A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin.

Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.     

Utilization of phosphate diester by the marine diatom Chaetoceros ceratosporus

Utilization of phosphate diester (PDE) and phosphodiesterase(PDEase) production by five marine phytoplankton species wereexamined in the laboratory to evaluate the contribution of PDEto the growth of phytoplankton. Among the five marine phytoplanktonspecies tested, only Chaetoceros ceratosporus was able to usethe PDE compound, bis(p-nitrophenyl) phosphate (bis-NPP), effectivelyas a sole phosphorus source. In addition, C. ceratosporus simultaneouslyproduced both PDEase and alkaline phosphatase (APase) at almostequal activity levels under the phosphate-deficient condition.These results suggest that PDE compounds presumably play animportant role as a phosphorus source for PDEase-producing phytoplanktonin coastal environments.     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)     

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.     

Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.