α-Hydroxylation of Fatty Acids in Brain: Characterization of Heat-Labile Factor

Abstract: The particulate fraction, heat-labile factor, heat-stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (α-hydroxylignoceric acid). The heat-labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD-Agarose affinity chromatography. The specific activity of the heat-labile factor was increased 105-fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS-polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat-labile factor was incubated with 1 mM- N -ethylmaleimide. This inhibition was prevented by preincubating the heat-labile factor with 1 mM-NADH. These observations indicate that the heat-labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.     

Mechanism and stereospecificity of alpha-hydroxylation of lignoceric acid in rat brain.

Cerebronic acid (2-hydroxytetracosanoic acid) is the major fatty acid component of cerebrosides and sulfatides in mammalian brain. Our previous communication demonstrated the synthesis of cerebronic acid from lignoceric acid (tetracosanoic acid) by a rat brain preparation in the presence of molecular oxygen and a reduced pyridine nucleotide (Hoshi, M., and Kishimoto, Y. (1973) J. Biol. Chem., 248, 4123–4130). The present'studies on the conversion of (RS)-[2-³H]-, (RS)-[3-³H]-, (R)-[2-³H]-, and (S)-[2-³H]lignoceric acids to cerebronic acid by rat brain preparations establish that the pro-R hydrogen at the α-carbon of lignoceric acid is replaced by a hydroxyl group with overall retention of configuration.     

α-Hydroxylation and oxidation of lignoceric acid in brain: The role of heat-stable and heat-labile factors

Our previous investigations disclosed that the heat-stable and heat-labile factors obtained from brain cytosol are required for -hydroxylation and oxidation of lignoceric acid by rat brain particulate fraction. The heat-stable factor was recently found to contain glucose-6-phosphate, N-acetylaspartate, glutamate, aspartate, glutamine, inorganic phosphate and low levels of adenosine nucleotide as active components. A combination of these compounds was as effective as the crude heat-stable factor for enzymic activity. Using these compounds, we reinvestigated the requirement for the heat-labile factor. With crude heat-stable factor there was an absolute requirement for the heat-labile factor; however, with various combinations of the individual components of the heat-stable factor, some degree of activity was obtained without the heat-labile factor. When aspartate or one of its derivatives, N-acetylaspartate or oxaloacetate, was used in place of the heat-stable factor, the activity was relatively low but highly stimulated by the addition of heat-labile factor. On the other hand, higher activity was obtained when glutamate or one of its derivatives, glutamine or -ketoglutarate, was used without heat-labile factor. The addition of heat-labile factor to this system did not stimulate the activity. When studying the aspartate family, we discovered that the requirement for the heat-labile factor varied in a descending order: N-acetylaspartate > aspartate > oxaloacetate. Lignoceric acid oxidation was further characterized with rat brain particulate fraction, NADPH, Mg²⁺, glutamate, inorganic phosphate, and AMP without heat-stable and heat-labile factors. It was found that the requirement for NADPH was also partially eliminated with glutamate but not aspartate. The effects of various inhibitors, such as inhibitors of the electron transfer system, oxidative phosphorylation, the enzymes involved in citric acid cycle, and glycolysis, suggest that the heat-stable factor is involved in producing ATP or other high energy compounds to be used for the activation of lignoceric acid. ATP added to the system in place of heat-stable factor resulted in less than one-half of the lignoceric acid oxidation.     

Alpha hydroxylation of lignoceric acid to cerebronic acid during brain development. Diminished hydroxylase activity in myelin-deficient mouse mutants.

Alpha Hydroxylation of lignoceric acid (n-tetracosanoic acid) to cerebronic acid (2-hydroxylignoceric acid) by postnuclear preparations of brains from developing rat, mouse, and several neurological mouse mutants was studied. The preparations of brains from jimpy and myelin synthesis deficiency (msd) mice were found to synthesize cerebronic acid at less than 10 percent of their control rates, and those from quaking and dilute-lethal approximately 30 and 50 percent, respectively. The apparent low rate of in vitro hydroxylation by brains of the mutant mice appeared to be due to decreased synthesis rather than increased oxidation of cerebronic acid. Mixing experiments eliminated the possibility of an inhibitor in the mutant or an activator in normal animals. The preparations of brains from wabbler-lethal, ducky, and weaver mice showed normal activity. The developmental pattern of the hydroxylase activity was examined in quaking, jimpy, and their control mice. In normal brains the hydroxylase activity was low in the immediate postnatal period, increased sharply between 10 and 20 days after birth, and fell to a low level following maturation of the brain. The hydroxylase activity in quaking mice changed similarly during brain development but at a much reduced level. The brains of jimpy mice had barely detectable hydroxylase activity which changed little with age and reached a peak at about 15 days postpartum. The subnormal hydroxylase activity in brains of quaking mice and the near absence in brains of jimpy and msd mice correlate with the observations that myelin deficiency is more severe in jimpy and msd than in quaking. These results suggest a close association of the synthesis of cerebronic acid with the synthesis of the characteristic myelin lipid that is cerebroside (N-acyl sphingosine beta-D-galactoside).     

Ceramide Synthesis from Free Fatty Acids in Rat Brain: Function of NADPH and Substrate Specificity

At the subcellular level, the synthesis of ceramide from free lignoceric acid and sphingosine in brain required reconstituted enzyme system (particulate fraction, heat-stable and heat-labile factors) and pyridine nucleotide (NADPH). The mitochondrial electron transfer inhibitors (KCN and antimycin A), energy uncouplers (oligomycin and 2,4-dinitrophenol), and carboxyatractyloside, which prevents the transport of ATP and ADP through the mitochondrial wall, inhibit the synthesis of ceramide in the presence of NADPH but have very little effect in the presence of ATP. Similar to the synthesis of ceramide, the synthesis of ATP from NADPH and NADH by the particulate fraction also required cytoplasmic factors (heat-stable and heat-labile factors). Moreover, ATP, but not its analog (AMP-CH2-P-O-P), can replace NADPH, thus suggesting that the function of the pyridine nucleotide is to provide ATP for the synthesis of ceramide. The cytoplasmic factors were not required for the synthesis of ceramide in the presence of ATP. The maximum velocity for synthesis of ceramide from free fatty acids of different chain lengths (C16-C26) was bimodal, with maxima around stearic acid (C18) and behenic acid (C22). The relative rate of synthesis of ceramide parallels the relative distribution of these fatty acids in brain cerebrosides and sulfatides.     

A novel synthesis of ceramide from lignoceric acid and sphingosine by rat brain preparation; the amide formation requires a pyridine nucleotide.

The conversion of free lignoceric acid and sphingosine to lignoceroyl sphingosine (ceramide) by rat brain particulate fraction and two cytosolic factors, one heat-stable and the other heat-labile, requires pyridine nucleotide. This enzymatic reaction is apparently different from two previously published enzymic reactions, microsomal sphingosine:acyl CoA acyltransferase and the reverse reaction of lysosomal ceramidase. The reaction is strongly inhibited by common respiratory chain inhibitors, KCN, Antimycin A and sodium azide, this indicates the involvement of an electron-transfer system. From these observations it appears that the brain ceramide synthesis described above is catalyzed by an enzyme system which involves a mechanism for amide formation which has not been previously characterized.     

The Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy

The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1 +/- 0.8 to 19 +/- 2 (nmol X cm-2 X min-1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.     

诺丽果与热带水果中氨基酸含量及组成对比分析

以诺丽果和16种海南常见热带水果为材料,对比分析了氨基酸含量及组成。结果表明:诺丽果含有18种氨基酸,种类齐全。其氨基酸总量、人体必需氨基酸含量和儿童必需氨基酸含量均居第1位。诺丽果的E/T值为37.13%,E/N值为0.59,符合理想蛋白质的要求。诺丽果中各种人体必需氨基酸,Val、Ile、Leu、Phe+Tyr、M et+Cys、Thr的含量占氨基酸总量的比例,与1973年FAO/WHO修订的人体必需氨基酸含量模式谱基本一致,仅Lys中度缺乏。诺丽果中鲜味类、芳香族、甜味类氨基酸含量居第1位。     

Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine, parasitic nematode, Ascaris suum

The acidic glycolipid fraction (AF) of the porcine, parasitic nematode, Ascaris suum , consisted of two subfractions. The major component AF II reacted with orcinol-sulfuric acid and molybdate, while the minor component AF I gave a positive reaction with azure-A, a cationic dye specific for sulfatides. Sugar constituent analysis, methanolysis, methylation analysis, matrix-assisted laser desorption/ionization time- of-flight mass spectrometry, liquid secondary-ion mass spectrometry, and gas-liquid chromatography/mass spectrometry specified AF II to be an unusual phosphoinositolglycosphingolipid (Galalpha1-Ins-P-1ceramide) and the minor component AF I to be a 3-sulfogalactosylcerebroside (HSO3- 3Galss1-1ceramide). The ceramide moiety of both components consisted of lignoceric (C24:0) and cerebronic (C24h:0) acids and mainly C17 iso- branched sphingosine. Immunohistochemical localization studies of the glycolipid-bound antigenic determinants with a polyclonal antiserum against AF II and an anti-sulfatide monoclonal antibody against AF I revealed the presence of the AF II-epitope in the intestine, whereas the AF I-epitope was found in the hypodermis, contractile zone of somatic muscle cells and the external musculature of the uterus. To our knowledge, this is the first report of the presence of a sulfatide in an invertebrate.      

Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii.

Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.     

超声波对谷氨酸结晶过程影响的研究

利用超声波这一能量场对谷氨酸结晶过程进行了研究,通过对谷氨酸溶液的表面张力和电导率测定,分析了超声波场影响谷氨酸溶液结晶机理。实验结果表明:超声波可提高谷氨酸溶液的结晶速率,并可改善谷氨酸晶体颗粒质地。在超声波功率为50 w条件下3 min内谷氨酸结晶速率可达到90%以上,比未经超声波处理同等条件下提高70%。     

产有机酸采油菌的筛选及产酸情况的分析

目的:从大庆油田原油样品中筛选出2株产有机酸量较高的菌株,并对其产物进行分析.方法:根据形态特征、生理生化性质和16S rDNA序列分析对菌株进行鉴定,并运用GC/MS法对发酵液进行分析.结果:经鉴定这两株为枯草芽孢杆菌,菌株T10 -3的发酵液中含有乙酸11.407％,异丁酸9.375％,丁二醇79.217％;菌株DH -2 -l发酵液的中含有异丁酸41.56％,丁二醇46.619％,异戊酸4.138％,异庚酸10.680％.结论:这两株细菌在微生物采油方面均有良好的应用前景.     

Inactivation of phosphorylase phosphatase by a factor from rabbit liver and its chemical characterization as glutathione disulfide.

A factor inactivating phosphorylase phosphatase was isolated from rabbit liver. The isolation procedure consisted of heat treatment at 85 degrees C, extraction with n-butyl alcohol, and chromatography on Dowex 1 and DEAE-cellulose columns. The purified factor was different from the known protein inhibitors and was shown to be tripeptide composed of equimolar amounts of glutamic acid, cysteine, and glycine. The NH2-terminal and COOH-terminal amino acids were determined as glutamic acid and glycine, respectively. The factor was finally identified as glutathione disulfide by high voltage paper electrophoresis, paper chromatography, and liquid column chromatography using an amino acid analyzer. Addition of the purified factor or glutathione disulfide converted phosphorylase phosphatase to a stable, less active enzyme species, the extent of conversion depending on the amount added. The inactivated phosphatase was completely reactivated by addition of both glutathione (or 2-mercaptoethanol) and Mn2+ and partially reactivated by adding glutathione alone. Injection of glutathione disulfide into the portal vein of rabbits caused a rapid increase in phosphorylase alpha activity in the liver. These results suggest that glutathione disulfide is involved in regulation of phosphorylase activity in vivo, by causing inactivation of phosphorylase phosphatase in the liver.     

Heat-stable proteins and abscisic Acid action in barley aleurone cells

[³⁵S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100°C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered.     

D-焦谷氨酸的研制

研究了Ｄ－焦谷氨酸的制备,提纯精制以及分析化验等方面的有关问题。     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Isolation of a biologically active low-molecular-mass chromium compound from rabbit liver

A low-molecular-mass chromium-binding substance (LMCr), which is recognized as a detoxification ligand of chromium, was isolated from the livers of rabbits injected intravenously with K2Cr2O7 (200 mumol Cr/kg body wt) as a biologically active form. LMCr appears as an anionic, organic Cr compound with a relative molecular mass of 1500. It is composed of glutamic acid or glutamine, glycine, cysteine and aspartic acid or asparagine with a Cr/amino-terminal residue ratio of 4:1. The purified LMCr (10-300 ng Cr/ml) shows in vitro activities comparable to those of glucose tolerance factor in relation to insulin action. In the presence of insulin it enhances [U-14C]glucose conversion to 14CO (23-30% up) in rat epididymal adipocytes above the value obtained with insulin alone. LMCr also stimulates the rate of [3-3H]glucose incorporation into lipid by 30-40% with insulin or by 15-23% without insulin, as compared with the basic value obtained with insulin alone or without insulin. These findings suggest that LMCr plays essential roles in both glucose metabolism and detoxification of invaded Cr in the body.     

Influence of Glutamic Acid Enantiomers on C‐mineralization

Seasonal dynamics in the mineralization of glutamic acid enantiomers in soils from selected ecosystems was determined and subjected to a range of treatments: ambient x elevated CO₂ level and meadow x dense x thinned forest environment. Mineralization of glutamic acid was determined by incubation of the soil with 2 mg L‐ or D‐glutamic acid g^‐1 of dry soil to induce the maximum respiration rate. Mineralization of glutamic acid enantiomers in soils fluctuates over the course of a vegetation season, following a similar trend across a range of ecosystems. Mineralization is affected by environmental changes and management practices, including elevated CO₂ level and thinning intensity. L‐glutamic acid metabolism is more dependent on soil type as compared to metabolism of its D‐enantiomer. The results support the hypothesis that the slower rate of D‐ compared to L‐ amino acid mineralization is due to different roles in anabolism and catabolism of the soil microbial community. Chirality 27:104–108, 2015. © 2014 Wiley Periodicals, Inc.