Journal of Muscle Research and Cell Motility 10, 326-330 (1989) Functional aspects of calcium transport in sarcopl reticulum vesicles derived from frog skeletal musc treated with saponin G. FANO*, S. BELIA, S. FULLE, P. A N G E L E L L A , F. P A N A R A , V. M A R S I L I a n d R. P A S C O L I N I ~ Istituto di Biologia Cellulare and rdi Anatomia Comparata, Universita' degli Studi di Perugia, 06100, Perugia, Italy Received in revised form 25 Match 1989 Summary To study the physiological aspects of the excitation-contraction cycle, saponin (10--100 ~g ml -1) was used agent on muscle and sarcotubular vesicles derived from fast muscles (sartorius and tibialis anterior) of Rana esculenta. The vesicles showed similar Ca2+-ATPase activity and similar protein profiles carried out by SDS--PAGE. C transport in untreated vesides and those treated with different concentrations of saponin seemed to have th quantitative and qualitative parameters if the saponin was used in a range between 10 and 50 ~g ml -1. Our results confirm that saponin may be considered to be a valid skinning agent for the external membran skeletal muscles. Introduction There are many open questions about the functional aspects of skeletal muscle fibre contraction. One of these is related to the complex mechanism in the E-C cycle which transfers the information from the T-tubule to sarcoplasmicreficulum (SR) (trigger mechanism) (Endo, 1977; Frank, 1982). The lack of a precise answer to this question may be due to the difficulty of getting to the SR through the sarcolemmasince the sarcolemma is a selective barrier even for small molecules or ions. To resolve this problem, a series of chemical procedur• has been developed over the last few years for the partial or total elimination of the sarcolemma (Stephenson, 1981; Mfller & Smith, 1985). Endo and Iino (1980) demonstrated that the use of saponin as skinning agent markedly reduced the mechanical response of the skeletal muscle. Saponin is a non-ionic detergent capable of changing the permeability of different cells by altering the cholesterol present in the membrane (Ohtsuki et ai., 1978). Since SR membranes have a lower cholesterol content than the sarcolemma (Martonosi, 1968), it is possible to obtain a selective perforation in the external membranes without changing the functional characteristics of the SR membranes (Endo & Iino, 1980). The aim of this work was to test whether the functional aspects of SR membranes were preserved after saponin treatment. A sarcotubular preparation derived from the frag- mentation of the SR of fast skeletal muscles of Rana esculenta was used. The parameters directly involved in the mechanism of calcium ion uptake and release were measured. Materials and methods Some experiments were earried out using the sartorius muscle of Rana esculenta while for other experiments the tibialis anterior was used. The frogs were kept for 3 days constant temperature (4 ~ C) before dissection. Mechanical procedures The muscles were immersed in Ringer solution, mount on a recording apparatus (Mancinelli et al., 1983) and incubated for 30 rein in a solution containing diffeie conientrations (10, 25, 50 or 100 ~g ml -1) of saponin. At different time intervals (1, 10, 20, or 30 rein), a series o twitches at a frequency of 1 Hz was obtained. Calcium transport Ca 2+ uptake and release were measured in purified vesi derived from the fragmentation of terminal cisternae skeletal muscle using the method of Saito and co-worke (1984). Ca 2+ uptake was measured according to Fitt a *Author to whom correspondence should be addressed. 0142-4319/89 $03.00 + .12 9 1989 Chapman and Hall Ltd.