ELSEVIER Biochimica et Biophysica Acta 1298 (1996) 31-36 BIOCHIMICA ET BIOPHYSICA ACTA BB I Thermal resistance of pantetheine hydrolase Giuseppina Pitari a,,, Giovanni Antonini a, Raffaella Mancini a, Silvestro Dupr~ b a Dipartimento di Biologia di Base e Applicata, Unieersit~t degli Studi L'Aquila, Via Vetoio-Coppito, 67100 L'Aquila, Italy b Dipartimento di Scienze Biochimiche, Universit~t di Roma 'La Sapienza ', Roma, Italy Received 18 June 1996; accepted 2 July 1996 Abstract Pantetheine hydrolase from pig kidney shows a very high resistance to denaturation with chemical denaturants, being unfolded at concentrations of guanidinium chloride higher than 6.5 M. On the contrary, chemical inactivation, followed by recording catalytic activity, occurs before conformational changes can be detected by fluorimetric or spectroscopic measurements. The enzyme resists temperatures as high as 80°C, as monitored by second derivative spectroscopy and circular dichroism. Activity increases with temperature to an optimum of about 70°C recording the initial velocity. The enzyme behaves very differently against chemical denaturants or against temperature denaturation. These results are unusual for a mesophilic protein. Keywords: Pantetheine hydrolase; Thermoinactivation; Thermostability 1. Introduction Studies on enzymes characterized by high ther- mostability have recently improved [1-4] and nowa- days the knowledge of mechanism of thermal stabil- ity is of great importance for biotechnological appli- cations [5-8]. The most studies concerning ther- mostability were performed on enzymes extracted from thermophilic microrganisms, whereas enzymes from mesophilic origin and in particular mammalian enzymes have seldom been studied from this point of view. Comparative studies were sometimes reported Abbreviations: GndHC1, guanidine hydrochloride; SDS, sodium dodecyl sulfate; CD, circular dichroism; pl, isoelectric point. * Corresponding author. Fax: + 39 862 433273. 1 This paper is dedicated to Professor Doriano Cavallini, on the occasion of his 80th birthday. between enzymes from thermophilic sources and the corresponding enzymes of mesophilic origin [3-9]. Data reported in the literature do not permit correla- tion between thermostability and specific character- istics peculiar to enzymes from thermophilic sources. The hydrolysis of D-pantetheine is catalyzed by the enzyme pantetheine hydrolase present in horse, rat and pig liver and kidney [10-12]. The pig and horse kidney enzymes have been characterized more accu- rately and many properties have been reported [12- 15]. The pig kidney enzyme is an acidic glycoprotein of M r = 57 kDa, with 11.8% glucides [12]. This enzyme shows a remarkable resistance to heat denaturation and this property has been successfully utilized for purification of the protein. The resistance to high temperatures and the presence of three disul- fide bridges [16] suggest the presence of a rather compact and resistant overall structure. In a previous paper [17] we reported the denaturation of panteth- 0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0167-4838(96)00112-4