216 Biochimica et Biophysica Acta, 954 (1988) 216-223 Elsevier BBA 33128 Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor Jests M. Fominaya, Juan M. Garcia-Segura and Joss G. Gavilanes Departamento de BioquJmica y Biologla Molecular, Facultad de Ciencias, Unioersidad Complutense, Madrid (Spain) (Received 20 July 1987) (Revised manuscript received 22 February 1988) Key words: Ribonuclease, intracellular; RNAase inhibiior; Ribonuclease-RNAase inhibitor complex; (Rat testis) The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated M r value is 48 000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ. Introduction Cytoplasmic inhibitor proteins for neutral and alkaline ribonucleases (RNAases I) have been de- scribed for a variety of sources (see Ref. 1 for a review). In spite of some reported differences, all of them are inactivated by SH-reagents (p-hy- droxymercuribenzoate is the reagent most em- ployed), and this inactivation releases the masked ribonuclease activity (latent RNAase) from the enzyme-inhibitor complex [2]. The current knowl- edge, at a molecular level, about these inhibitors is restricted to that from mammalian species [3-5]. Abbreviations: RI, RNAase inhibitor; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Correspondence: J.G. Gavilanes, Departamento de Bioquimica y Biologla Molecular, Facultad de Ciencias, Universidad Com- pultense, 28040 Madrid, Spain. Thus, the ribonuclease-RNAase inhibitor interac- tion has only been characterized for the human placenta inhibitor-RNAase A model system [6-8]. Kinetic studies on the inhibition mechanism have also been reported [3,4,9,21] and a tight binding between both proteins has been found. This strong interaction may suggest the in vivo involvement of some modulator in order to release the RNAase activity from the enzyme-inhibitor complex. Nev- ertheless, the in vivo functionality of these inhibi- tors remains unknown. The involvement of these inhibitor proteins in the regulation of the cyto- plasmic RNA catabolism [11] and consequently in protein biosynthesis has been proposed, but fur- ther studies must be performed on this topic. These inhibitor proteins have mainly been studied for biological tissues exhibiting high levels of these molecules. This seems adequate for molecular studies when significant amounts of protein are required. However, tissues char- acterized by a high RNA turnover (active cell 0167-0838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)