Józef Zborowski

Phosphatidylserine decarboxylase is located on the external side of the inner mitochondrial membrane.

It is shown that the trypsin‐treatment of rat liver mitochondria, depleted of the outer membrane, causes a strong inactivation of phosphatidylserine decarboxylase. This inactivation is dependent on trypsin concentration and the time of digestion in a similar manner as the inactivation of cytochrome oxidase. Under these conditions only a moderate inactivation of succinate dehydrogenase is observed. Phosphatidylserine decarboxylase is thus localized in the outer leaflet of the inner mitochondria membrane or, at least, is accessible from the outer surface of the inner membrane.

Phospholipid synthesis in rat liver mitochondria

Abstract 1. 1. Synthesis of phospholipids in isolated rat liver mitochondria was studied by measuring the incorporation into mitochondrial lipids of glycerol 3-[32P]phosphate and [14C]palmitic acid. 2. 2. Both precursors were incorporated chiefly into phosphatidic acid. Under experimental conditions this incorporation was much higher in mitochondria than in microsomes. 3. 3. Maximum esterification of glycerol 3-[32P]phosphate was obtained with palmitic acid and was less pronounced with acids of shorter or longer carbon atom chains. Elaidic acid was inhibitory. 4. 4. ATP either added or generated intramitochondrially was required. However, in the presence of carnitine esters of long-chain fatty acids the incorporation of glycerol 3-[32P]phosphate was independent of ATP. 5. 5. With internally formed ATP, the synthesis of phosphatidic acid was inhibited by atractyloside but was unaffected by this glycoside with external ATP or with palmitoyl carnitine. 6. 6. Outer and inner mitochondrial membranes were separated by two different procedures. The esterification of glycerol 3-[32P]phosphate by free fatty acids in the presence of ATP and CoA was much higher, on a protein basis, in the outer than in the inner membrane fractions. A substantial esterification by palmitoyl carnitine occurred, however, only in unfractionated membranes and was low in both the outer and the inner membrane fractions examined separately.

Exchange of phospholipids between microsomes and mitochondrial outer and inner membranes

Abstract 1. 1. 32P-labelled phospholipids are transferred from rat liver microsomes to rat liver mitochondria when the particles are incubated in the presence of the soluble cytoplasmic fraction. Labelled phospholipids are incorporated mainly into the outer mitochondrial membrane, and only after disruption or detachment of the outer membrane could a substantial uptake of the label by the inner membrane be observed. The exchange of phospholipids between microsomes and the outer mitochondrial membrane was essentially complete within 15–20 min under experimental conditions, whereas the exchange between microsomes and exposed inner mitochondrial membranes proceeded for at least 60 min. 2. 2. Free fatty acids added to the mitochondrial suspension in tracer amounts could easily penetrate across the outer membrane and be bound by the inner membrane. 3. 3. Liver slices incubated in the presence of [14C]choline incorporated the label into both microsomes and mitochondria. A chase applied after 60 min labelling produced a decrease of the specific activity of microsomal lecithin, but the specific activity of mitochondrial lecithin continued to increase. [14C]Choline was mainly incorporated into lecithin of the outer mitochondrial membrane, about as little as 20 % being found in the inner membrane. 4. 4. The results are interpreted in the sense that the exchange of phospholipids between the inner and outer membranes of intact mitochondria either does not occur or is qualitatively different and quantitatively much slower than a direct exchange between microsomes and the outer mitochondrial membrane.

NET TRANSFER OF PHOSPHATIDYLINOSITOL FROM MICROSOMES AND MITOCHONDRIA TO LIPOSOMES CATALYZED BY THE EXCHANGE PROTEIN FROM RAT LIVER

It is now well established that an exchange of phospholipids can occur between intracellular organellae [l-6] (for review see [7] ). This exchange is catalyzed by special exchange proteins found in the cytosol of several tissues [8-l 51. These proteins seem to be rather specific toward individual phospholipids [9, 1 l-l 3, 151 but unspecific with respect to membranes, i.e. they can catalyze the exchange of phospholipids between different biological as well as artificial membraneous structures, e.g. liposomes [16-l 91. The exchange protein for PC* contains one molecule df PC per molecule of protein [7,171. Kagawa et al. [20] provided an indirect evidence for a net transfer of PC from liposomes to reconstituted energy-coupling vesicles, as mediated by the exchange protein from beef heart, and Ehnholm and Zilversmit [ 1 l] showed a unidirectional transfer of sphingomyelin. Recently, Wirtz and his colleagues [21,22] have directly demonstrated a net transfer of PI between microsomes and liposomes catalyzed by the transfer protein isolated by these authors from beef brain [ 151. Independently, we have briefly reported [23] on a net transfer of PI from microsomes and mitochondria to liposomes lacking this phospholipid as catalyzed by the transfer protein(s) from rat liver. The present paper describes these studies in more detail.

The metabolism of CDP-diacylglycerol and phosphatidylinositol in the microsomal fraction of rat liver. Effects of chlorpromazine, magnesium and manganese.

1. The metabolism of CDPdiacylglycerol and phosphatidylinositol was measured using substrates bound to the microsomal membranes of rat liver. 2. Chlorpromazine inhibited the degradation of [14C]CDPdiacylglycerol and the concomitant inositol-independent release of 14C in water-soluble products in the presence of various concentrations of Mg2+ and Mn2+. 3. The activity of CDPdiacylglycerol inositol phosphatidyltransferase was measured by determining the rate of incorporation of [3H]inositol into phosphatidylinositol, and by the inositol-dependent release of water-soluble 14C from [14C]CDPdiacylglycerol. Both of these parameters were inhibited by chlorpromazine in incubations that contained rate-limiting concentrations of Mg2+. However, chlorpromazine stimulated the reaction when 20 mM Mg2+, 0.5 mM Mn2+, 2 mM Mn2+ or 20 mM Mn2+ was added to the incubations. 4. Low concentrations of chlorpromazine increased an inositol-exchange reaction in the presence of 0.5 mM Mn2+ whereas higher concentrations of chlorpromazine inhibited. Chlorpromazine had relatively less effect on the inositol-exchange reaction at higher concentrations of Mn2+. 5. The action of chlorpromazine in decreasing the breakdown of CDPdiacylglycerol and in stimulating its conversion to phosphatidylinositol could explain part of the mechanism by which this compound and other amphiphilic cations increase the synthesis of acidic phospholipids.

Intramitochondrial distribution and transport of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine. Application of pyrene-labeled species

To investigate the mechanism of intramitochondrial translocation of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, the distribution of these lipids between the outer (OM) and inner (IM) mitochondrial membranes, as well as their transversal and lateral distribution in OM were studied. Fluorescent, pyrenyl derivatives of phosphatidylserine (PyrxPS) and phosphatidylethanolamine (PyrxPE) species were employed because they allow: (i), direct monitoring of PS (and PE) loading to the mitochondria; (ii) assay of PS decarboxylation by high-performance liquid chromatography with fluorescence detection and (iii), determination of the lateral distributions of PS and PE within the mitochondrial membranes. All PyrxPS species tested were efficiently decarboxylated by the solubilized decarboxylase and thus the distribution of the endogenous PE could be also studied. When the PyrxPS species were loaded to isolated mitochondria very little, if any, of the loaded PyrxPS or of the PyrxPE product was found in IM independent of the time and temperature of incubation, strongly suggesting that these lipids either never enter IM or their residence there is only transient. When mitochondria preloaded with Pyr4PS were incubated with an excess of acceptor vesicles in the presence of the lipid transfer protein, 80% of Pyr4PS and 30-40% of the Pyr4PE product were transported to the acceptor vesicles, indicating that at least corresponding fractions of these lipid were located in, or were in rapid equilibrium with the outer leaflet of OM. Since the decarboxylase is located in the inner membrane, these results signify that both PS and PE must be able to move readily across OM. Determination of the excimer to monomer ratio as the function of pyrenyl lipid concentration in mitochondria (i.e., OM) gave parallel results for PyrxPS and -PE species suggesting the lateral distribution of PS and PE in OM is similar and thus there is no specific enrichment of PS to the contact sites. To investigate the mechanism of PS transport from the outer leaflet to the decarboxylation site, the influence of PyrxPS hydrophobicity, i.e., pyrenylacyl chain length, on the rate of decarboxylation was determined. The variation of the length of the pyrenyl acyl chain from 4 to 12 carbons did not significantly affect the rate of PyrxPS decarboxylation in intact mitochondria, indicating that the transport of PS from the outer leaflet of OM to the site of decarboxylation takes place by lateral diffusion rather than by spontaneous or protein-mediated transport. The implications of these findings on the mechanism of intramitochondrial transport of PS and PE are discussed in terms of alternative models.

Effect of hyper- and hypothyroidism on phospholipid fatty acid composition and phospholipases activity in sarcolemma of rabbit cardiac muscle☆

Lipid content and composition of fatty acids esterified to phospholipids of cardiac sarcolemma isolated from hyperthyroid, hypothyroid and control rabbits were analysed. Hyperthyroidism resulted in a significant reduction of the cholesterol to phospholipid molar ratio as compared to control animals, while hypothyroidism exerted the opposite effect. Complex changes in composition of phospholipid fatty acids observed in hyperthyroid state led to an elevation of the fatty acid unsaturation index over the control value. The unsaturation index value was, however, not affected in the hypothyroid state. Thyroxine hormone administration increased phospholipase A1 and decreased phospholipase A2 activity. The opposite effect was observed in thyreodectomized animals. The effect of changes in sarcolemmal bulk phospholipids upon thyroxine administration or deficiency on regulation of activity of membrane-bound enzymes is discussed.

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.

Membrane lateral pressure as a modulator of glycerol-3-phosphate dehydrogenase activity

Michaelis‐Menten kinetics of glycerol‐3‐phosphate dehydrogenase activity in proteoliposomes from brown adipose tissue mitochondria with exogenously added phospholipids or cholesterol was measured. It was shown that changes in membrane lipid composition affected the membrane lateral pressure and therefore modulated the enzyme activity, namely V max value. Contrarily, changes in surface charge caused by minute amounts of phosphatidylserine or charged organic substances influenced only the apparent K m value. The role of bulk phospholipids in regulation of glycerol‐3‐phosphate dehydrogenase is discussed.

Transport of phosphatidic acid within the mitochondrion

Transfer of phosphatidic acid from the outer to the inner membrane within intact rat liver mitochondria was assessed by measuring the ratio of lipid 32P to the marker enzyme of the outer membrane, rotenone-insensitive NADH-cytochrome c reductase, in the outer and inner membrane fractions obtained after incubation of mitochondria under conditions for net synthesis of [32P]phosphatidic acid. This transfer was found to proceed with time, to occur only under high ionic strength of the external medium and to be insensitive to N-ethylmaleimide and factors reducing the number of contact sites between the two mitochondrial membranes. These results are interpreted as supporting the idea that phosphatidic acid transport within the mitochondrion occurs as free diffusion through the aqueous phase and not being mediated by phospholipid transfer protein(s).