exchange column; (4) pooled eluant from the ATP affinity column (purified Hsc70 preparation); (5) 0.1µg of an Hsc70 standard purified from bovine brain. (B) Western blot detection of purified 70kDa heat-shock proteins using a rat monoclonal anti-Hsp70 antibody. Lane 1, 0.1µg of bovine brain Hsc70 standard; lane 2, 5µg of Hsc70 purified from G. mirabilis.

View larger version (16K): [in a new window]   Fig.3. Prevention of thermal denaturization of luciferase at 38°C by Gillichthys mirabilis Hsc70. Samples of luciferase (0.2nmoll-1) were incubated at 38°C for 40min, and 10µl aliquots were removed and placed in 50µl of LAR (Promega; relative light units were measured in a luminometer). The percentage of initial activity remaining after the indicated incubation period is given on the y-axis. BSA, bovine serum albumin.

View larger version (14K): [in a new window]   Fig.4. Effects of substrate on the ATPase activity of Hsc70 purified from Gillichthys mirabilis. On the x-axis, data sets 1–3 represent ATPase activity measurements for three independent Hsc70 preparations. Duplicate samples were incubated with 3.7x105Bq of [-32P]ATP at 23°C, and the amount of ATP hydrolysis was measured after 30min by thin-layer chromatography. Values are the mean pixel absorbance volume (background-corrected) + variance (N=2). Black, dark grey and light grey columns represent assays composed of 80µmoll-1 RCMLA in assay buffer (AB), 2µg of Hsc70 in AB and 2µg of Hsc70 plus 80µmoll-1 RCMLA in AB, respectively. See Materials and methods for a full description of the assay buffer components.

View larger version (17K): [in a new window]   Fig.5. Effects of temperature on the ATPase activity of unstimulated and RCMLA-stimulated Hsc70 purified from Gillichthys mirabilis white muscle. Assays, containing 2µg of Hsc70 plus 3.7x105Bq of [-32P]ATP in assay buffer, were incubated in the absence (open circles) or presence (triangles) of 80µmoll–1 RCMLA at 5°C increments in a temperature gradient that ranged from 10 to 45°C. Following a 60min incubation, triplicate 1µl samples were removed from each assay tube and analyzed for ATP hydrolysis by thin-layer chromatography. The black circles represent a control assay containing only 80µmoll-1 RCMLA. Each data point represents the mean pixel absorbance volume (background-corrected) ± S.D. (N=3). See Materials and methods for a full description of the assay buffer components.

View larger version (19K): [in a new window]   Fig.6. Thermal stability of the ATPase activity of Gillichthys mirabilis Hsc70. Native Hsc70 was incubated in assay buffer at 50, 60, 62.5, 65 and 80°C for 40min. During the 40min incubation period, 2µg samples were taken in triplicate every 10min, combined with 3.7x105Bq of [-32P]ATP in 50µl of assay buffer and placed in a water bath at 23°C. The amount of ATP hydrolysis was measured after 15min by thin-layer chromatography. Values are mean pixel absorbance volume (background-corrected) ± S.D. for N=3 samples for each time point. See Materials and methods for full a description of the assay buffer components.