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1.
Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic microsomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [35S]Met-tRNAi demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits. 相似文献
2.
The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl
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Incorporation of leucine-C14 into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C14 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions. 相似文献
3.
The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate 总被引:2,自引:1,他引:1
P. C. Jocelyn 《The Biochemical journal》1970,117(5):951-956
1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase. 相似文献
4.
S. Yokota 《Histochemistry and cell biology》1973,37(2):149-159
Summary Distribution of urate oxidase in subcellular components such as nuclei, mitochondria, lysosomes, microsomes, and cell sap, was investigated by both enzymatic and immunochemical methods. The subcellular components were prepared from mouse liver homogenate by differential centrifugation and the resulting microbody-rich mitochondrial fraction was fractionated by sucrose density gradient centrifugation. The enzymatically determined urate oxidase was distributed mainly in mitochondrial and lysosome fractions. The immunochemically assayed urate oxidase antigen was localized in mitochondrial, lysosome, and microsome fractions. The antigen to enzyme ratio was 1.0 in the mitochondrial and lysosome fractions, and about 2.0 in the microsome fraction.Sucrose density gradient centrifugation of the mitochondrial fraction indicated that the urate oxidase antigen was distributed around three density bands of 1.07, 1.15, and 1.24. The main band (1.24) was consistent with the microbody fraction. From these results, it was suggested that a precursor protein (proenzyme) might be located in the microsome fraction.This work was supported in part by a grant 777007 from the Ministry of Education, Japan, in 1972. 相似文献
5.
Various cell fractions from rat liver were tested for their ability to convert nitrosopyrrolidine (NO-PYR) to products which were mutagenic to E. coli in liquid-incubation assays. Microsomes alone produced only a small number of tyr+ revertants, approximately 40/10(8) survivors), while the S100 supernatant produced none at all. However, the S8 Fraction or combinations of microsomes and the S100 supernatant, yielded 300-400 tyr+ revertants/10(8) survivors. Neither products of the microsomal, nor microsome + supernatant reactions were mutagenic in the absence or presence of cellular fractions. These results suggest that bacterial mutagens are formed during the microsomal metabolism of NO-PYR to 2-hydroxytetrahydrofuran by alpha-hydroxylation, but not during the metabolism of 2-hydroxytetrahydrofuran by the S100 supernatant enzymes. Possible roles of the supernatant enzymes in the formation of mutagenic intermediates during the initial alpha-hydroxylation of NO-PYR are discussed. 相似文献
6.
Biological Activity of Fractions Obtained by Differential Centrifugation of the Bovine Adrenal Cortex
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This study is an attempt to correlate adrenocortical hormonal activity with various cell fractions obtained by homogenization and differential centrifugation of bovine adrenocortical tissue. Bioassays involving the decrease in circulating eosinophils and sodium retention were employed for the estimation of the glucocorticoids and mineralocorticoids respectively. The supernatant was the most active fraction in both bioassays, while the microsome and mitochondrial fractions demonstrated varying degrees of activity. The total nitrogen, lipide phosphorus, and cholesterol contents of the fractions were determined. The microsome fraction contained a relatively higher amount of lipide phosphorus and a marked amount of cholesterol. 相似文献
7.
Mouse follicles were labeled with [3H]uridine and then cultured in vitro for 3 days. When oocytes were disrupted, about 40% of the total radiolabeled RNA could be sedimented at 9,000g. Fractionation of this RNA on poly(U)-Sepharose revealed that about 30% and 60% of the total amount of radiolabeled poly(A)- and poly(A)+ RNA, respectively, were in the pellet fraction. Treatments that disrupt protein structure reduced the amount of 9,000g sedimentable RNA and affected to the same extent the distribution of Poly(A)- and poly(A)+ RNA in the pellet and supernatant fractions. CsCl centrifugation of formaldehyde-fixed pellets revealed that virtually all of the radiolabeled RNA had a density significantly lower than that of ribosomes. The sedimentable RNA appeared not to be polysomal, membrane bound or associated wih a cytoskeleton. Agarose gel electrophoresis after poly(U)-Sepharose fractionation of either the pellet or supernatant revealed the presence of 28S, 18S, 5S + 4S, and heterodisperse poly(A)+ RNA. The size of distribution of poly(A)+ RNA in the pellet and supernatant fractions was fairly similar. Pulse-chase experiments revealed that the stability of poly(A)- RNA in the pellet and supernatant fractions was the same within the experimental error and a similar situation was found for poly(A)+ RNA. RNA in pellet translated in vitro coded for discrete size classes of protein. Since the relative band intensities were similar for both total and pellet RNA translated in vitro there seemed to be no major partitioning of specific size classes of mRNA into the pellet fraction. These results are discussed in terms of a possible composition of the lattice structures that accumulate during mouse oocyte growth and have been postulated to be a storage form for ribosome (Burkholder et al., '71). 相似文献
8.
The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g. 相似文献
9.
The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g. 相似文献
10.
Rats were divided into high protein diet and low protein diet fed groups. Their livers were removed, homogenized, and fractionated into nuclei, mitochondria, microsome and supernatant fraction.Amount of total- and phospho-lipids in each fraction was measured, and simultaneously, total nitrogen was measured. Then, the ratio of total- and phospho-lipids to nitrogen was calculated. The influence of low protein diet on this calculated value was seen in the mitochondrial and supernatant fraction but was not recognized in other fractions.The relationship between this phenomenon and energy efficiency of diets was discussed. 相似文献
11.
本文报道柞蚕卵巢亚细胞组分中核酸和蛋白质的分布与含量变化。首先以差速离心盼方法从卵巢匀浆中分离出细胞核、重线粒体、轻线粒体、重微粒体、轻微粒体和105,000g上清六个组分,然后分别测定了各种组分中的DNA、RNA和蛋白质的含量。 结果表明,卵巢DNA主要分布在细胞核组分中(约占75%以上);RNA在佩粒体中含量较离(约占20—51%);蛋白质则主要分布于105,000g上清组分中(约占56—83%)。卵巢中核酸和蛋白质含量(毫克/头)在滞育蛹期很低,随着卵巢的发育迅速增长。DNA在发育蛹4期最高,比滞育蛹期增长61倍;RNA在发育蛹5期最高,比滞育蛹期增长144倍;蛋自质在成虫期达到高峰,约比滞育蛹期增长490倍。并讨论了核酸和蛋白质的含量变化与卵巢细胞分裂分化之间的关系。 为了获得进一步的生化资料,对不同时期的柞蚕卵巢105,000g上清组分中的蛋白质进行了聚丙烯酰胺凝获也泳和SDS-电泳的分析。初步推测柞蚕卵巢中卵黄蛋白亚基之一的分子量大约为200,000道尔顿。 相似文献
12.
By means of differential centrifugation, cytosol fraction and microsome were prepared from maize roots which have been grown in dark for 4 d. Highly purified plasma membranes were isolated from the microsome in two-phase aqueous system which is composed of 6.9 % (W/W) Dextran T500 and PEG 3350. The tonoplast was collected from the interface between 1% and 8% (W/W) Dextran T70 after gradient centrifugation. Electron microscopic observation and marker enzyme activities analysis proved that these fractions contained very few other membranes. Microvolume radioactivity ligand binding assay indicated that the specific binding sites of ABA in maize root microsome were mainly distributed on tonoplast and plasma membrane fractions. Their specific binding activity was 2485.4 and 1257.3 fmol/mg protein, respectively, the specific binding activity of cytosol fraction being the lowest (one order of magnitude lower). The dissociation constant (KD) of ABA-BP in plasma membrane was 1.57 nmol/L. 相似文献
13.
Free and membrane-bound polysomes from rat liver. 1. Improvements of subcellular fractionation 总被引:2,自引:0,他引:2
Substantial improvements of cellular fractionation in ionic conditions allowing preservation of polysome structure and polysome-membrane interactions are reported. They consist primarily in minimizing the lysosomal content of fractions containing endoplasmic reticulum by isolating a lysosome-rich fraction with little loss (6%) of RNA. Endoplasmic reticulum membranes were recovered in high yield, mainly in association with mitochondria, the remainder being found in the post-lysosomal supernatant. The latter also contained practically all the free polysomes, as judged by metrizamide gradient analyses. The distributions of various constituents (RNA, DNA, protein and marker enzymes) among cell fractions is presented. 相似文献
14.
We have investigated the abilities of a cell-free supernatant of splenocytes or thymocytes, which have been incubated with myelin basic protein (MBP), and of membranes prepared by lysing these cells, to stimulate proliferation of a Lewis rat anti-MBP T lymphocyte line in vitro. The supernatant fraction, obtained by low-speed centrifugation, is thought to contain shed membrane fragments bearing class II MHC protein (Ia) and processed antigen. Almost all of 67 preparations of supernatant fraction and about a third (26/70) of the membrane preparations stimulate proliferation of the line cells in the absence of other antigen-presenting cells and antigen. Some membrane preparations bearing the synthetic peptide S69 (residues 69-89 of MBP), containing the immunodominant encephalitogenic determinant for the Lewis rat, instead of processed MBP could also stimulate proliferation. Those membrane preparations bearing either processed MBP or synthetic S69, which do not stimulate proliferation, induce a state of unresponsiveness in which the cells do not proliferate but produce inositol phosphate. Stimulation of proliferation and induction of unresponsiveness were both inhibited by anti-Ia antibody. Addition of cyclosporin A prevents induction of unresponsiveness. Addition of allogeneic splenocytes or the cell-free supernatant fraction of syngeneic or allogeneic splenocytes or thymocytes, prevents induction of unresponsiveness by providing a necessary costimulatory signal. Further fractionation of the cell-free supernatant by high-speed ultracentrifugation showed that the costimulatory signal resided in a particulate fraction which sedimented and not in the supernatant. These results indicate that the encephalitogenic peptide can induce anergy in T cells when presented on class II MHC in the absence of the costimulatory signal. Tolerizing forms of the membrane preparations which lack the costimulatory signal may be useful for in vivo treatment of autoimmune response. 相似文献
15.
Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases. 相似文献
16.
Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.
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The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion. 相似文献
17.
Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system. 总被引:31,自引:6,他引:25
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We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno-isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. The G-protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno-isolated on a solid support using as antigen the cytoplasmic domain of the G-protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno-isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G-protein. The immuno-isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno-isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell-free assay using both a bio-chemical and a morphological detection system. These well defined endosomal vesicles were immuno-isolated with the G-protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post-nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion-specific iodination of the G-protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G-protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G-protein internalization. 相似文献
18.
Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method. 相似文献
19.
The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid 总被引:20,自引:17,他引:3
1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture. 相似文献
20.
S Moorkerjea 《Canadian journal of biochemistry》1979,57(1):66-71
Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction. Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100. Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yold lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase. 相似文献