[Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine].

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.

Intra-thoracic blood volume measurement by contrast magnetic resonance imaging.

The intra-thoracic blood volume (ITBV) is a cardiovascular parameter related to the cardiac preload and left ventricular function. Its assessment is, therefore, important for diagnosis and follow-up of several cardiovascular dysfunctions. Nowadays, the ITBV can be accurately measured only by invasive indicator dilution techniques, which require a double catheterization of the patient. In this study, a novel technique is presented for ITBV assessment by dynamic magnetic resonance imaging after intravenous injection of a small bolus of gadolinium chelate. The dose was chosen on the basis of in vitro calibration. The bolus first pass is detected from a simultaneous dynamic image series of the right and left ventricles. Two indicator dilution curves are derived and used to inspect the transpulmonary dilution system. Various mathematical models for the interpretation of the measured indicator dilution curves are compared. The ITBV is assessed as the product of the transpulmonary mean transit time of the indicator and the cardiac output, obtained by phase contrast magnetic resonance angiography. In vitro measurements showed a correlation coefficient larger than 0.99 and preliminary tests with volunteers proved the feasibility of the method, opening new possibilities for noninvasive quantitative cardiovascular diagnostics.

Pathways of fatty acid metabolism in human platelets.

The metabolic fate of (14)C-labeled fatty acids which have been incubated with human platelets, has been traced. The following has been shown. (a) Intact platelets have a considerable capacity to oxidize fatty acids. (b) When tracer amounts of four of the most common fatty acids in normal plasma were incubated with platelets, each showed a distinctive pattern of uptake among neutral lipids and phospholipids. With regard to the latter, it was shown that these distribution patterns were, in most cases, similar to those of the fatty acids found in natural platelet phospholipids. (c) By increasing the time of incubation or the amount of added oleic acid, the distribution of oleic acid uptake between lecithin and other phosphoglycerides was altered so that a larger share was incorporated into the latter. (d) The effects of added lysolecithin or lysoethanolamine phosphoglycerides on oleic acid incorporation into platelet phosphoglycerides are quite variable. At low concentrations, added lysolecithin functions chiefly as a reaction partner for oleic acid. Added adenosine triphosphate and CoASH augment the incorporation of oleic acid into lecithin over a wide range of added lysolecithin (12.5-500 mumoles/liter). At higher concentrations of added lysolecithin, in the absence of ATP and CoASH, oleic acid incorporation into lecithin is considerably reduced. Also, added lysolecithin and lysoethanolamine phosphoglycerides, in the absence of ATP and CoASH, are able, at certain concentrations, to stimulate oleic acid incorporation into all except the serine phosphoglycerides. (e) Platelets appear to have a de novo pathway for renewal of lecithin.

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

Localization of enzymes involved in glycero-ether bond formation in rat liver.

Studies on the localization of alkyldihydroxyacetone-phosphate synthase in rat liver are described. Less than 5% of the total activity was found in the cytosolic fraction, suggesting that the enzyme is membrane-bound. The ratio of the enzymatic (specific) activity over that of dihydroxyacetone-phosphate acyltransferase, a peroxisomal enzyme, is 10-fold higher in the microsomal fraction, when compared to the peroxisomal fraction. Studying the distribution of the enzyme in a linear density gradient, two activity peaks were found in the peroxisomal and the microsomal fraction indicating a bimodal localization of alkyldihydroxyacetone-phosphate synthase. Rabert et al. (Rabert, U., Völkl, A. and Debuch, H. (1986) Biol. Chem. Hoppe-Seyler 367, 215-222) have presented evidence that the activity in the microsomal fraction was mainly caused by a different enzyme that preferentially converted acyldihydroxyacetone to alkyldihydroxyacetone. We also found a radioactive product, different from alkyldihydroxyacetone-phosphate, upon incubation of microsomal protein in the presence of [14C]hexadecanol. However, it was shown that this product was formed independently of the presence of acyldihydroxyacetone. The product yielded [14C]hexadecanol upon alkaline hydrolysis, clearly showing that it did not contain an ether-bond. Upon incubation of microsomal protein with [14C]palmitic acid and hexadecanol the product was also observed and its chromatographic behaviour resembled that of a synthetically prepared palmitoyl ester of hexadecanol. From these data it was concluded that the product formed is most likely a wax and that the enzyme responsible for this conversion is clearly different from the alkyldihydroxyacetone-phosphate synthase. The implication is that acyldihydroxyacetone-phosphate cannot be replaced by acyldihydroxyacetone in the process of glycero-ether bond formation.

Rat liver dihydroxyacetone-phosphate acyltransferase: enzyme characteristics and localization studies.

Peroxisomes were isolated from rat liver by pelleting a light mitochondrial (L) fraction over a 30% (w/v) Metrizamide layer. Peroxisomes were recovered as a loose pellet from the bottom of the tube and the purity of the peroxisomal fraction was calculated to be about 90%. The characteristics of dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in the light mitochondrial fraction and the purified peroxisomal fraction were compared. The behaviour of the enzyme in the two fractions was very similar, except for the effect of sodium fluoride, which stimulated the activity in the L fraction 5-10-fold and in the peroxisomal fraction only 1.6-fold. This difference could be explained by the action of fluoride-sensitive acid phosphatases present in the L fraction that dephosphorylate palmitoyl-coenzyme A, a substrate for DHAP-AT. The localizations of DHAP-AT and alkyldihydroxyacetone-phosphate synthase in the rat liver peroxisomal membrane were studied. It is shown that in intact peroxisomes, DHAP-AT and alkyl-DHAP synthase are resistant to proteolytic inactivation by trypsin, as is fatty acid beta-oxidation activity, which served as a marker for the intactness of the peroxisomal membrane. Catalase was found not to be a suitable marker to assess peroxisome intactness in view of its relative insensitivity to trypsin. In 1-lauroyllysophosphatidylcholine-permeabilized peroxisomes, DHAP-AT, alkyl-DHAP synthase and beta-oxidation activities were rapidly inactivated by trypsin. It is concluded that in rat liver peroxisomes, at least the active sites of the integral membrane proteins DHAP-AT and alkyl-DHAP synthase are localized exclusively at the inner surface of the peroxisomal membrane.

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques.

1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. Microsomal lysophospholipase activity was completely inhibited by [3H]diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrohporesis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [125I]iodosulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.

Topography of ether phospholipid biosynthesis.

The enzyme, dihydroxyacetone-phosphate acyltransferase (DHAP-AT) is localized at the inner surface of the peroxisomal membrane but shows no latency. The product, i.e., acyl-DHAP, is the first lipidic intermediate in ether lipid biosynthesis that is converted by alkyldihydroxyacetone-phosphate synthase into alkyl-DHAP. This step and the reductions of acyl-DHAP and alkyl-DHAP to the glycerol 3-phosphate analogs can still take place in peroxisomes. The further conversions of these intermediates into ether-linked choline or ethanolamine glycerophospholipids or plasmalogens take place in the endoplasmic reticulum. In this paper we describe studies to localize acyl-DHAP in the transversal plane of the peroxisomal membrane. To enable these studies, the usually employed assay conditions for DHAP-AT were modified by omission of BSA and by lowering the temperature and the palmitoyl-CoA concentration. Using these modified conditions, we were able to label peroxisomes with endogenously generated acyl-DHAP, with retention of catalase within the peroxisomal membrane. Endogenously generated acyl-DHAP was rapidly extractable from the outer surface of peroxisomes with BSA at 0 degree C, suggesting that even at this temperature the product was transported very fast across the membrane. Trypsin treatment of peroxisomes did not affect this behaviour. Only after short incubation periods, an increase in the proportion of non-extractable acyl-DHAP was observed. In large unilamellar vesicles made from peroxisomal phospholipids no transmembrane movement of acyl-DHAP was found. Despite the apparently rapid transbilayer movement of acyl-DHAP in the presence of BSA, it could still serve as a substrate for the enzyme alkyl-DHAP synthase, which is also localized at the inner surface of the peroxisomal membrane. In addition it was shown that endogenously generated substrate is used at a higher efficiency compared to exogenously added acyl-DHAP, suggesting a close interaction of the two enzymes in the peroxisomal membrane.

Cytosol-stimulated remodeling of phosphatidylcholine in rat lung microsomes.

When 600 X g supernatants of 10% (w/v) rat lung homogenates were incubated with CDP[Me-14C]choline both saturated and unsaturated species of phosphatidylcholine were formed from endogenous diacylglycerols. The percentage radioactivity in the disaturated species of total phosphatidylcholine increased with time from 12% after 5 min to 30% after 60 min incubation. In similar experiments with 20 000 X g supernatants, the increase in the disaturated species of microsomal phosphatidylcholine was from 25 to 37% over the same time period. In incubations of isolated microsomes in buffer, the percent of 14C label in disaturated phosphatidylcholine remained constant at a level of 25%. To investigate a possible role of cytosolic factor(s) in the increase in the percentage of disaturated phosphatidylcholine with time, microsomes were prelabeled by incubation in buffer with CDP[Me-14C]choline to give a fixed ratio of radioactive saturated and unsaturated phosphatidylcholine species. When the reisolated microsomes were incubated in buffer, the distribution of radioactivity over saturated and unsaturated species remained constant. In contrast, incubation of prelabeled microsomes in the presence of cytosol caused an increase in the percent radioactivity in saturated phosphatidylcholines from a starting value of 18 to 30% after 60 min incubation, while leaving total phosphatidylcholine radioactivity unaffected. These results indicate a remodeling of phosphatidylcholine under the influence of a cytosolic factor(s). Evidence is presented that suggests that Ca2+-independent-cytosolic phospholipase A2 activity as well as a microsomal ATP-independent CoA-mediated acyltransferase activity might contribute to this remodeling. The cytosol donates the necessary CoA for this acyl transfer as well as saturated acyl-CoA for the reacylation of lysophosphatidylcholine.

Cholesterol in the year 2000.

Cholesterol research was one of the key areas of scientific investigation in the 20th century. Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland in the first part of the century. The structure of cholesterol was completely elucidated in 1932. With the development of isotopic tracers in the 1930s studies on cholesterol biosynthesis were initiated. In 1942 K. Bloch and D. Rittenberg showed that deuterium-labeled acetate was incorporated into the ring structure and side chain of cholesterol. Another important discovery from Bloch's laboratory was that squalene was a precursor of cholesterol. In 1956, the main elements of the biosynthetic pathway became known when isopentenyl pyrophosphate was discovered as a precursor. In 1966, J. Cornforth and G. Popjak predicted that there were 16234 possible stereochemical pathways by which mevalonate could be converted into squalene. They subsequently showed which of these pathways was correct. In the 1970s and 1980s K. Bloch was able to provide intriguing evidence for an evolutionary advantage of cholesterol over lanosterol or some of the intermediates in the conversion of lanosterol to cholesterol. The last quarter of the 20th century was when M. Brown and J. Goldstein showed that the low density lipoprotein receptor was a key regulator of cholesterol homeostasis. They have also demonstrated that cholesterol balance in the cell is transcriptionally regulated via the sterol regulatory element binding protein. In the later part of the 20th century drugs were developed that effectively lower plasma cholesterol and lessen the risk of atherosclerosis and cardiovascular disease.

Does de novo synthesis of lysophosphatidylcholine occur in rat lung microsomes?

Incubation of rat lung microsomes with CDP[Me-14C]choline resulted in formation of radioactive lysophosphatidylcholine and phosphatidylcholine. Evidence is provided which suggests that lysophosphatidylcholine formation cannot be ascribed completely to phospholipase. A degradation of phosphatidylcholine. Lysophosphatidylcholine production can be stimulated by addition of monoacylglycerol or diacylglycerol. It is suggested that diacylglycerol is partly hydrolyzed to monoacylglycerol and subsequently converted to lysophosphatidylcholine. A direct transfer of phosphocholine from CDPcholine to monoacylglycerol is demonstrated by equimolar incorporation of 1(3)-[9,10-3H2]palmitoylglycerol and phospho[Me-14C]choline into lysophosphatidylcholine.

Alkyl-dihydroxyacetonephosphate synthase.

Mammalian ether phospholipids are characterized by a glycero-ether linkage at the sn-1-position of the glycerol backbone. In humans this type of phospholipid species occurs mainly in the ethanolamine and choline phosphoglycerides comprising an estimated 15% of total phospholipids. The glycero-ether linkage is synthesized by replacement of the acyl chain in acyl-dihydroxyacetonephosphate by a long-chain alcohol that donates the oxygen for the ether linkage. Both the enzyme that forms acyl-dihydroxyacetone phosphate (see Chapter II of this volume) and the one that introduces the glycero-ether linkage. i.e. alkyl-dihydroxyacetonephosphate synthase, are located in peroxisomes. The deficiency of ether phospholipids in human inborn errors of metabolism, caused by defects in peroxisome biogenesis, has clearly delineated the indispensable role of peroxisomes in ether phospholipid synthesis. The most characteristic enzyme of ether lipid synthesis is alkyl-dihydroxyacetonephosphate synthase. Its discovery and some of its properties, including mechanistic studies, have been discussed in recent reviews. This review recapitulates these findings and focuses on the new insights into the structure and properties of the enzyme that have recently been obtained resulting from the purification and subsequent cloning and expression of the cDNA encoding this peroxisomal enzyme.

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.

A comparison of acyl-oxyester and acyl-thioester substrates for some lipolytic enzymes.

1. A comparison of 2-hexadecanoylthio-ethane-1-phosphocholine and 3-hexadecanoylthio-propane-1-phosphocholine and their oxyester counterparts as substrates for some lipolytic enzymes was made. 2. The critical micelle concentration and the transition temperature of the synthetic substrates were compared with the values for 1-hexadecanoyl-sn-glycero-3-phosphocholine. 3. All above-mentioned compounds were deacylated by lysophospholipases. Phospholipase A2 hydrolyzed only the acyl- sulfur- and oxygenester bond in 2-hexadecanoyl-ethane-1-phosphocholine. 4. Kinetic parameters, Km and V, for hydrolysis of these substrates were determined. Km values for thioester substrates were 5--10 fold lower than for the corresponding oxyesters. Maximal hydrolysis rates were 2--5 times higher for the thioesters. 5. Hydrolysis of thioesters by phospholipase A2, lipase and lysophospholipase was shown to proceed by an S-acyl cleavage mechanism. 6. Beef liver lysophospholipase II was rapidly and stoichiometrically inactivated by diisopropylfluorophosphate and bis(p-nitrophenyl) phosphate. Inactivation by the latter inhibitor showed burst-like kinetics. 7. Attempts to show burst-kinetics during the pre-steady state hydrolysis of 2-hexadecanoylthio-ethane-1-phosphocholine by lysophospholipase II were negative. These results are interpreted to indicated that a step prior to deacylation of the enzyme is rate-determining.

Studies on lysophospholipases. IV. The subcellular distribution of two lysolecithin-hydrolyzing enzymes in beef liver.

1. In a previous paper (Biochim. Biophys. Acta (1974) 369, 50-63) the purification of two proteins with lysophospholipase activity (EC 3.1.1.5), provisionally denoted lysophospholipase I and lysophospholipase II, has been described. The subcellular localization of both enzymes was investigated by cell fractionation studies. 2. For each subcellular fraction the total lysophospholipase activity, after solubilization by n-butanol treatment, was separated into a lysophospholipase I and II contribution by DEAE-Sephadex ion exchange chromatography. 3. Lysophospholipase I was found to be a soluble enzyme with a bimodal distribution. Highest relative specific activities were measured in the mitochondrial and the cytoplasmic fraction. Evidence is presented indicating that this enzyme is present in the mitochondrial matrix fraction. 4. Lysophospholipase II appeared to be a membrane-bound enzyme with highest relative specific activity in the microsomal fraction.

Characterization of reconstituted partially purified glycerophosphate acyltansferase from Escherichia coli.

A modification of the method of Snider and Kennedy (J. Bacteriol. (1977) 130, 1072-1083) was worked out to solubilize sn-glycero-3-phosphate acyltransferase from whole cells by Triton X-100. The solubilized preparation was used for a systematic study of the reconstitution of enzymatic activity as observed by addition of phospholipid vesicles. Although enzymatic activity was regained by addition of vesicles and not by addition of multilayered liposomes, subsequent Sepharose 4B chromatography revealed the enzyme to be incorporated in large lipid aggregates of undefined structures. Incorporation of glycerophosphate acyltransferase in single bilayer vesicles composed of phosphatidylcholine and phosphatidylglycerol (4:1) was obtained after removal to Triton X-100 from the enzyme solution, co-dispersion of enzyme and phospholipids with cholate and Sephadex G-50 gel filtration of this mixture to remove cholate. The optimal conditions for this reconstitution procedure with respect to phospholipid/protein and phosphatidylcholine/phosphatidylglycerol ratio were established. The active site of glycero-3-phosphate acyltransferase in the reconstituted system was localized for at least 90% at the outside surface of the vesicle, as revealed by proteolysis experiments under conditions of vesicle intactness as shown by C-NMR experiments. The reconstituted systems produced only lysophosphatidate from sn-[14C]glycero-3-phosphate and palmitoyl-CoA and showed identical apparent Km for sn-glycero-3-phosphate and identical pH- and temperature-dependencies as the enzyme in isolated Escherichia coli membranes.

Lysophospholipase activity of bovine adrenal medulla. A reevaluation.

1. Chromaffin granule preparations isolated from bovine adrenal medulla hydrolyzed endogenous lysophosphatidylcholine (lyso-PC) and generated lysophosphatidylethanolamine when dialyzed at pH 7.5. 2. Undialyzed granule preparations hydrolyzed exogenously added [1-14C]palmitoyl-lyso-PC maximally (12-16 nmol/min per mg) at pH 7.5. At a given concentration of protein, activity increased with increasing concentrations of substrate lyso-PC to a maximum beyond which substrate inhibited activity up to 95%. 3. More than 95% of the lysophospholipase activity of fresh granule preparations toward exogenously added lyso-PC was inactivated irreversibly by dialysis. 4. By contrast, fresh microsomal preparations from adrenal medulla had similar substrate requirements for maximal lysophospholipase activity, but more than 35% of the activity was retained at high substrate concentrations or after extensive dialysis. 5. We conclude that adrenal organelles, other than microsomes, contain potent membrane-associated lysophospholipase activity that is inactivated preferentially by high detergent-substrate concentrations and/or dialysis. These observations suggest that lysophospholipase activity (and perhaps phospholipase A activity) is more widely distributed in organelle membranes of the adrenal medulla than was reported previously.