Short Communication Heat shock protein 70 purification and characterization from Cyprinion macrastomus macrastomus and Garra rufa obtusa Yusuf Tutar a,b,c,n ,S - ule Okan c a Department of Biochemistry, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey b CUTFAM Research Center, Cumhuriyet University, Sivas 58140, Turkey c Department of Chemistry, Faculty of Science, Cumhuriyet University, Sivas 58140 Turkey article info Article history: Received 22 July 2011 Accepted 2 November 2011 Available online 11 November 2011 Keywords: Hsp70 Protein folding Protein aggregation Luciferase ATPase abstract Organisms resist stress factors by common mechanisms at cellular and physiological levels. Heat shock proteins (Hsps) play an essential role as a protective mechanism against stressors, and Hsp70 has a central role in the heat shock response. Although several investigations have been made on the cellular functions of Hsps, very little is known about extreme temperature effects on organisms like fish. To address this point, we have studied two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, whose habitat is the Kangal hot spring in Turkey. The hot spring has multiple stress factors; extremely hot temperature, food deprivation, infectious medium and low levels of oxygen. Like other Hsp70s, two Cyprinidae species Hsp70 showed similar ATPase and substrate protein folding activities in vitro. These proteins also showed relatively higher thermal stability. Biochemical characterization of two Hsp70, suggested a Hsp mechanism capable of protecting the cell against stressors even under adverse conditions and in the presence of multiple stress factors. & 2011 Elsevier Ltd. All rights reserved. 1. Introduction Fish are convenient models to study the effects of stress in the intact organism. Cyprinion macrostomus macrostomus (so called striker) and Garra rufa obtuse (so called licker) are two Cyprinidae species found in hot springs in Kangal, 98 km Southeast of Sivas city, Turkey. These species are adopted to live at temperatures up to 40 1C, as opposed to their relatives that live in the river at temperatures of up to 25 1C. Average temperatures of the hot spring are 35 1C throughout the year. In this environment, several challenges are faced, such as high altitude, low oxygen concen- tration, extreme heat, food deprivation, the presence of heavy metals and infections leading to continuous physiological stress. All these effects compromise body metabolism and lead to the loss of tissue and cell homeostasis. Moreover, these effects result in the activation of free radical production and induction of heat shock protein production (O ¨ zer et al., 1987; Al-Habib and Al-Habib, 1979; G¨ oz ¨ ukara and C - avas, 2001). Patients suffering from skin diseases like psoriasis and eczema use the hot spring. Limited food sources in the pools make fish species aggressive, the two fish species strike and lick the psoriatic plaque. This process clears the wound area and it has been proposed that dissolved selenium in water plays a critical role for healing (Undar et al., 1990; Kuru 1979; Demir 2009). Little information is available for persistent stressors on organisms and further research is needed to elucidate how the species respond when exposed to multiple stressors as seen in this hot spring. Several families of heat shock proteins (Hsps) are indicators of stress response at the cellular level. The cell responds to stressors through changing the concentration of different classes of Hsps (Craig and Gross, 1991; Fung et al., 1996; Tutar and Tutar, 2010). There are several Hsp families, which are named based on the molecular weight of the protein; Hsp70, Hsp40, Hsp90, Hsp100, Hsp60, nucleotide exchange fac- tors and small Hsps. Hsp70 has been extensively studied (Shorter and Lindquist, 2008; Bukau et al., 2006; Summers et al. 2009). Hsp70 is expressed constitutively to maintain cellular home- ostasis. Hsp70 is also expressed inductively under stress condi- tions. Cognate and inductive heat shock proteins were shown by different nomenclature in the literature; however we used Hsp70 for both types in this paper for convenience. Since Hsp70 expressed under stress conditions, one can assume that most Hsp70 originates from inductive form (Tutar 2009; Tutar et al., 2006; Song et al., 2005). Hsp70 assist folding of nascent and unfolded peptides. Hsp70 has two main domains; ATPase and substrate binding domain. The two domains communicate, and energy from ATP hydrolyzes drives peptide to reach its native folded state (McCarty et al., 1995). The substrate binding domain has two sub-domains; a hydrophobic core and a lid. The Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jtherbio Journal of Thermal Biology 0306-4565/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2011.11.002 n Corresponding author at: Department of Biochemistry, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey. Tel.: þ90 346 219 1010x1075; fax: þ90 346 219 1186. E-mail addresses: yusuf.tutar@nih.gov, ytutar@cumhuriyet.edu.tr (Y. Tutar). Journal of Thermal Biology 37 (2012) 95–99