Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers.

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only. The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A₂ from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only.The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A₂ from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm. It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm.     

Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.

In this study we have determined the fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis of rat plasma very low density lipoprotein (rat VLDL). The experiment was carried out in vitro with lipoprotein lipase purified from bovine milk, VLDL labeled with [(14)C]palmitate, [(3)H]cholesterol, [(32)P]phospholipids, and (125)I-labeled apolipoprotein C and in plasma-devoid systems. Triglyceride hydrolysis ranged between 0 and 98.6%. [(32)P]Phospholipids, unesterified [(3)H]cholesterol, and (125)I-labeled apolipoprotein C were removed from the VLDL (d < 1.019 g/ml) during lipolysis. About one-third of the [(32)P]phosphatidylcholine was hydrolyzed to lysolecithin, and was transferred to the fraction d > 1.21 g/ml. The other two-thirds of the phospholipids were removed unhydrolyzed, mainly to the fraction d 1.04-1.21 g/ml. With the progression of the lipolysis, unesterified [(3)H]cholesterol was removed from VLDL at increasing rates, predominantly to the fraction d 1.04-1.21 g/ml. (125)I-Labeled apolipoprotein C removed from the VLDL partitioned between the fraction of d 1.04-1.21 g/ml and d > 1.21 g/ml. Negative-staining electron microscopy of the fraction d 1.04-1.21 g/ml (containing phospholipids, unesterified cholesterol, and apolipoprotein C) revealed many discoidal lipoproteins. [(3)H]Cholesteryl esters remained associated with the VLDL even when 70-80% of the triglycerides were hydrolyzed. These observations suggest that during in vitro lipolysis of VLDL, surface constituents leave the lipoprotein concomitantly with the hydrolysis of core triglycerides. The process of removal of surface constituents is independent of the presence of an acceptor lipoprotein and may occur in the form of a surface-fragment particle. -Eisenberg, S., and T. Olivecrona. Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.     

Phospholipase A2 activity on mixed lipid monolayers: Inhibition and activation of phospholipases A2 from porcine pancreas and Crotalus adamanteus by anionic and neutral amphiphiles

The effects of anionic and neutral amphiphiles on porcine pancreatic and Crotalus adamanteus phospholipases A2 were studied in a monolayer system as a function of surface pressure. The insoluble amphiphile, dicetyl phosphate (DCP), inhibited the hydrolysis of didecanoylphosphatidylcholine (DDPC) by both enzymes below their normal cutoff pressures with pure DDPC. DCP, however, enhanced enzyme penetration and thus activated the pancreatic enzyme above its normal cutoff pressure. The soluble surfactants, 3,5-dibromo- and 3,5-diiodosalicyclate, acetyl salicylate, and salicylic acid, had similar effects. 1,2-Didecanoin inhibited the hydrolysis of DDPC below the normal cutoff pressures and increased the cutoff pressures for both enzymes. Zwitterionic detergents, N-dodecyl- and N-tetradecyl-N,N-dimethyl-3-aminopropanesulfonate, were found to be potent inhibitors of the pancreatic enzyme on DDPC monolayers. Relative substrate specificities for both enzymes were determined as a function of surface pressure with phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid. Pancreatic phospholipase A2 was more active and penetrated to higher pressures with the anionic phospholipids, while the venom enzyme was more active with phosphatidylcholine.     

Membrane-perturbing activity of Viperidae myotoxins: an electrostatic surface potential approach to a puzzling problem

Phospholipase-like myotoxins are a class of proteins present in Viperidae venom. Despite the high level of amino acid and structural homology with soluble phospholipases A(2), myotoxins are devoid of enzymatic activity and share cytolytic activity by means of a totally unknown mechanism involving the lipid bilayer perturbation. The distribution of electrostatic surface potentials of four myotoxins and seven phospholipases A(2) has been compared. The charge distribution is similar in all active non-cytolytic phospholipases with a strongly positive side corresponding to the domain interacting with the micellar substrate and with the opposite side negatively charged. In contrast, all myotoxins examined are positively charged on both sides. Myotoxin III, the only known example of a myotoxin sharing enzymatic activity, displays the same electrostatic surface potential as other related toxins. Using liposomes made with non-hydrolysable phospholipids, we demonstrate that myotoxin III perturbs the lipid bilayer like other myotoxins. Based on these results, a molecular model for myotoxin-membrane perturbing activity is proposed. In this model, potential double-face binding of myotoxic phospholipases A(2) to lipid surfaces could trigger a lipid bilayer destabilization and could generate a stable fusion pore, probably because of the presence of hydrophobic moieties that flank the cationic sites.     

Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis

In order to determine the effects of a plasma phospholipid transfer protein on the transfer of phospholipids from very low density lipoproteins (VLDL) to high density lipoproteins (HDL) during lipolysis, biosynthetically labeled rat 32P-labeled VLDL was incubated with human HDL3 and bovine milk lipoprotein lipase (LPL) in the presence of the plasma d greater than 1.21 g/ml fraction or a partially purified human plasma phospholipid transfer protein (PTP). The addition of either the PTP or the d greater than 1.21 g/ml fraction resulted in a 2- to 3-fold stimulation of the transfer of phospholipid radioactivity from VLDL into HDL during lipolysis. In the absence of LPL, the PTP caused a less marked stimulation of transfer of phospholipid radioactivity. Both the d greater than 1.21 g/ml fraction and the PTP enhanced the transfer of VLDL phospholipid mass into HDL, but the percentage transfer of phospholipid radioactivity was greater than that of phospholipid mass, suggesting stimulation of both transfer and exchange processes. Stimulation of phospholipid exchange was confirmed in experiments where PTP was found to augment transfer of [14C]phosphatidylcholine radioactivity from HDL to VLDL during lipolysis. In experiments performed with human VLDL and human HDL3, both the d greater than 1.21 g/ml fraction and the PTP were found to stimulate phospholipid mass transfer from VLDL into HDL during lipolysis. Analysis of HDL by non-denaturing polyacrylamide gradient gel electrophoresis showed that enhanced lipid transfer was associated with only a slight increase in particle size, suggesting incorporation of lipid by formation of new HDL particles. In conclusion, the plasma d greater than 1.21 g/ml fraction and a plasma PTP enhance the net transfer of VLDL phospholipids into HDL and also exchange of the phospholipids of VLDL and HDL. Both the transfer and exchange activities of PTP are stimulated by lipolysis.     

Surface diffusion in human serum lipoproteins

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.8 X 10(-8) cm2/s, respectively. These values are similar to values reported for diffusion of phospholipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-2H3]phosphatidylcholine incorporated into HDL2 yielded a value similar to that determined by 31P (DT = 1.9 X 10(-8) cm2/s). Slower diffusion coefficients were measured for LDL2 and VLDL. VLDL had a value DT = 9.1 X 10(-9) cm2/s. The diffusion coefficient for LDL2 was 1.4 X 10(-9) cm2/s. Thus, diffusion of phospholipids in LDL2 is a full order of magnitude slower at 25 degrees C than diffusion of phospholipids in the HDLs.     

Is dipalmitoylphosphatidylcholine a substrate for convertase?

Convertase has homology with carboxylesterases, but its substrate(s) is not known. Accordingly, we determined whether dipalmitoylphosphatidylcholine (DPPC), the major phospholipid in surfactant, was a substrate for convertase. We measured [(3)H]choline release during cycling of the heavy subtype containing [(3)H]choline-labeled DPPC with convertase, phospholipases A(2), B, C, and D, liver esterase, and elastase. Cycling with liver esterase or peanut or cabbage phospholipase D produced the characteristic profile of heavy and light peaks observed on cycling with convertase. In contrast, phospholipases A(2), B, and C and yeast phospholipase D produced a broad band of radioactivity across the gradient without distinct peaks. [(3)H]choline was released when natural surfactant containing [(3)H]choline-labeled DPPC was cycled with yeast phospholipase D but not with convertase or peanut and cabbage phospholipases D. Similarly, yeast phospholipase D hydrolyzed [(3)H]choline from [(3)H]choline-labeled DPPC after incubation in vitro, whereas convertase, liver esterase, or peanut and cabbage phospholipases D did not. Thus convertase, liver esterase, and plant phospholipases D did not hydrolyze choline from DPPC either on cycling or during incubation with enzyme in vitro. In conclusion, conversion of heavy to light subtype of surfactant by convertase may require a phospholipase D type hydrolysis of phospholipids, but the substrate in this reaction is not DPPC.     

Phospholipid asymmetry in renal brush-border membranes

The topological distribution of phospholipids between the inside and the outside of rabbit kidney brush-border membranes has been investigated by incubating membrane vesicles with sphingomyelinase, phospholipases A2 from bee venom and hog pancreas, phospholipases C and D, and trinitrobenzene sulfonate. Orientation and integrity of vesicles upon phospholipase treatment was determined by using two monoclonal antibodies recognizing an extracytoplasmic and a cytoplasmic domain, respectively, of the neutral endopeptidase (EC 3.4.24.11). It is shown that the transbilayer distribution of phospholipids is highly asymmetrical in kidney brush-border membranes: sphingomyelin accounted for 75% of the phospholipids present in the external leaflet, whereas phosphatidylethanolamine and phosphatidylserine plus phosphatidylinositol were found to comprise the majority of the inner layer of the membrane.     

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids.

2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did not inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition.

3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols.

4. It is concluded that mitochondrial transhydrogenase, catalyzing the non-energy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.     

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases

The artificial 1,3-diacyl-glycero-2-phosphocholines (1,3-PCs), which form similar aggregate structures as the naturally occurring 1,2-diacyl-sn-glycero-3-phosphocholines (1,2-PCs), were tested as substrates for different classes of phospholipases such as phospholipase A₂ (PLA₂) from porcine pancreas, bee and snake venom, and Arabidopsis thaliana, phospholipase C (PLC) from Bacillus cereus, and phospholipase D (PLD) from cabbage and Streptomyces species. The regioisomers of the natural phospholipids were shown to bind to all investigated phospholipases with an affinity similar to the corresponding naturally occurring phospholipids, however their hydrolysis was reduced to different degrees (PLA₂s and PLC) or even abolished (PLDs belonging to the PLD superfamily). The results are in accordance with binding models obtained by docking the substrates to the crystal structures or homology models of the phospholipases.     

Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.     

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.     

Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [J.J. Agren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223-6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.     

Changes in the phospholipid catabolism of mitochondria and microsomes during the development of rat liver.

The lipolytic activities of mitochondrial and microsomal fractions ('microsomes') isolated from foetal, suckling and adult rat liver were compared. The catabolism of endogenous phospholipids was followed by measuring the loss of phospholipids and the appearance of non-esterified fatty acids and lysophosphatides. The rate of mitochondrial phospholipid catabolism does not change significantly during development, but the rate of lipolysis of microsomal phospholipids increases 3-fold during development. Balance studies showed that, in mitochondria and microsomes of foetal, suckling and adult rat liver, fatty acid formation is greatly in excess of the fatty acids that can be accounted for by measuring phospholipid disappearance and lysophosphatide appearance. The hypothesis that this excess fatty acid formation resulted from the lipolysis of mitochondrial and microsomal triacylglycerols were tested and confirmed by preliminary experiments. Mitochondria and microsomes isolated from all developmental ages investigated had phospholipases with A1 and A2 activities. The degree of unsaturation of the fatty acids derived from the phospholipids of mitochondria did not vary significantly during development.     

Asymmetry of arachidonic acid metabolism in the phospholipids of the human platelet membrane as studied with purified phospholipases

Human platelets were incubated with high density lipoproteins (HDL) doubly labelled with either free [14C]arachidonate/[3H]arachidonoylphosphatidylcholine or free [14C]oleate/[3H]oleoylphosphatidylcholine. Whereas [14C]arachidonate was incorporated at a 10-15-times higher rate than [14C]oleic acid, the exchange of both species of phosphatidylcholine occurred to the same extent. In both cases, free 3H-labelled fatty acids were generated during the labelling procedure, indicating phospholipase A2 hydrolysis. A redistribution of radioactivity to other phospholipids was noted after exchange of [3H]arachidonoylphosphatidylcholine only. (2) The exchange of phosphatidylcholine to platelets was confirmed using [14C]choline-labelled dipalmitoyl-and 1-palmitoyl-2-arachidonoylphosphatidylcholines. (3) Non-lytic degradation of platelet phospholipids by phospholipases revealed that free fatty acids were incorporated at the inside of the cells, whereas exchange was taking place on the platelet outer surface. However, 2-arachidonoylphosphatidylcholine displayed a more rapid movement towards the cell inside. The above findings suggest a topological asymmetry for the two pathways (acylation and exchange) of fatty acid renewal in platelets. The possible mechanisms and physiological relevance of the translocation of the external arachidonic acid pool across the membrane are discussed.     

Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions

Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.     

The role of intramembrane Ca2+ in the hydrolysis of the phospholipids of Escherichia coli by Ca2+-dependent phospholipases

Ca2+-dependent phospholipases A require Ca2+ concentrations in the millimolar range for optimal activity toward artificial substrates. Because Ca2+-dependent phospholipases A2 degrade the phospholipids of Escherichia coli, treated with the membrane-active antibiotic polymixin B equally well with and without added Ca2+ (Weiss, J., Beckerdite-Quagliata, S., and Elsbach, P. (1979) J. Biol. Chem. 254, 11010-11014), we have examined the possibility that intramembrane Ca2+ can provide the Ca2+ needed for phospholipase action. We studied the effect of Ca2+ depletion on the hydrolysis of the phospholipids of polymixin B-killed E. coli by 1) added pig pancreas phospholipase A2 in E. coli S17 (a phospholipase A-lacking mutant) and 2) endogenous Ca2+-dependent phospholipase A1 in the parent strain E. coli S15. Transfer of E. coli from nutrient broth (Ca2+ concentration approximately 3 X 10(-5) M) to Ca2+-depleted medium (Ca2+ concentration less than 10(-6)M) reduced polymixin B-induced hydrolysis by 50-75%, in parallel with a reduction of bacterial Ca2+ from 19.6 +/- 2.8 to 3.9 +/- 0.6 nmol (mean +/- standard error) per 3 X 10(10) bacteria. The bacterial Ca2+ content was repleted and the sensitivity of the bacterial phospholipids to hydrolysis by both exogenous phospholipase A2 (E. coli S17) and endogenous phospholipase A (E. coli S15) was restored by adding Ca2+ back to the suspensions. Complete restoration occurred at low Ca2+ levels in the reaction mixture (3 X 10(-5) - 10(-4) M) and required time, suggesting that hydrolysis was restored because bacterial Ca2+ stores were gradually replenished and not because extracellular Ca2+ concentrations were raised to levels that were still at least 10X lower than needed for optimal phospholipase A activity. This conclusion is supported by the finding that Ca2+ depletion or addition caused respectively decreased and increased release of lipopolysaccharides by EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), suggesting that the bacterial Ca2+ pool bound to lipopolysaccharides in the outer membrane shrinks or expands depending on extracellular Ca2+ levels. Thus, the cationic membrane-disruptive polymixin B, thought to compete with Mg2+ and Ca2+ for the same anionic sites on lipopolysaccharides, may liberate the Ca2+ near where the phospholipids are exposed to phospholipase.     

Age‐Related Changes in Fatty Acids in Obese Offspring of Streptozotocin‐Induced Diabetic Rats

Objective: The long‐term effects of fetal hyperinsulinemia, time course of changes in liver and very‐low‐density lipoprotein (VLDL) lipid levels and fatty acid compositions were investigated in obese offspring of streptozotocin‐induced mildly diabetic rats. Research Methods and Procedures: Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on day 5 of gestation. Control pregnant rats were injected with citrate buffer. Liver and VLDL lipids and fatty acids were analyzed in offspring at different ages. Results: At birth, obese pups had higher VLDL triglyceride levels, saturated fatty acids, and C20:4n‐6. They also had lower C18:2n‐6 proportions in VLDL triglycerides, phospholipids, and cholesteryl esters than controls pups. In 1‐month‐old male and female obese rats, VLDL and liver lipid amounts were similar to those in their respective controls; however, high levels of C18:2n‐6 and C20:4n‐6 were noted in liver and VLDL lipids. At the age of 2 months, liver and VLDL triglyceride levels were higher in obese females than in control females. Fatty acid abnormalities seen in obese rats included low C18:3n‐3 and high C22:6n‐3 proportions in liver triglycerides and phospholipids. At the age of 3 months, obese rats, both males and females, compared with control animals, had higher VLDL and hepatic lipids with reduced C20:4n‐6 levels and polyunsaturated/saturated fatty acids ratios in hepatic and VLDL triglycerides and phospholipids. Discussion: Fetal obesity, associated with alterations in VLDL lipid fatty acid composition, represents an important risk factor for adult obesity and diabetes.