Copyright © Physiologia Plantarum 2001 PHYSIOLOGIA PLANTARUM 113: 203 – 209. 2001 Printed in Ireland —all rights resered ISSN 0031-9317 Monophenolase activity of latent Terfezia claeryi tyrosinase: Characterization and histochemical localization Manuela Pe ´rez-Gilabert a , Asuncio ´n Morte b , Mario Honrubia b and Francisco Garcı ´a-Carmona a, * a Departamento de Bioquı ´mica y Biologı ´a Molecular -A, Facultad de Biologı ´a, Uniersidad de Murcia, Campus de Espinardo, E-30080, Murcia, Spain b Departameno de Biologı ´a Vegetal (Laboratorio de Micologı ´a), Facultad de Biologı ´a, Uniersidad de Murcia, Campus de Espinardo, E-30080, Murcia, Spain *Corresponding author, e -mail: gcarmona@um.es Received 7 February 2001 concentrations. The presence of catalytic concentrations of The monophenolase activity of Terfezia claeryi tyrosinase o -diphenols affected the lag period but not the steady-state (EC 1.14.18.1) is described for the first time. This enzyme is rate. By increasing the concentration of o -diphenols, it was fully latent and can only be detected if SDS is present in the possible to evaluate the enzyme activation constant, K act , reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed ki- which showed a value of 7.2 M. The experimental results are compatible with the mechanism previously described for ty- netic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady- rosinases from other sources. state rate are affected by pH and the enzyme and substrate desert truffles have mainly been morphological (Moreno et al. 1986, Honrubia et al. 1992) and bromatological (Ahmed- Ashour et al. 1981, Al-Shabibi et al. 1982, Sawaya et al. 1985, Bokhary and Parvez 1993) and virtually no biochemi- cal investigations into their metabolism have been performed. It has been known for some time that the copper-contain- ing enzyme tyrosinase or polyphenol oxidase (monophenol, dihydroxy-L-phenylalanine: oxygen oxidoreductase; EC 1.14.18.1) is essential for melanization (Mayer and Harel 1979, Lerch 1981). The first biochemical investigations were carried out in 1895 on the mushroom Russula nigricans, whose cut flesh turns red and then black upon exposure to air. Although it is widely distributed on the phylogenetic scale, most of the studies with this enzyme have been carried out with mushroom (Agaricus bisporus ) and Neurospora crassa tyrosinase. The enzyme catalyzes two different reac- tions in the presence of molecular oxygen. The first, and only specific reaction catalyzed by tyrosinase, is the hydrox- ylation of monophenols to o -diphenols, a reaction that is usually termed cresolase or monophenolase activity (for a Introduction Desert truffles are a complex concept of mycorrhizal hypo- geous fungi including several species of the genera Picoa, Balsamia, Tuber, Tirmania and Terfezia, whose distribution is limited to semi-arid and arid conditions (Honrubia et al. 1992). Their ecological value is derived from their position in desert ecosystems as a symbiotic ectendomycorrhizal fungi associated with annual and perennial species of the genera Cistus and Helianthemum of the Cistaceae family. The most important species of desert truffle are those in- cluded in the genera Terfezia and Balsamia. Their organoleptic qualities are highly appreciated, particularly in Mediterranean countries and the Arabic Peninsula, where they are proving to be an interesting agricultural alternative, helping to improve the social and economical level of these dry regions. The diversity of desert truffles, and particularly of Terfezia claeryi Chatin, can be preserved by pure cul- ture, mycorrhization, host-plant micropropagation and molecular characterization (Morte and Honrubia 1992, 1995, 1997a,b, Gutie ´rrez et al. 1996) but more studies are needed to select the strains showing the best organoleptic characteristics. The studies carried out up to date with Abbreiations – CMC, critical micellar concentration; L-DOPA, L-dihydroxyphenylalanine; K act , activation constant; SDS, sodium dodecyl sulfate; TX-114, Triton X-114. Physiol. Plant. 113, 2001 203