Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.

Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

Glycerophosphate acyltransferase (acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase, EC 2.3.1.15) solubilized from Escherichia coli membranes was highly activated by phosphatidylglycerol. Phosphatidylethanolamine, cardiolipin and 1,2-diacyl-sn-glycerol 3-phosphate showed no effect. The Km of the enzyme for sn-glycerol 3-phosphate was increased 20-fold by solubilization. The value could not be restored by the addition of phospholipids. Temperature-sensitive regulation of the synthesis of either 1-palmitoyl- or cis-vaccenoyl-sn-glycerol 3-phosphate by the solubilized enzyme was identical with that by the membrane-bound enzyme in vivo and in vitro. The proportion of the molecular species of 1-acyl-sn-glycerol 3-phosphate varied when the ratios of palmitoyl-CoA and cis-vaccenoyl-CoA were changed, but changes in the sn-glycerol 3-phosphate concentration had no effect on selective acylation by both the solubilized and membrane-bound enzymes.

Temperature-sensitive formation of the phospholipid molecular species in Escherichia coli membranes.

Phospholipids in the membranes of Escherichia coli grown at 37 degrees C are composed of different proportions of molecular species than are those at 17 degrees C. The 1,2-disaturated and 1-saturated-2-unsaturated molecular species increased at 37 degrees C, but the 1,2-diunsaturated species increased at 17 degrees C. When the growth temperature was lowered from 37 to 17 degrees C during the middle exponential growth phase, phosphatidylethanolamine and phosphatidylglycerol composed of proportions of molecular species similar to those found at 17 degrees C were immediately synthesized. By using various membranes composed of different compositions of the phospholipid molecular species, the temperature-sensitive formation of the phospholipid molecular species was found to be independent of the composition of the membrane phospholipids and to be dependent on changes in the specificities of membrane-bound sn-glycerol 3-phosphate acyltransferase against the acyl-CoAs, due to temperature changes.

Function of phosphatidylglycerol molecular species in membranes. Activation of membrane-bound sn-glycerol 3-phosphate acyltransferase in Escherichia coli.

SN-Glycerol 3-phosphate acyltrasferase (EC 2.3.1.15) bound to the elaidate enriched membranes of an unsaturated fatty acid auxotroph of Escherichia coli had a lower specific activity than the acyltrasferase associated with the wild-type membranes. The 1-saturated-2-cis-unsaturated and 1,2-di-cis-unsaturated molecular species of phosphatidylglycerol activated this enzyme. However, these molecular species did not change the original temperature profile obtained by Arrhenius plots of the enzyme activity bound to the elaidate-enriched membranes.

Phosphatidylethanolamine molecular species of fatty acid auxotroph of Escherichia coli grown with elaidate.

Monoacetyldiglycerides derived from the phosphatidylethanolamine molecular species of the fatty acid auxotroph of Escherichia coli grown with elaidate at 37 degrees C were fractionated on thin-layer plates of silica impregnated with silver nitrate and were identified by gas chromatography-mass spectrometry with an OV-17 column and gas chromatography with a Silar-10C column. Phosphatidylethanolamine was made up of the following molecular species: 1-16 : 0-2-16 : 0 (1.2%), 1-14 : 0-2-trans-16 : 1 (1%), 1-16 : 0-2 trans-16 : 1 (3.5%), 1-16 : 0-2-trans-18 : 1 (26.4%), 1-16 : 0-2-cis-16 : 1 (3.8%), 1-trans-18 : 1-2-trans-16 : 1 (13.2%), 1-trans-18 : 1-2-trans-18 : 1 (44.9%), 1-trans-18 : 1-2-cis-16 : 1 (4.5%) and trans-18 : 1-cis-18 : 1 (1.5%).

Spin-labeling of Escherichia coli membrane by enzymatic synthesis of phosphatidylglycerol and divalent cation-induced interaction of phosphatidylglycerol with membrane proteins.

Escherichia coli membrane particulate fraction has been spin-labeled by incubating with sn-glycerol-3-phosphate, CTP, palmitoyl CoA and 12-nitroxide stearoyl CoA. Incorporation of the spin-labeled acyl chain into phosphatidyl-glycerol was confirmed. ESR spectrum of the spin-labeled phosphatidylglycerol in E. coli membrane consisted at least of two components; one due to the labels undergoing rapid anisotropic motions and the other due to strongly immobilized labels (the overall splitting value, approx. 58 G). The relative intensity of the two components was dependent on the concentration of divalent cations. The immobilized component decreased on treatment of the membrane with EDTA and increased on addition of Mg2+ or Ca2+. The spectrum at 1 mM Mg2+ or Ca2+ consisted almost only of the immobilized component. Spin-labeled phosphatidylglycerol in total lipid membrane showed ESR spectrum due to mobile labels and the spectrum was not affected appreciably by the divalent cations. The results suggest the divalent cation-mediated interaction of phosphatidylglycerol with proteins in E. coli membrane. Phosphoenolpyruvate-dependent uptake of methyl-alpha-D-glucoside was markedly accelerated by Mg2+. Ca2+ was not effective for the enhancement. The divalent cation-induced interaction of phosphatidylglycerol with proteins was discussed in relation to the sugar transport.

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels.

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.

Differential effect of phosphatidylethanolamine molecular species on glycerophosphate acyltransferase activity of Escherichia coli.

The effect of phosphatidylethanolamine (PE) molecular species on the reconstitution of partially purified glycerophosphate acyltransferase of Escherichia coli was investigated. The acyltransferase activity was abolished by 1,2-di-unsaturated (U-U) PE, but not by 1-saturated-2-unsaturated (S-U) PE or 1-saturated-2-cyclopropanoyl PE. Since both the U-U and S-U PE used in the present work are in a fluid state at temperatures above about 30 degrees C, the differential effect cannot be accounted for in terms of the membrane fluidity. Therefore, the inactivation of the reconstituted enzyme was attributed to the large amount of the 1,2-di-cis-vaccenoyl species of PE.

Calorimetric study on a dipalmitoylphosphatidylcholine-propylgallate mixture.

The effect of propylgallate (PrG, an antioxidant) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) was studied by means of differential scanning calorimetry. A DPPC/PrG mixture displayed distinctive thermotropic behavior that was significantly different from that of a DPPC/cholesterol or DPPC/vitamin E mixture. Although the enthalpy of the phase transition (delta H) for DPPC decreased at a low concentration of the PrG and the transition peak became broadened, delta H increased again and the peak became sharper on the addition of more PrG. The same was observed for DPPC/methylgallate and DPPC/ethylgallate mixtures, but not for a DPPC/butylgallate mixture. On the other hand, the transition temperature (Tm) of the DPPC/gallate derivative mixtures decreased with an increase in the chain length of the acyl moiety of the gallate derivatives. The pre-transition and subtransition of the DPPC/PrG mixture were eliminated on the addition of a PrG, and Tm of the DPPC/PrG mixture approached about 26 degrees C. These results suggested that the chain length of the acyl moiety must be C1 to C3 for the unique effect of the gallate derivatives described above, and that DPPC forms a complex with PrG as a pure component.

Distribution of phospholipid molecular species in outer and cytoplasmic membrane of Escherichia coli.

The outer membrane of Escherichia coli K-12 contained a smaller proportion of phospholipid molecular species with two unsaturated fatty acyl chains than did the cytoplasmic membrane. Proportions of phospholipid molecular species in the outer and cytoplasmic membranes changed in response to temperature changes. As the temperature increased, the content of 1-palmitoyl-2-cis-9,10-methylenehexadecanoyl species increased. Translocation of phospholipids from the cytoplasmic membrane to the outer membrane and synthesis of various molecular species were observed.

Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen.

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.

Perturbation of phospholipid metabolism by erucic acid in male Sprague-Dawley rat heart.

Erucic acid was incorporated into cardiac phosphatidylserine and hepatic and cardiac sphingomyelin of male Sprague-Dawley rat. The fatty acid compositions of mitochondrial and microsomal phospholipids were similar in both liver and heart. The effect of a low fat diet and a diet containing erucic acid on the fatty acid composition of mitochondrial and microsomal phospholipids was also similar, except for the effect on sphingomyelin. However, the diet containing erucic acid influenced the metabolism of phosphatidylcholine of the heart but not of the liver, indicating that the turnover of 1-stearoyl-2-arachidonoyl phosphatidylcholine in the heart was inhibited by the diet containing erucic acid. On the other hand, the proportion of erucic acid in the free fatty acid was higher in the heart than in the liver.

Kinetics of selective acylation of sn-glycerol 3-phosphate in the synthesis of phospholipid molecular species.

The kinetics of the sn-glycerol 3-phosphate acyltransferase [EC 2.3.1.15] reaction support the view that the selective acylation primarily depends on the differences in the affinity of the enzyme for acyl-CoAs.

Effect of propylgallate on a dipalmitoylphosphatidylglycerol and calcium mixture.

Effect of propylgallate (PrG) on the thermotropic behavior of mixtures of dipalmitoylphosphatidylglycerol (DPPG) and Ca2+ was studied by means of differential scanning calorimetry (DSC). In the case of DPPG or DPPG/Ca (molar ratio, 15 : 1), the transition temperature (Tm) of the main transition and the subtransition decreased from 40 degrees C to 29 degrees C and from 29 degrees C to 20 degrees C, respectively, with an increase in the concentration of PrG. The addition of PrG to the DPPG/Ca mixture induced a shoulder on the high temperature side in the reheating scan. Neither PrG nor low concentrations of Ca2+ bind to the Lc phase of DPPG. When the molar ratio of DPPG to Ca was 1 : 1, the subtransition did not occur, that is, only the main transition (Tm = 90 degrees C) appeared. The Tm of the main transition was slightly affected by PrG. On the addition of PrG, another metastable endothermic transition peak (Tm = 78 degrees C) appeared. It is concluded that Ca2+ and PrG inhibit each other's binding.

Clinical survey of syphilis at the Dermatological Clinic of Nippon Medical School Hospital from 1984 to 1988, with special reference to the recent clinical manifestations and evaluation of IgM antibodies to Treponema pallidum.

One hundred eighty-one patients with syphilis were seen from May 1, 1984, to April 30, 1988 at the Dermatological Clinic of Nippon Medical School Hospital. The incidence of syphilis has increased gradually year by year. The number of early infectious syphilis cases was almost twice as high as late latent syphilis ones. As a source of infection, female prostitutes were noteworthy. Among primary syphilis cases, multiple chancres were observed in 29.2%. The frequency of ulcus durum was much higher than initial sclerosis. A relationship with oral sex is suggested. Among secondary syphilis cases, pruritus was observed in 23.9%, prominently on volar lesions. Psoriasiform papular and macular syphilide were the commonest features. Secondary syphilis with persisting chancres were seen in 41.3% and is gradually increasing. JH reactions were observed in 26.3%. The frequency was highest in late primary and in early secondary stages. IgM-TPHA and IgG-TPHA were tested in 94 sera by gel-filtration and 77 by HPLC. IgM-TPHA tests were reactive in virtually all the sera from untreated syphilis cases. The titres in untreated syphilis were higher than in treated cases. IgM-TPHA/IgM-TPHA + IgG-TPHA was higher in early syphilis than in late syphilis. Fifty-eight untreated cases were tested at frequent intervals after treatment for up to 12 months. IgM antibodies disappeared in 53 patients within 12 months. Non-treponemal antibodies measured by the CF test disappeared within 15 patients and TPHA tests remained positive after 12 months in all patients. IgM-TPHA may support a diagnosis of active syphilis.

Daily intakes of fatty acids, sterols, and phospholipids by Japanese women and serum cholesterol.

The daily intakes of various lipids by 72 Japanese women (40-59 years of age) were measured directly from mock samples of food actually consumed (this method is similar to the duplicate portion method). One sample was collected from each of 12 subjects every 2-months for a period of 1 year. The daily intakes of total fatty acid (FA), cholesterol, plant sterol, phospholipid (PL), and phosphatidylcholine (PC) were 37.9 g, 300 mg, 152 mg, 3.1 g, and 1.7 g, respectively. No effect of the sampling period was found for any lipid measured. The combined eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids showed marked individual variation, and ranged from 0 to 4.3 g, and the average was 0.8 g. The n6/n3 polyunsaturated fatty acids (PUFA) ratio ranged from 0.9 to 19.1 (average, 4.2). There was a strong correlation only between cholesterol and PL intakes (r = 0.796, p < 0.05). The mean serum cholesterol, which was known in 42 subjects was 190 mg/dl, and showed no relation to their daily intakes of any of the measured lipids.

[Daily intakes of nitrate and nitrite in middle-aged men by the duplicate portion method].

Daily intakes of nitrate and nitrite of middle-aged men (30-59 years of age, n = 100) in Hiroshima Prefecture were estimated directly by the duplicate portion method. The daily intake of nitrate was 190.8 +/- 128.5 mg. The daily intake of nitrate/kg body weight was 2.87 +/- 2.00 mg, which is about 78% of the acceptable daily intake (ADI). The daily intake of nitrate tended to increase with increasing age. The daily intake of nitrite was 3.837 +/- 3.647 mg. The daily intake of nitrite/kg body weight was 0.057 +/- 0.050 mg, which is about 95% of the ADI. In the case of nitrite, there was no age-related difference. The proportions of men, whose daily intakes of nitrate and nitrite were above the ADI, were 27% and 34%, respectively. The proportion of men above the ADI of both nitrate and nitrite was 10%.