Temperature differentially affects adenosine triphosphatase activity in Hsc70 orthologs from Antarctic and New Zealand notothenioid fishes

Abstract

To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0 degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0 degrees C to 10 degrees C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.

Fig 1.

(A) Silver-stained gel of purified Hsc70 from each species. For silver staining, 5 μg total protein was applied to lanes 1–3 and separated by electrophoresis on a 10% sodium dodecylsulfate (SDS)–polyacrylamide gel. Lanes are as follows: (1) silver stain protein molecular weight standards; (2) Hsc70 from Trematomus bernacchii white muscle; (3) Hsc70 from Notothenia angustata white muscle. (B) Western blot detection of purified 70-kDa heat shock proteins using a rat monoclonal anti-Hsp70 antibody. Lanes are as follows: (1) 0.1 μg bovine brain Hsc70 standard; (2) 5 μg Hsc70 purified from T bernacchii; (3) 5 μg Hsc70 purified from N angustata

Fig 2.

Prevention of thermal denaturation of luciferase at 38°C by T bernacchii Hsc70 and N angustata Hsc70. Samples of luciferase were incubated at 38°C for 45 minutes, 10 μL aliquots were removed, placed in 50 μL of luciferase assay reagent (LAR) (Promega), and relative light units were measured in a luminometer. The circles, triangles, squares, and diamonds represent, luciferase alone, 2 μM bovine serum albumin (BSA), 1 μM T bernacchii Hsc70 and 0.2 μM Hsp40, and 1 μM N angustata Hsc70 and 0.2 μM Hsp40, respectively

Fig 3.

Effects of temperature on unstimulated (A) and reduced carboxymethylated α-lactalbumin (RCMLA)–stimulated (B) Hsc70 from T bernacchii and N angustata. The samples were incubated for 1 hour in the presence or absence of 80 μM RCMLA at the indicated temperatures and the amount of adenosine triphosphate (ATP) hydrolysis was measured by thin-layer chromatography. Values are relative mean density (background corrected) ± standard error of the mean (SEM) (n = 3 replicate assays). Levels not connected by the same letter are significantly different (analysis of variance [ANOVA]; P < 0.01)

Fig 4.

Thermal stability of the adenosine triphosphatase (ATPase) activity of T bernacchii Hsc70 (A) and N angustata Hsc70 (B). Native Hsc70 was incubated in the assay buffer at the indicated temperatures for 40 minutes. At indicated times during the incubation period, 2 μg aliquots were removed and combined with 10 μCi of [α-³²P]– adenosine triphosphate (ATP) in 50 μL of assay buffer and placed in a 0°C ice bath for T bernacchii Hsc70 and 10°C water bath for N angustata. The amount of ATP hydrolysis was measured after 15 minutes by thin-layer chromatography (TLC). Values are relative mean density (background corrected) ± standard error of the mean (SEM) (n = 3 replicate assays)