Yoshimasa Nishihara

Selective interaction of cytoskeletal proteins with liposomes

The initial step in cell activation by a ligand is believed to be its binding to specific receptors on the cell surface [I], and a ligand-induced redistribution or cross-linking of the receptor proteins is considered to be a step in the generation of a transmembrane signal [2]. It has been proposed that transmembrane surface proteins associate with components of the cytoskeletal network at the inner face of the cell membrane [3]. Therefore, a network of cytoskeletal structures on the inner face of membrane could interfere with the topology of receptor moieties on the outer surface of membrane [4]. In this case, it is considered that the hydrophobic interaction between membrane lipids and cytoplasmic proteins has an important role in the generation of the transmembrane signal. In attempting to understand this role, the interaction between phospholipid vesicles and cytoplasmic proteins was studied and it was found that many of the cytoskeletal proteins such as tubulin [5], actin, a-actinin and myosin associated strongly with liposomes made from dimyristoy1 or dipalmitoyl phosphatidylcholine vesicles [6]. In this report, we describe the association of actomyosin complex of polymorphonuclear leukocytes (PMN) and purified muscle actin with phospholipid vesicles and the formation of protein-liposome recombinants depending on the disorder of the phospholipid matrix. Liposomes were made with dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol, cetylamine and dicetylphosphate as described in [6]. Unilamellar vesicles containing carboxyfluorescein (CF) were prepared by the method of Klausner et al. [lo] for the measurement of phase transition release (PTR). Phospholipids were dissolved in chloroform-methanol (2: 1) and the thin filmed lipids were hydrated by vortex-mixing and sonicated with a sonicator (Branson, type 185) in the presence of CF (0.1 M) at 25’C for DMPC and 45°C for DPPC. The CF-containing vesicles were centrifuged at 100 X g for 5 min and unilamellar vesicles were fractionated in a Sepharose 4B column (1.5 X 25 cm) with a 0.1 M NaCl-20 mM phosphate buffer (pH 6.8) eluant at 4°C.

Studies on the response of isolated rat liver mitochondria to polychlorinated biphenyls (Kanechlors

A comparative study was made of the effects of biphenyl and polychlorinated biphenyls (Kanechlors®, KCs) on the respiratory and energylinked activities of rat liver mitochondria. With αketoglutarate/malate as the substrate, biphenyl acted as the strongest inhibitor of state 3 respiration. The inhibition of state 3 respiration was decreased as chlorine content increased. The stimulation of state 4 respiration was greatest in KC-200, 300, and 400; intermediate in biphenyl and KC-500; least in KC-600. When succinate was the substrate, from the results of this and a previous study (Nishihara 1983) the action of biphenyl as an inhibitor of state 3 respiration was least effective. The inhibitory action on state 3 respiration was strengthened by the chlorination of aromatic rings (KC-200), reaching a peak where maximum inhibition was observed (KC-300, 400, and 500), further increases in chlorine content (KC-600) repressed the inhibitory action. The stimulating ability of state 4 respiration was greatly diminished with increases in chlorination. Biphenyl and KCs induced the K+ -release from mitochondria, thereby dissipating membrane potential across the mitochondrial membranes in the order; biphenyl > KC-200 > KC-300 > KC-400 > KC-500 > KC-600. It is concluded that with α-ketoglutarate/malate (NAD+ -linked substrate), KCs act as uncouplers by increasing the permeability of mitochondrial inner membranes to ions, and that when succinate is the substrate, KCs act as inhibitors rather than uncouplers of oxidative phosphorylation of mitochondria by inhibiting the electron transport chain.

Lipid-dependent interaction of d-β-hydroxybutyrate dehydrogenase with cellular membranes

A mechanism of selective localization of membrane-bound enzymes was examined by studying the interaction between D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and native cellular membranes in which the lipid components were altered. (1) The catalytic activity of the purified lipid-free enzyme could be restored by the re-interaction with microsomal and mitochondrial membranes, whereas with erythrocyte membranes or liposomes from lipids of erythrocyte membranes this activity could not be restored (Miyahara, M., Utsumi, K. and Deamer, D.W. (1981) Biochim. Biophys. Acta 641, 222-231). In the erythrocyte lipid components, only lysophosphatidylcholine markedly inhibited the enzyme reactivation. (2) The inhibitory effect of lysophosphatidylcholine was confirmed in microsomes in which the lysophosphatidylcholine contents had been increased, by phospholipase A2 treatment, to the levels in erythrocyte membranes. (3) Selective digestion by phospholipase C of phosphatidylcholine in the microsomes was accompanied by a lowering of the level of reactivation in the membranes. (4) The presence of lipophilic alkyl compounds such as cetylamine and cetyltrimethylammonium bromide, which contain the ammonium group, in the membranes also inhibited the enzyme reactivation. However, negatively charged and neutral alkyl compounds were less suppressive. The results above suggested that the interaction of D-beta-hydroxybutyrate dehydrogenase with native cellular membranes is dependent on the amounts of phosphatidylcholine and lysophosphatidylcholine exposed on the membrane surface. It was also suggested that the presence of the ammonium group of non-diacyl compounds is unfavorable for the effective interaction of the enzyme.