Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena

Abstract

The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs' protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins.The inactivation of a Mg2"-requiring, K+- MATERIALS AND METHODSPlant materials used and procedures for detergent solubilization and gel filtration, as well as ATPase and protein assays used, were described previously (9). Polar Lipid Dispersion. PC,2 PE, and PI from soybean, PG from egg lecithin, and PS from bovine brain, all 99.9% purity, were obtained from Sigma Chemical Company. A mixture of lipids (approximately 50% PC from soybean) was also obtained from Sigma. The lipids were dispersed as described before (9)

Mechanism of Photoinactivation of Plant Plasma Membrane Atpase

“…3). This confirms a relationship previously observed between ATPase activity and tryptophan fluorescence excited at 290 nm (Imbrie and Murphy, 1984b). With the UVsensitive ATPase irradiated at 365 nm, there was proportionally a higher loss of absorption than loss of enzyme activity.…”

“…The inactivation by ultraviolet light of an Mg2+requiring, K+-stimulated ATPase activity associated with the plasma membrane of suspension cultured rose cells has biphasic kinetics (Imbrie and Murphy, 1982). Using detergent-solubilized enzyme preparations fractionated on a Sephadex G-150 column, we have shown that the plasma membrane contains two distinct ATPase activities separable not only by UV sensitivity but also by molecular weight (Imbrie and Murphy, 1984a). The action spectra for inactivation of the solubilized and fractionated ATPase activities are very similar to the spectra for inactivation of ATPase in vesicles, suggesting that the proteins themselves contain the UV chromophores.…”

“…Plasma membrane vesicles were prepared from 4-day old suspension cultured cells of rose (Rosa damascena) based on the method of Hodges et al (1972) modified as described by Imbrie and Murphy (1982). The vesicle preparation was solubilized with a 1% solution 243 of sodium cholate (Sigma) and fractionated on a Sephadex G-150 column (Imbrie and Murphy, 1984a). The solubilized enzymes were reconstituted in a mixture of lipids (approximately 50% L-a-phosphatidylcholine) from soybean (Sigma) dispersed with a 15-s pulse from a sonicator (BIOSONIK, Bronwill Scientific, Rochester NY with a small probe set at an intensity of 60) in a solution containing 2 mM histidine, 0.1 mM ethylenediaminetetraacetic acid, 2 mM N-2hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (pH 6.7) and 50 pg per me butylated hydroxytoluene to give a final phosphorous concentration of 10 pM or higher (Imbrie and Murphy, 1984a).…”

Selective Delipidation of the Plasma Membrane by Surfactants

“…This suggests that there is no absolute requirement for phospholipids for ATPase activity. This might seem to contradict the frequent observations that after surfactant treatment, ATPase activity can be at least partially restored by addition of phospholipids (2,8,9,17,19). Studies with other membrane-bound ATPases have shown, however, that as long as an adequate hydrophobic environment with sufficient fluidity is maintained around the ATPase, by either surfactants or lipids, activity is maintained or enhanced depending on anomalies in that environment (4,11,16,23,27).…”

Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

“…UV-irradiation of unwashed cells increased the specific activity of the ATPase in the plasma membranes prepared from those cells by 33%. UV clearly had effects on ATPase other than direct inactivation (such as was described for in vitro irradiation by Imbrie & Murphy (1984)), and these effects were modulated by the washing procedure.…”