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Y Kamisaka

Publications and source records attributed to Y Kamisaka.

At least 19 recordsLinked to original sources
PubMed2004

Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence.

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24-0.47 microg/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5-10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2-5000 microg-lipid/ml-broth.

Biomass↗
PubMed2001

Purification and characterization of thiol-reagent-sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus.

Glycerol-3-phosphate acyltransferase (GPAT), responsible for the first committed, rate-limiting, step of glycerolipid synthesis, was purified to homogeneity from the membrane fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the membrane fraction by pretreatment with 0.05% Triton X-100 and treatment of the resulting pellet with 0.3% Triton X-100. The enzyme was subsequently purified by column chromatography on heparin-Sepharose, Yellow 86 agarose, a second heparin-Sepharose column, Superdex-200 and hydroxylapatite Bio-Gel. Enzyme activity was finally enriched 1308-fold over that of the starting membrane fraction. SDS/PAGE of the purified fraction revealed a single band with a molecular mass of 45 kDa. Native PAGE showed a major band that corresponded to GPAT activity. Enzyme activity was inhibited by thiol reagents, suggesting that it originated from microsomes rather than mitochondria. Purified GPAT depended on exogenous oleoyl-CoA and sn-glycerol-3-phosphate, with the highest activity at approx. 50 and 250 microM, respectively, and preferred oleoyl-CoA 5.4-fold over palmitoyl-CoA as an acyl donor. Anionic phospholipids, such as phosphatidic acid and phosphatidylserine, were absolutely required for activity of the purified enzyme, and their ability to activate GPAT was influenced by the purity of the GPAT preparation. Bivalent cations, such as Mg(2+) and Ca(2+), inhibited purified GPAT activity, whereas 5 mM Mn(2+) elevated activity approx. 2-fold. These results provide new insights into the molecular characterization of microsomal GPAT, which has not been well characterized compared with mitochondrial and plastidic GPAT.

Cell Membrane↗
PubMed2001

Ontogeny of cholecystokinin-immunoreactive cells in the digestive tract of Atlantic halibut, Hippoglossus hippoglossus, larvae.

The appearance and distribution of cholecystokinin (CCK)-producing cells were investigated in the digestive tract of developing larvae of cultured Atlantic halibut, Hippoglossus hippoglossus. The CCK-producing cells were detected immunohistochemically, by use of a primary antiserum against CCK cloned for the Japanese flounder, Paralichthys olivaceus. No CCK-immunoreactive (IR) cells were detected in first-feeding larvae (33 days after hatching, DAH). Forty-five DAH or 12 days after first feeding, there were a few scattered CCK-IR cells in the epithelium of the anterior midgut in about 30% of the examined larvae. All larvae older than 52 DAH had CCK-IR cells in the anterior midgut, particularly frequent in the most anterior region adjacent to the pyloric caeca. No CCK-IR cells were detected in the foregut, the hindgut, or the midgut posterior to the first curvature. The CCK-IR cells spanned the intestinal epithelium from the basal lamina to the lumen and were triangular in shape, with the nucleus in the basal part and a thin apex toward the lumen. The mechanisms controlling release of bile, pancreatic enzymes, and peristalsis during the 12 days between first feeding and the first detection of CCK-IR cells remain to be clarified.

Animals↗
PubMed2001

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora.

Exogenous fluorescent phosphatidic acid (PA) and phosphatidylcholine (PC) were transported into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora [Kamisaka et al. (1999) Biochim. Biophys. Acta 1438, 185-198]. We further investigated the processes of fluorescent PA and PC transport into lipid bodies in this fungus by changing culture conditions. Lowering incubation temperature decreased lipid body labeling by 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-PA (C5-DMB-PA), but fluorescence did not accumulate in organelles other than lipid bodies. C5-DMB-PC transport into lipid bodies was blocked at temperatures below 15 degrees C and fluorescence accumulated in intracellular membranes, presumably endoplasmic reticulum membranes. The low-temperature block of C5-DMB-PC transport enabled us to do pulse-chase experiments in which fungal cells were pulse-labeled at 15 degrees C with C5-DMB-PC and chased at 30 degrees C. The results clearly depicted transport of C5-DMB-PC and its derivatives from intracellular membranes to lipid bodies. Transport was temperature-dependent and ATP-dependent, although microtubules and actin filaments were not substantially involved. Experiments using 14C-labeled fatty acids and glycerol instead of C5-DMB-PC under the same conditions suggested that transport depicted by fluorescence agreed with metabolism and transport of PC containing native fatty acids. Furthermore, the transport mechanism preferred PC containing unsaturated fatty acids such as linoleic acid. This study dissect lipid transport of PA and PC into lipid bodies and reveal regulatory steps for lipid body formation in this fungus.

Biological Transport↗
PubMed2000

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies: use of fluorescent lipids to study lipid-body formation in an oleaginous fungus.

Fluorescent phosphatidic acid and phosphatidylcholine were used to characterize lipid-transport pathways into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Several characteristics of the lipid transport such as temperature dependence and ATP dependence were evaluated. The transport depicted by these fluorescent lipids was consistent with metabolism of radiolabelled lipids, indicating that fluorescent lipids are useful to study lipid-body formation in this fungus. The results dissect lipid transport of phosphatidic acid and phosphatidylcholine into lipid bodies and reveal regulatory steps for lipid-body formation in this fungus.

Adenosine Triphosphate↗
PubMed1999

Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora.

Mortierella ramanniana var. angulispora accumulates triacylglycerol (TG) in lipid bodies. Studies on lipid transport into lipid bodies are essential for elucidating mechanisms of lipid body formation. We used fluorescent dyes and fluorescent lipid analogs to visualize lipid body formation with a confocal laser scanning microscope. Different sizes of lipid bodies were stained by Nile red, a lipid body marker - one with a diameter of about 1 micrometer and the other with a diameter of about 2-3 micrometers. Lipid bodies matured into larger ones with culture. To metabolically monitor lipid bodies, we used 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-phosphatidic acid (C5-DMB-PA), and C5-DMB-phosphatidylcholine (C5-DMB-PC). These were taken up into fungal cells and incorporated into intracellular organelles at 30 degrees C. C5-DMB-PA was quickly incorporated into lipid bodies while C5-DMB-PC was initially incorporated into internal membranes, presumably endoplasmic reticulum membranes, and fluorescence was then gradually transported into lipid bodies. The transport of fluorescent lipids accompanied their metabolism into diacylglycerol (DG) and TG, which, taken together with the fluorescence distribution, suggested that conversion to TG was not necessary for transport into lipid bodies. It is likely that the synthesized DG was mainly located in lipid bodies and the conversion to TG took place in lipid bodies. C5-DMB-PA and C5-DMB-PC were converted to DG and TG in the membrane and lipid body fractions of this fungus, which agreed with in vivo metabolism of these fluorescent lipids and in vitro enzyme activity related to PA and PC metabolism. These results indicate that transport and metabolism of C5-DMB-PA and C5-DMB-PC represent two different routes for lipid body formation in this fungus.

Animals↗
PubMed1999

Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase.

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an approximately 38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an approximately 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of approximately 10,000-fold purified Golgi GCS with the same reagents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.

Amino Acid Sequence↗
PubMed1998

Characterization of triacylglycerol biosynthesis in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora.

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT<LPAAT approximately PAP<DGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.

Acyl Coenzyme A↗
PubMed1997

Purification and characterization of diacylglycerol acyltransferase from the lipid body fraction of an oleaginous fungus.

Diacylglycerol acyltransferase (DGAT) [EC 2.3.1.20] was purified to apparent homogeneity from the lipid body fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the lipid body fraction with 0.1% Triton X-100, and purified by subsequent column chromatography on Yellow 86 agarose, Superdex-200, Heparin-Sepharose, second Superdex-200, and second Yellow 86 agarose. The enzyme activity was finally enriched 4,802-fold over that of the starting 1,500X g supernatant. The apparent molecular mass of the enzyme was 53 kDa on SDS polyacrylamide gel electrophoresis. The purified enzyme did not exhibit glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, lipase, transacylase, or acyl-CoA hydrolase activities, although 2-monoolein was acylated with about a half of the enzyme activity toward 1,2-diolein. The purified DGAT depended on exogenous sn-1,2-diolein and oleoyl-CoA, with the highest activity at about 200 and 20 microM, respectively. Purified DGAT utilized a broad range of molecular species of both diacylglycerol and acyl-CoA as substrates. The highest activity was observed with sn-1,2-diolein and lauroyl-CoA. Anionic phospholipids such as phosphatidic acid (PA) activated the purified enzyme, as found for the Triton X-100 extract. Sphingosine dose-dependently inhibited DGAT activity activated by PA and basal activity without PA. These results provide a basis for further studies on the molecular mechanism of triacylglycerol biosynthesis and lipid body formation, in which DGAT plays an important role.

Acyltransferases↗
PubMed1996

Oleate 6-hydroxylase activity in the membrane fraction from an oleaginous fungus, Mortierella ramanniana var. angulispora.

Conversion of [1-14C]oleoyl-CoA (OA-CoA) in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora (IFO 8187) was investigated. The membrane fraction actively catalyzed the transformation of OA-CoA into one metabolite, which was identified as 6-hydroxy oleic acid (6-HOOA) by GC-MS analysis. The enzyme activity to form 6-HOOA was dependent on OA-CoA concentrations and increased linearly with incubation time and protein concentrations under appropriate experimental conditions. Since the enzyme activity required molecular oxygen and reduced pyridine nucleotides (NADH and NADPH), it was most probable that the formation of 6-HOOA was ascribed to direct hydroxylation to the C6 position, oleate 6-hydroxylase. 6-HOOA was formed from free oleic acid (OA) and OA-NH4 salt as substrate as well as OA-CoA, but not from OA covalently bound to phosphatidylcholine or triacylglycerol. Since CoA stimulated the hydroxylation of free OA and OA-NH4 salt but not that of OA-CoA, OA may be converted to OA-CoA and then hydroxylated. The enzyme activity was distinctly reduced by addition of lysophosphatidic acid, phosphatidic acid and dithiothreitol. Since hydroxy fatty acids are very minor components in this fungus, it may raise the possibility that the 6-hydroxylase activity works for other functions such as comprising one step for desaturation as well as forming the hydroxy fatty acids.

Acyl Coenzyme A↗
PubMed1996

Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver Golgi membranes.

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS) from a rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment > 10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis. which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-spingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.

Animals↗
PubMed1996

Activation of detergent-solubilized diacylglycerol acyltransferase by anionic phospholipids.

Diacylglycerol acyltransferase (DGAT), which catalyzes the final step in triacylglycerol (TG) biosynthesis, is crucial for lipid accumulation and formation of lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Since solubilization of DGAT in the lipid body fraction from this fungus with 0.5% Triton X-100 gave very low recovery of the activity, some activation factors for solubilized DGAT activity were investigated. Addition of phospholipids, especially anionic phospholipids such as phosphatidic acid and phosphatidylserine, to the assay mixture greatly increased DGAT activity. The activation by these phospholipids was most prominent when 0.2% Triton X-100 was added to the assay mixture. The effect of phosphatidic acid was reproduced using DGAT fraction obtained by 0.5 M KCl elution on Mono S column chromatography. The results provide new insight on activation of DGAT during TG accumulation as well as optimal DGAT assay conditions for solubilized fractions.

Acyltransferases↗
PubMed1994

Characterization of the diacylglycerol acyltransferase activity in the lipid body fraction from an oleaginous fungus.

Diacylglycerol acyltransferase (DGAT) was examined as a key enzyme for triacylglycerol (TG) accumulation of an oleaginous fungus, Mortierella ramanniana var. angulispora. Subcellular fractionation of the fungus showed that DGAT activity was highest in the lipid body fraction, which occupied 77% of the recovered DGAT activity. DGAT activity in the lipid body fraction was much higher than that in the membrane fraction in terms of both total activity and specific activity. Similar results were obtained with another homogenization method. After repeated washing of the lipid body fraction, DGAT activity in the lipid body fraction was still larger than those in other fractions. The lipid body fraction contained larger amounts of lipids, especially TG and diacylglycerol. Moreover, the lipid body fraction had a specific set of polypeptides at 24, 29, and 59 kDa. These analyses of lipid and polypeptide composition suggested that the lipid body fraction represented a specific intracellular structure, presumably the lipid body. DGAT activity in the lipid body fraction had a similar characteristics to that in the membrane fraction, although some differences in sensitivity to SH-reagents were observed. Increase in DGAT activity in the lipid body fraction was observed when lipids were accumulated in the fungus. On the other hand, DGAT activity in the lipid body fraction decreased when lipids were accumulated with an increase in carbon to nitrogen ratio in media.

Acyltransferases↗
PubMed1990

Inhibitory effects of mold oil including gamma-linolenate on platelet thrombus formation in mesenteric microvessels of the rat.

Diet including mold oil from a lipid accumulative fungus, containing gamma-linolenic acid, showed an inhibitory effect on thrombus formation in the microvessels of rats by the light/dye method of the authors. Male Wistar rats were fed for 3 to 4 weeks with two series of experimental diets and were examined for thrombus formation. The thrombus formation times to totally occlude, ts, were 347 sec for (mold + soybean)-oil and 236 sec for (palm + soybean)-oil in the first series of diets and 1288 sec for mold oil, 538 sec for olive oil and 575 sec for safflower oil in the second series of diets. Fatty acid composition of plasma, erythrocyte and liver lipids showed an increase in arachidonate content with the diet including the mold oil. Higher arachidonate content seem favorable in inhibiting thrombus formation with increasing PGI2 formation. In terms of the level of lipid hydroperoxides, indicated as a desaturation index of constituent fatty acids, the higher desaturation index with safflower oil gave shorter ts, which suggested some oxygen derived free radicals from polyunsaturated fatty acids were involved in the mechanism of thrombogenesis study by this method.

Animals↗