Konrad S. Famulski

Influence of the surface potential on the Michaelis constant of membrane-bound enzymes: effect of membrane solubilization.

Detergents used at sufficiently high concentrations solubilize biological membranes, setting free their integral hydrophobic proteins. During this process the proteins may undergo serious alterations (review [I]). Mild detergents, especially those of non-ionic character, interact less drastically and therefore are routinely used for isolation of membrane-bound enzymes [2]. Nevertheless, even in these cases catalytic properties of the enzymes may be changed due to several factors such as alterations of secondary and tertiary structures, depletion of essential lipids, and presence of detergent molecules associated with the enzyme. This paper points to the abolition of the membrane surface potential as a factor influencing the activity of membrane enzymes after solubilization. It is a continuation of our studies in which the effect of the surface potential on Michaelis constants of a series of membrane-bound enzymes has been demonstrated [3-51. substrate. Glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was assayed with phenazine methosulphate as the primary electron acceptor and 2,6-dicbloroindophenol as the secondary acceptor as in [3]. The activity of monoamine oxidase (EC 1.4.3.4) was determined as in [lo] with dopamine as substrate by measuring oxygen uptake in the presence of 1 mM KCN. Acetylcholinesterase (EC 3.1 .1.7) was determined by measuring liberation of thiocholine from acetylthiocholine [ 111. NADPHcytochrome c reductase (EC 1.6.2.4) was measured according to [ 121. Glucose&phosphatase.(EC 3 .1.3.9) and the pyrophosphate-glucose phosphotransferase activity of this enzyme were assayed as in [ 131.

Phosphorylation of mitochondrial membrane proteins: effect of the surface potential on monoamine oxidase

Proteins of mitochondrial membranes become phosphorylated when liver mitochondria are incubated with ATP in the presence of Mg2+. This is accompanied by an increase of the negative surface potential of mitochondrial membranes, as calculated from the dissociation constant of fluorescent probes, 8‐anilino‐1‐naphthalene sulphonate and ethidium bromide, and by a decrease of the apparent K m‐value of mitochondrial monoamine oxidase. Protein phosphorylation, the increase of the negative surface potential and the decrease of apparent K m of monoamine oxidase are greatly potentiated by cytoplasmic protein kinases in the presence of cyclic AMP.

Thyroid hormones control lipid composition and membrane fluidity of skeletal muscle sarcolemma.

Sarcolemma membrane lipid phase of skeletal muscles of hyperthyroid animals was compared to that of control (euthyroid) ones. Hyperthyroidism caused 15% decrease in cholesterol and 70% increase in the phospholipid content of the membrane. This was accompanied by the alterations in proportions between individual phospholipid classes, and was followed by changes in the composition of phospholipid fatty acids. The calculated fatty acid unsaturation index was higher for membrane lipid phase of hyperthyroid animals than of euthyroid ones. Thyroxine-induced alterations in the lipid composition of sarcolemma caused changes in the membrane fluidity and the activity of calmodulin-stimulated (Ca(2+)-Mg(2+)-ATPase. Measurements of the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the lipid phase transition of membrane vesicles occurred at 25.9 degrees C and at 28.9 degrees C for preparations isolated from hyperthyroid and euthyroid rabbits, respectively. Arrhenius plot break-point temperature for CaM-stimulated (Ca(2+)-Mg(2+)-ATPase activity was lower in membrane preparations isolated from hyperthyroid (26.9 degrees C) than from euthyroid ones (30.0 degrees C). Thus, the increase of the membrane fluidity presumably caused that the enzyme was characterized by the lower activation energy value. This phenomenon may be viewed as a supplementary mechanism for activation of the enzyme by thyroid hormones to previously reported elevation of the amount of (Ca(2+)-Mg(2+)-ATPase protein exerted by hyperthyroidism (Famulski et al. (1988) Eur. J. Biochem., 171, 363-368; Famulski and Wrzosek (1988) in The Ion Pumps-Structure, Function and Regulation (Stein, W.D., ed.), pp. 355-360, Alan R. Liss, New York).

Effect of the phosphorylation of microsomal proteins on the surface potential and enzyme activities.

It Ins been demonstrated [I ] that altering the surface charge density of biological membranes changes the; activity of membrane-bound enzymes at suboptimal substrate concentrations. This was shown by an increase or a decrease of the app. K;, values, the activity at infinite substrate concentration remaining unchanged_ It has been postulated that a change of the app. Kn, reflects an alteration of the charged substrate concentration in the immediate vicinity of the membrane, due to electric attraction or repuision. Manipulation of the surface charge density was performed using divalent cations, natural surfactants, as oleate and pahnitoyl-CoA, and commercial detergents. Since intracellular concentrations of fatty acids, acyl-CoA and divalent cations vary under specific conditions, it may be spe8culated that the surface charge plays a certain regulatory role. Looking for other physiological mechanisms of changing the surface charge density of cellular membrane;, -we focussed our attention on phosphorylaticla ofmembrane proteins by protein kinases. It has been shown [2-93 that several membraneous structures, like the endoplasmic and the sarcoplasmic reticnlum, mite. chondria, synaptosomes and the plasm:. membrane, can be phosphorylated by solubie hnd membranebound (endogenous) protein kinases. The present investigation she-ws that such phosphoryl@icril alters the activity of certain membrane enzymes in a similar way as a change of t!le surface charge L-y means of surfactants or divalent cations.

Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane

Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Pikułla, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes.