Interaction of ruthenium red with isolated sarcolemma

Abstract

Summary. The stain ruthenium red binds very strongly to isolated sarcolemma and the maximal binding is about 125 nmoles/mg protein, decreasing slightly in lipid-extracted membranes. The binding is half maximal when the free stain concentration is about 1.0 ~tM, for both intact and lipid-depleted material. The nucleotide, ATP, reduces markedly the binding and the apparent affinity of the membranes for the stain. Minute concentrations of ruthenium red (10 laM) inhibit by 80 to 90% the Ca+ +-binding by sarcolemma. The inhibition does not depend on the Ca + + concentration and is similar in both intact and lipid-extracted preparations. Ruthenium red inhibits the ATPase activity of sarcolemma. The inhibition is decreased by increasing the ATP concentration in the medium.The primary events of excitation-contraction coupling in muscle occur at the plasma membrane in the sarcolemma [27,28]. Much work has been done to clarify the mechanism which links the electrical activity of the plasma membrane to the contractile machinery inside the cell [15,29,30,35,37], but the detailed events have not yet been elucidated.The sarcoplasmic reticulum is the main muscular structure in skeletal muscle which regulates the intracellular Ca + § concentration [11,12], and therefore, the contractile activity [43,44]. However, in some types of muscle, which are relatively poor in sarcoplasmic reticulum, other structures may work as Ca + § regulators, such as mitochondria [5,16] and, possibly, sarcolemma [1]. A fraction of Ca + + involved in the contraction-relaxation cycle may enter or leave the muscle cell through sarcolemma [24,27,32].Furthermore, we must necessarily consider an energetic efflux of Ca ++ through the sarcolemma to maintain a constant intracellular concentration of Ca + + since the concentration of free Ca + + in the extraceUular medium is much greater than that inside the muscle cell [23,25,32]. Although sarcoplasmic reticulum [6,11,12,44] and possibly mitochondria [5,16,18] may account for most Ca + + sequestration during relaxation of the muscle cell, there is a constant invasion of the cell by an inward net diffusion of

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

“…DMPC and egg phosphatidylcholine were tried without success. The total lipid extract of sarcoplasmic reticulum is a mixture of phospholipids cm2/s at about 36 OC to a value of (3. containing mainly phosphatidylcholine, with small amounts of phosphatidylethanolamine and other minor lipids (Madeira & Antunes-Madeira, 1976). The translational diffusion of SR-ATPase reconstituted into bilayers of total sarcoplasmic reticulum lipids at a molar ratio of lipid/protein of about 5000/ 1 was studied in the temperature range between 13 and 36 "C. No phase transitions occur in bilayers of this lipid in this range of temperature.…”

“…All of the above chemicals and lipids were used as received. A total lipid extract of rabbit muscle sarcoplasmic reticulum was prepared as described by Madeira & Antunes-Madeira (1976). NBD-DMPE was a gift from Dr. D. Hallmann, MaxPlanck-Institut, Gottingen, FRG.…”

Modulation of sarcoplasmic reticulum calcium pump activity by membrane fluidity

“…Fragmented sarcoplasmic reticulum from rabbit white muscles was prepared according to Madeira & Antunes- Madeira (1976), although isolation and ressuspension media always contained 2.5 mM DTT and 10 pM PMS. Protein was normally determined by the biuret method (Gornall et al, 1951).…”

“…Lipids of SR were extracted by the method of Madeira & Antunes- Madeira (1976). Phospholipids were quantitated by measuring the inorganic phosphate (Bartlett, 1959), after hydrolysis of extracts at 180 OC in 70% HCIOl (Bottcher et al, 1961).…”

Calcium-binding of synaptosomes isolated from rat brain cortex

“…It has been well documented that ruthenium red inhibits the binding or uptake of Ca / + by subcellular organelle such as mitochondria (Moore, 1971;Vasington, Gazzotti, Tiozzo & Carafoli, 1972;Rossi, Vasington & Carafoli, 1973), synaptic plasma membrane (Madeira & Antunes-Madeira, 1973;Kamino, Inouye, Ogawa, Uyesaka&Inouye, 1975a) and sarcolemma (Madeira & Antunes-Madeira, 1974). Furthermore, Sottocasa, Sandri, Panfili, de Bernard, Gazzotti, Vasington&Carafoli (1972) and Gomez-Puyou, de Gomez-Puyou, Becker and Lehninger (1972) demonstrated the inhibitory effect of the dye on Ca / +-binding by some Ca 2 +binding protein.…”