Studies on (K+ + H+)-ATPase IV. Effects of phospholipase C treatment

(1) The total phospholipid content of a gradient purified ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase and K⁺ stimulated $\text{p-}\text{nitrophenylphosphatase}$ activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP and temperature dependence of the gastric ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase, but slightly decreases the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for K⁺. (5) Phospholipase C treatment lowers the $\text{Ado}\text{PP[}\text{NH}\text{]P}$ binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.     

Decreased iodination of the red cell surface following phospholipase C treatment

Human red blood cells were treated with phospholipase C from Clostridium welchii. Lipase concentrations which produced <1% hemolysis and 10–15% hydrolysis of the membrane phospholipids reduced markedly ($\text{>80\%}$) the accessibility of mambrane proteins to the external surface as measured by lactoperoxidase-catalyzed iodianation.     

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A₂ produced residual membrane preparations with very low $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate.Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and sphingomyelinase C (Staphylococcus aureus) resulted in 90–95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50–100% of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane

After incubation of human erythrocytes at 37 °C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A₂ from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes.Pancreatic phospholipase A₂, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, $\text{o-}\text{iodosobenzoate}$ and hydroquinone) even render susceptible to pancreatic phospholipase A₂ phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Transfer of phospholipids between the endoplasmic reticulum and mitochondria in rat hepatocytes in vivo

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling $\text{in}\text{vivo}$. With [³H]glycerol microsomal phosphatidylethanolamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occurred only after about 5 hours. It appears that the $\text{in}\text{vivo}$ transfer of these phospholipids between the two membrane compartments is a relatively slow process.     

Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C

The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine: diphosphatidylglycerol: phosphatidylinositol, 90:5:5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70–75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–7}\text{days}$) appeared to be greater than that for phosphatidylinositol ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8–60}\text{days}$). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Operation of an electrogenic Na-pump in mammalian red muscle fibre

The resting membrane potential in ‘Na-rich’ soleus muscle fibres, obtained from the K-depleted rats fed a K-free diet, was rapidly hyperpolarized beyond the theoretical value derived from the ionic theory or even beyond the potential measured in ‘fresh’ muscle fibres, when 2.5 to 15 mM-K was added to the bathing solution at 37°C. The K-sensitive hyperpolarization was abolished after cooling to $\text{ca.}$ 4°C or adding ouabain. Therefore, the observed membrane potential exceeding the calculated potential during hyperpolarization was attributed to an electrogenic Na-pump.     

Role of proteins and lipids in non-linear Arrhenius plots of Drosophila mitochondrial succinate-cytochrome c reductase studied by rebinding experiments

Abrupt changes in the Arrhenius activation energy of membrane-bound enzymes have often been correlated with changes in the physical state of membrane phospholipids. Similar changes in activation energy have also been found in soluble enzymes. The possibility exists, therefore, that in some of the membrane-bound enzymes the changes might reflect intrinsic changes of the proteins independent of changes in the membrane phospholipids. This hypothesis was investigated using Drosophila mitochondria isolated from wild type and the mutant Ocd^ts-1. In this mutant it has been shown that succinate-cytochrome $\text{c}$ reductase exhibits a change in Arrhenius activation energy at 18°C which is not found in the wild type (Sondergaard, L., Nielsen, N.C. and Smillie, R.M. (1975) FEBS lett. 50, 126–129). A quantitative thin-layer chromatographic analysis of mitochondrial phospholipids showed sphingomyelin to be more abundant in the wild type than in the mutant (5.2% and 4.3% of the total phospholipids, respectively). Since it was shown that the succinate-cytochrome $\text{c}$ reductase had a lipid requirement for full activity, reciprocal rebinding experiments were done. These experiments showed that the reconstituted membranes exhibited the change in activation energy at 18°C only when the protein moiety came from mutant mitochondria, that is, the change was independent of the source of the phospholipids used.     

Interaction of DDT (1,1,1-trichloro-2,2-BIS(p-chlorophenyl)-ethane with liposomal phospholipids

The influence of $\text{1,1,1-}\text{trichloro}\text{-2,2-}\text{bis}\text{(p-}\text{chlorophenyl}\text{)}\text{ethane}$ (DDT) and several other pesticides on the physical state of membrane phospholipids was investigated using model lipids. The thermal dependence of fluorescence intensity of the probe parinaric acid in dipalmitoylphosphatidylcholine liposomes and lipid vesicles of mixed composition were recorded. DDT was incorporated into the liposomal bilayer. The insecticide lowered the phase transition temperature and broadened the temperature range of the transition. The effects were concentration-dependent.The results may be interpreted as a sort of blurred and facilitated phase transition of bilayer lipids caused by intercalation of DDT between fatty acyl chains of membrane phospholipids.     

Phospholipase A inhibition of acetylcholine receptor function in Torpedo californica membrane vesicles

A protein isolated from $\text{Naja naja siamensis}$ venom on the basis of its phospholipase A activity inhibits acetylcholine receptor function in post-synaptic membrane vesicles from $\text{Torpedo californica}$. Specifically, the phospholipase A prevents the large increase in sodium efflux that can normally be induced by carbamylcholine, a receptor agonist. The phospholipase A inhibition shows the following properties: 1) it occurs at concentrations 50 times lower than the concentrations required for inhibition by α-neurotoxins; 2) the phospholipase A has no effect on the binding properties of the receptor; 3) the inhibition is abolished by removal of calcium ions; and 4) some phospholipid hydrolysis accompanies inhibition. It is suggested that the phospholipase A acts enzymatically to uncouple ligand binding from ion permeability in the receptor containing membrane vesicles.     

Calorimetric studies of the structural transitions of the human erythrocyte membrane. Studies of the B and C transitions

Differential scanning calorimetry has been used to study several structural transitions of the human erythrocyte membrane. Earlier studies have shown that one of these transitions (the A transition) is due to the thermal unfolding of spectrin on the membrane. In this paper, it is shown that two of the other transitions (B and C) exhibit a high sensitivity to a local anesthetic, benzyl alcohol. Increasing the ionic strength of the suspending medium results in a splitting of the B transition into two independent transitions (B₁ and B₂). It is found that one of these (B₂) is associated with titrating groups, since the midpoint for the transitions shifts by about 20°C, with an apparent $\text{p}\text{K}$ near 7.5. Extensive bilateral proteolysis by papain causes a drastic decrease in the size of all transitions except the C transition, which remains unaltered. On the other hand, treatment with phospholipase A₂ largely affects the C transition, causing its disappearance. Because of the lack of sensitivity to proteolysis and the high sensitivity to phospholipase, it appears that the C transition has a large extent of ‘lipid involvement’. It might result from the melting of a small fraction of phospholipid which exists in a crystalline state under physiological conditions. Alternatively, the C transition could arise from changes in protein-lipid interactions or from lipid-dependent changes in protein-protein interactions, providing one assumes that only protease-resistant portions of membrane proteins are participating.     

Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18:50:29:2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17,17,6,5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either $\text{N-}\text{acetylglucosamine}$ or $\text{N-}\text{acetylgalactosamine}$) in the molar ratio of 1:2:1:1.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.