Maintenance of respiratory control by beef heart mitochondria incubated at 25 degrees C: response to protective agents and to protective agents and to prior stress.

Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphoethanolamine and 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphocholine to yield 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1-¹⁴C]hexadecyl-sn-glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate was ruled out. Neither 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine nor 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg²⁺, is inhibited by Ca²⁺, and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem., 249, 1742–1746).     

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803^T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803^T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.     

Properties of microsomal phospholipases in rat liver and hepatoma.

Phospholipase A1, A2 and lysophospholipase activities in microsomes of Novikoff hepatoma host rat liver and regenerating rat liver were compared using 1-[9', 10'-3H2]palmitoyl-2-[1'-14C] linoleoyl-sn-glycero-3-phosphoethanolamine, 1-[1' -3H-]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and 1-[9', 10'-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca2+ for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca2+ for activity at both pH values. 3. Phospholipase A1 activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A1 activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using 1-[1'-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2+ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.     

Aromatic ring cleavage of β-O-4 lignin model dimers without prior demeth(ox)ylation by lignin peroxidase

Methyl oxalate of arylglycerol was formed as an aromatic ring cleavage product in degradation of arylglycerol-β-aryl ether (β-O-4) type lignin substructure model dimers by extracellular lignin peroxidase of Phanerochaete chrysosporium. The enzymatic cleavage of arylglycerol-β-(o-[²H₃]methoxyphenyl) ether indicated that the methyl group of the methyl ester was derived from the methoxy group of the β-O-4 model dimer. It is thus concluded that demeth(ox)ylation was not essential for the enzymatic aromatic ring cleavage of the methoxylated aromatic substrates, β-O-4 lignin substructure models.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Enzymatic esterification of vitamin a in the pigment epithelium of bovine retina

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-³H]retinol and [³H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 μg protein and for the first 10–15 min. The apparent K_m values were 16.6 · 10^?6 and 5.5 · 10^?6 M for [³H]retinol and bound [³H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic stransformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated.Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, onlt intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

Conversion of diglyceride to triglyceride by rat liver microsomes: a requirement for the 105,000 x g supernatant

This paper describes a requirement for the 105,000 × g supernatant of rat liver for the synthesis of triglyceride from diglyceride and palmityl coenzyme A by rat liver microsomes. ATP and magnesium chloride are also required. The incorporation of both [1-¹⁴C]-palmityl coenzyme A and [1-¹⁴C]-diolein into triglyceride has been observed. The 105,000 × g supernatant has no enzymatic activity for this reaction when incubated in the absence of microsomes. The supernatant contains a soluble, essential protein which is nondialyzable, heat sensitive, and destroyed by trypsin. Net synthesis of triglyceride has been demonstrated by chemical analysis.     

Synthesis of 1,3,4,6-tetra-O-acetyl-2-[3-alkyl-(aryl)-thioreido]-2-deoxy-α-d-glucopyranoses and their transformation into 2-alkyl(aryl)amino-(1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazolines

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-isothiocyanato-α-d-glucopyranose, produced from 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α-d-glucopyranose hydrochloride, thiophosgene, and calcium carbonate, was condensed with alkyl- and aryl-amines in ether to afford the crystalline 1,3,4,6-tetra-O-acetyl-2-[3-alkyl(aryl)-thioureido]-2-deoxy-α-d-glucopyranoses (2). Compounds 2 and the β anomers 3 were converted in high yield into 2-alkyl(aryl)amino-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline hydrobromides (4) by hydrogen bromide-promoted cyclisation. The O-deacetylated thiazoline hydrobromide 5 was also isolated and converted into 2-[N-(4-methoxyphenyl)acetamido]-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline (8). Conformational studies of 4 and 8 were made by ¹H-n.m.r. spectroscopy.     

Kinetics of protein-mediated transfer of rat pancreatic microsomal phosphatidylinositol to liposomes

Pancreatic microsomes were isolated from fasted and pilocarpine-injected rats and the microsomal phosphatidylinositol radiolabelled with myo-[2-³H]inositol by isotopic exchange. A standard reaction mixture was established in which partially purified rat liver phosphatidylinositol exchange proteins sustain a maximal rate of phosphatidylinositol transfer from rat pancreatic microsomes to liposomes. Determination of the transfer kinetics shows (1) that pancreatic microsomal phosphatidylinositol is partitioned approximately equally between a non-exchangeable and a single exchangeable pool and (2) that cholinergic stimulation does not significantly change the relative sizes of the two pools nor the exchange half-life of the latter pool.     

Sugars from Sphacelia sorghi honeydew

Several unusual oligosaccharides have been isolated from the honeydew of Sphacelia sorghi McRae. These include 1-O-β-D-fructofuranosyl-D-mannitol, 5-O-β-D-fructofuranosyl-D-arabinitol, 1,6-di-O-β-D-fructofuranosyl-D-mannitol, 1,5-di-O-β-D-fructofuranosyl-D-arabinitol, and 1-O-β-D-fructofuranosyl-6-O-[β-D-fructofuranosyl-(2→6)-β-D-fructofuranosyl]-D-mannitol. In addition to these oligosaccharides, D-glucose, D-fructose, D-arabinitol, D-mannitol, sucrose, and 6-O-β-D-fructofuranosyl-D-glucose were also found in the honeydew. The structures of the previously undescribed oligosaccharides were determined by periodate oxidation studies, their cleavage by β-D-fructofuranosidase, optical rotation measurements, and methylation analysis by combined gas-liquid chromatography-mass spectrometry. The position of linkage in the arabinitol-containing disaccharide was determined by incorporation of D-[1-³H]-arabinitol into a β-D-fructofuranosyl-D-arabinitol in vivo. The release of tritium-labeled formaldehyde during periodate oxidation of the product demonstrated that the β-D-fructofuranosyl moiety was linked to position 5 of the D-[1-³H]-arabinitol.     

Stereochemical aspects of the hydration of trans-anethole epoxide in the rat

Racemic trans-anethole epoxide [1-(4′-methoxyphenyl)-propane-1,2-oxide] was incubated with water, buffers, and rat liver microsomes and cytosol and the stereochemistry of the diols produced was determined by HPLC as their dicamphanyl esters. The diol metabolites were isolated by HPLC from the urine of rats administered [1′-¹⁴C] trans-anethole and their stereochemistry determined after derivatization to their camphanyl esters. The stereochemical course of the metabolism of trans-anethole by rat liver microsomes and cytosol is discussed. © 1995 Wiley-Liss, Inc.     

Galactosyltransferase activities in pancreas, liver and gut of the developing rat

Two galactosyltransferase activities (1 and 2) were measured in the pancreas, liver and gut of the developing rat embryo. 1. N-Acetylglucosamine:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylglucosamine → [¹⁴C]galactosyl-β-(1 → 4)-N-acetylglucosamine + UDP. 2. N-Acetylgalactosamine-protein:Galactosyltransferase. UDP [¹⁴C]galactose + N-acetylgalactosamine-protein → [¹⁴C]galactosyl-β-(1 → 3)-N-acetylgalactosamine-protein + UDP. Galactosyltransferases 1 and 2 increased in the pancreas, about 10- and 40-fold in specific activity, respectively, from 11 to 12 days in utero to birth. During this period the activities of both transferases in the liver were somewhat variable, but showed no definite trend. A drop in the level of galactosyltransferase 1 in the pancreas occurred at birth or shortly thereafter. The “Golgimarker” enzyme for liver, galactosyltransferase 1, may be absent or present at low levels in adult rat pancreas.Zymogen granule membrane preparations apparently are devoid of these galactosyltransferase activities. Bromodeoxyuridine, which inhibits the development of the synthetic capability of the specific exocrine proteins, had essentially no effect on the normal accretion of the galactosyltransferase activities in organ cultures of pancreatic rudiments from 13-day rat embryos.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

Metabolic activation of hexamethylmelamine and pentamethylmelamine by liver microsomal preparations

Incubation of [¹⁴C]-ring labeled hexamethylmelamine and pentamethylmelamine with rat and mouse liver microsomal preparations results in metabolic activation of both drugs as measured by covalent binding of radiolabel to acid-precipitable microsomal macromolecules. Covalent binding is dependent on viable microsomes, NADPH, and molecular oxygen. Binding of HMM (280 pmol/mg protein/15 min) was approximately 5 times greater than that observed for PMM (60 pmol/mg protein/15 min), and represents 0.22% of incubated material. Similar results were found with [¹⁴C]-methyl labeled substrates. Pretreatment with phenobarbital increased covalent binding while addition of SKF 525-A, addition of glutathione, or incubation in an 80% carbon monoxide atmosphere reduced covalent binding.