Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

Phospholipid transfer activity in synchronous populations of Rhodobacter sphaeroides

Studies of intracytoplasmic membrane biogenesis employing steady-state synchronously dividing populations of Rhodobacter sphaeroides reveal that the translocation of pre-existing phospholipid into the growing membrane is concurrent with cell division (Cain, B.D., Deal, C.D., Fraley, R.T. and Kaplan, S. (1981) J. Bacteriol. 145, 1154–1166), yet the mechanism of phospholipid movement is unknown. However, the discovery of phospholipid transfer protein activity in R. sphaeroides (Cohen, L.K., Lueking, D.R. and Kaplan, S. (1979) J. Biol. Chem. 254, 721–728) provides one possible mechanism for phospholipid movement. Therefore the level of phospholipid transfer activity in cell lysates of synchronized cultures was measured and was shown to increase stepwise coinciding precisely with the increase in cell number of the culture. Although the amount of transfer activity per cell remained constant throughout the cell cycle, the specific activity of the phospholipid transfer activity showed a cyclical oscillation with its highest value coincident with the completion of cell division. Purified intracytoplasmic membrane can be used as phospholipid acceptor in the developed phospholipid transfer assay by employing either cytoplasmic membrane or liposomes as the phospholipid donor. Intracytoplasmic membrane isolated from the cells prior to division (high protein to phospholipid ratio) served as a better phospholipid acceptor in the phospholipid transfer system when compared with membranes derived from the cells following cell division (low protein to phospholipid ratio).     

Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.     

Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts

The effects sterol carrier protein-2 (SCP-2) expression on L-cell phospholipid levels and fatty acyl composition was assessed using L-cells transfected with the murine cDNA encoding for either the 15 kDa proSCP-2 or 13.2 kDa SCP-2. Expression of these proteins reduced total phospholipid mass (nmol/mg protein) by 24% and reduced the cholesterol to phospholipid ratio 60 and 28%, respectively. In 15 kDa proSCP-2 expressing cells, individual phospholipid class masses, excluding sphingomyelin (CerPCho), were reduced as follows: phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) > ethanolamine glycerophospholipid (EtnGpl) > choline glycerophospholipid (ChoGpl). Furthermore, ethanolamine plasmalogen mass was decreased 25%, while choline plasmalogen mass was elevated 30% in 15 kDa proSCP-2 expressing cells. In 13.2 kDa SCP-2 expressing cells, phospholipid class mass was decreased as follows: PtdIns and PtdSer > ChoGpl. These changes in phospholipid mass resulted in altered cellular phospholipid composition. Expression of either protein differentially altered the type of fatty acid esterified onto the phospholipids. These effects included a greater proportion of polyunsaturated fatty acids and a reduction in saturated fatty acids, although 15 kDa proSCP-2 expression had a more robust effect on these parameters than did 13.2 kDa SCP-2 expression. In summary, expression of SCP-2 reduced individual phospholipid class mass, except for CerPCho, and altered the fatty acid composition of each phospholipid class examined.These results clearly demonstrate that SCP-2 expression altered basal phospholipid levels, suggesting that SCP-2 can alter the function of endoplasmic reticulum phospholipid synthetic enzymes.     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.     

A convenient and sensitive fluorescence assay for phospholipid vesicles using diphenylhexatriene

When phospholipid vesicles are added to an aqueous solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) a fluorescence enhancement of up to several hundredfold is observed which can be used for a determination of phospholipid concentration. Fluorescence enhancement of 2 μm DPH is proportional to the phospholipid concentration over a wide range. As little as 0.7 nmol (～0.5 μg of phospholipid) can be determined to within ±10%. The fluorescence is a function of the type of phospholipid used, salt concentration, and time of incubation. Protein and detergents also enhance DPH fluorescence but to a much smaller extent. Optimal conditions for the assay are presented. Use of this assay to detect phospholipid vesicles fractionated by size on a Sepharose 4B column is illustrated. In this case the method compares favorably to more classical methods of analysis in terms of sensitivity, accuracy, and time involved.     

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein.

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.     

Phospholipid specificity of bovine heart bc1 complex

Bovine heart bc1 complex was reversibly inactivated by a new simple and effective chromatographic delipidation method. Upon phospholipid replenishment, catalytic activity increased from values near zero to values 2-6-times higher than those of the original preparation. Compared to original preparations maximally activated by additional phospholipid, the degree of reactivation was up to 100%. By this delipidation method, the 6.4-kDa protein subunit was removed with the phospholipid. The loss of this protein neither diminished electron transport activity nor abolished proton translocation. Two requirements were necessary to obtain quantitative data: (a) only bc1 complexes, homogeneously dissolved before and after relipidation had to be used and (b) the phospholipid bound to the complex had to be determined. The correlation of catalytic activity to bound phospholipid was studied in the range of low phospholipid/protein ratios, which had previously been insufficiently resolved. Catalytic activity increased linearly with added phospholipid up to a molar ratio of 80-100 lipid molecules/dimeric complex. This corresponds to the number of phospholipid molecules that complete a single bilayer annulus. The activating effect of phospholipid is not merely due to a hydrophobic phase effect, since it strongly depends on the nature of the polar head group of the added phospholipid. Of the three major phospholipids bound to the bc1 complex, only phosphatidylethanolamine and phosphatidylcholine activated when added as sole phospholipid. Tightly bound diphosphatidylglycerol was needed for preservation of the native complex structure.     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties

Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein of Mr 27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and pI 5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 micrograms/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1 was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 degrees C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7 and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being on Vmax rather than Km. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 . 10(6) cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18--40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1-(14C)]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.     

In vivo intermembrane transfer of phospholipids in the photosynthetic bacterium Rhodopseudomonas sphaeroides.

The kinetics of accumulation of phospholipids into the intracytoplasmic membrane of Rhodopseudomonas sphaeroides have been examined. We have previously demonstrated that accumulation of phospholipids in the intracytoplasmic membrane is discontinuous with respect to the cell cycle. In this study we demonstrated a sevenfold increase in the rate of phospholipid incorporation into the intracytoplasmic membrane concurrent with the onset of cell division. Pulse-chase labeling studies revealed that the increase in the rate of phospholipid accumulation into the intracytoplasmic membrane results from the transfer of phospholipid from a site other than the intracytoplasmic membrane, and that the transfer of phospholipid, rather than synthesis of phospholipid, is most likely subject to cell cycle-specific regulation. The rates of synthesis of the individual phospholipid species (phosphatidylethanolamine, phosphatidyglycerol, and an unknown phospholipid) remained constant with respect to one another throughout the cell cycle. Similarly, each of these phospholipid species appeared to be transferred simultaneously to the intracytoplasmic membrane. We also present preliminary kinetic evidence which suggested that phosphatidylethanolamine may be converted to phosphatidycholine within the intracytoplasmic membrane.     

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

Inhibition by colchicine of phospholipid secretion induced by lung distension

The effect of colchicine, a microtubule disruptor, on phospholipid secretion stimulated by distension of fetal rabbit lungs was investigated. After colchicine injection and breathing for 45 min, pups were killed and their lungs were lavaged with colchicine. Controls were injected and lavaged with saline. All lungs were given static air inflation and a final lavage, and the returns were analyzed for phospholipid DNA, and lactate dehydrogenase. The first lavage after breathing yielded 33% less phospholipid with colchicine, 3.83 compared with 5.72 mg/g dry lung wt (P less than 0.05). The postinflation phospholipid yield was also significantly reduced with colchicine from 1.04 to 0.70 mg/g dry lung wt (P less than 0.05). The postinflation DNA was significantly reduced with colchicine, from 1.26 to 0.44 micrograms (P less than 0.01), suggesting reduced alveolar macrophages. Colchicine did not change the recovery by lavage of exogenous radioactive phospholipid. As reflected by ATP and lactate levels, tissue metabolism was well maintained. The results are interpreted to mean that colchicine reduced simultaneously lavage-associated phospholipid secretion, inflation-produced phospholipid secretion, and macrophage migration.     

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-naphthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS—phospholipid micelle formation. The results for a phospholipid—cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.     

Role of sarcoplasmic reticulum phospholipids in calcium ion binding activity

The relationship between sarcoplasmic reticulum phospholipid and Ca(2+) binding by sarcoplasmic reticulum membranes was explored. Ca(2+) bound in the absence of ATP was defined as "ATP-independent Ca(2+) binding," and the additional amount of Ca(2+) bound in the presence of ATP was defined as "ATP-dependent Ca(2+) binding." The latter was found to be very sensitive to the loss of sarcoplasmic reticulum phospholipid; the amount of Ca(2+) bound was reduced when as little as 3% of the phospholipid was destroyed by phospholipase C. Further destruction of membrane phospholipid up to a 40% loss caused little or no further reduction of this Ca(2+) binding. However, when the destruction of phospholipid exceeded 40%, further loss of this Ca(2+) binding occurred, and there was an almost complete loss of this function when more than 60% of the sarcoplasmic reticulum phospholipid was destroyed.     

The indispensability of phospholipid and ubiquinone in mitochondrial electron transfer from succinate to cytochrome c.

The indispensability of phospholipid and ubiquinone (Q) in mitochondrial electron transfer was studied by depleting phospholipid and Q in succinate-cytochrome c reductase and then replenishing the depleted enzyme. More than 90% of phospholipid and Q was removed by repeated ammonium sulfate-cholate fractionation. The depleted succinate-cytochrome c reductase showed no enzymatic activity for succinate leads to c or QH2 leads to c and yet retained most of the succinate leads to Q activity. All enzymatic activity was restored upon the addition of Q and phospholipid. Restoration required the addition of Q prior to the addition of phospholipid. Reversing the addition sequence or addition of a mixture of phospholipid and Q resulted only in a small restoration of activities. The conditions for restoration are given in detail. Removal of phospholipid from succinate-cytochrome c reductase resulted in reduction of cytochrome c1 in the absence of exogenous electron donor. Replenishing the preparation with phospholipid brought about the reoxidation of cytochrome c1 in the absence of electron acceptor or oxygen.     

Chronic and acute effects of thyrotropin on phosphatidylinositol turnover in cultured porcine thyroid cells

The 32P incorporation into phospholipids of isolated porcine thyroid cells, cultured for 1-4 days, has been studied in subsequent 2-h incubations. Along with culture ageing, decreased 32P incorporation into total phospholipid of control cells was observed. The presence of 40 munits/ml TSH during the 2 h incubation yielded a relative increase in labelling of phosphatidylinositol, named 'acute phospholipid effect'. A chronic treatment of the cells with TSH concentration ranging from 0.1 to 10 munits/ml ensured the maintenance of a high turnover rate of total phospholipids. The analysis of individual phospholipids revealed that 1-day culture cells in the presence of 0.1 munits/ml TSH presented a strong increase of phosphatidylinositol labelling. This 'chronic phospholipid effect' of TSH can be reproduced by a chronic treatment with dibutyryl cyclic AMP (10(-3)M) or prostaglandin E2 (10(-6)M), which did not evoke a classical phospholipid effect in a 2 h incubation. If TSH (40 munits/ml) is added to the cells in a 2 h incubation, control cells show the classical phospholipid effect whereas cells chronically treated with TSH, dibutyryl cyclic AMP or prostaglandin E2 presented a 'reverse phospholipid effect' i.e. a relative decrease in phosphatidylinositol labelling. 10(-4)M cycloheximide presence during the last 12 h of culture prevented the establishment of the 'chronic phospholipid effect' and of its consequence, 'the reverse phospholipid effect'. On the basis of these results a scheme is proposed in keeping with current hypotheses concerning phosphatidylinositol metabolism.