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PubMed2002

Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in 3LL Lewis lung carcinoma cells.

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.

Animals↗
PubMed1992

Oleic acid (18:1) in plasma, liver and brain myelin lipid of piglets fed from birth with formulas differing in 18:1 content.

Recent studies have shown reduced 18:1 fatty acids in brain total lipid of piglets fed formula with 17% 18:1 fatty acids rather than sow milk providing 37% 18:1. Because 18:1 is a major fatty acid in brain myelin lipid and is rapidly deposited during myelination, it is important to know if this reflects deposition of myelin lipid containing reduced 18:1 and/or delayed myelination, or is related to changes in other brain membranes. Therefore, these studies determined the myelin total lipid, phosphatidylethanolamine and plasma and liver phospholipid fatty acids in piglets fed from birth to 15 d with formula containing (g/100 g fatty acids): 18:1, 17 and 18:3(n-3), 0.8; 18:1, 38 and 18:3(n-3), 0.6 fatty acids; or 18:1, 40 and 18:3(n-3), 4; or sow milk. Different levels of formula 18:3(n-3) fatty acids were studied because of the known effect of essential fatty acid deficiency on myelination. The brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity and cerebroside concentration were also determined and used as indicators of myelination. Piglets fed the low 18:1 formula had lower 18:1 fatty acids in their plasma and liver phospholipid than sow milk-fed piglets. Formula providing a similar level of 18:1 to sow milk resulted in higher 18:1 fatty acids in the piglet plasma and liver phospholipid than in the sow milk-fed piglets. The brain myelin monoenoic fatty acids, CNPase activity and cerebroside concentration were similar in sow milk- and formula-fed piglets, irrespective of the formula 18:1 or 18:3(n-3) content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Sphingolipids in food and the emerging importance of sphingolipids to nutrition.

Eukaryotic organisms as well as some prokaryotes and viruses contain sphingolipids, which are defined by a common structural feature, i.e. , a "sphingoid base" backbone such as D-erythro-1,3-dihydroxy, 2-aminooctadec-4-ene (sphingosine). The sphingolipids of mammalian tissues, lipoproteins, and milk include ceramides, sphingomyelins, cerebrosides, gangliosides and sulfatides; plants, fungi and yeast have mainly cerebrosides and phosphoinositides. The total amounts of sphingolipids in food vary considerably, from a few micromoles per kilogram (fruits) to several millimoles per kilogram in rich sources such as dairy products, eggs and soybeans. With the use of the limited data available, per capita sphingolipid consumption in the United States can be estimated to be on the order of 150-180 mmol (approximately 115-140 g) per year, or 0.3-0.4 g/d. There is no known nutritional requirement for sphingolipids; nonetheless, they are hydrolyzed throughout the gastrointestinal tract to the same categories of metabolites (ceramides and sphingoid bases) that are used by cells to regulate growth, differentiation, apoptosis and other cellular functions. Studies with experimental animals have shown that feeding sphingolipids inhibits colon carcinogenesis, reduces serum LDL cholesterol and elevates HDL, suggesting that sphingolipids represent a "functional" constituent of food. Sphingolipid metabolism can also be modified by constituents of the diet, such as cholesterol, fatty acids and mycotoxins (fumonisins), with consequences for cell regulation and disease. Additional associations among diet, sphingolipids and health are certain to emerge as more is learned about these compounds.

Cell Division↗
PubMed1988

Long-term follow-up of the first successful bone marrow transplantation in Gaucher disease.

A 9-year-old girl with juvenile Gaucher disease underwent splenectomy and allogeneic bone marrow transplantation. Her HLA-identical brother with normal cerebroside-beta-glucosidase activity served as donor. One month after transplantation, cerebroside-beta-glucosidase activity in the lymphocytes were normal. Plasma glucosylceramide normalized already after splenectomy and further decreased after marrow transplantation. Glucosylceramide in the erythrocytes was normal around a year after transplantation. The enlarged liver normalized in size by 2 years. Gaucher cells were still present in the bone marrow 1 year after transplantation but had completely disappeared at 3 years. The patient has grown 29 cm during the 5 years that have passed after transplantation compared to 1 cm/year during 3 years before. The patient has a slight obstructive ventilatory impairment, and chest deformities have appeared. Wechsler intelligence scale performance has slowly decreased after transplantation. This may be caused by continued neuronal storage of glucosylceramide. Otherwise, this patient is active and healthy 5 years after bone marrow transplantation.

Bone Marrow↗
PubMed1991

Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance.

Plasma membranes were isolated from oat (Avena sativa) roots by the phase-partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane.

Journal Article↗
PubMed1996

Opioid receptor affinity of multivalent ligand system consisting of polymerized liposome.

A multivalent ligand system, in which enkephalin/phospholipid conjugates were immobilized on a polymerized liposome, has been prepared. A hydrophilic spacer chain, -(Sar-Sar-Pro)2-, was placed between Tyr-D-Ala-Gly-Trp-Leu (an enkephalin part) and a phospholipid part to diminish steric hindrance of the liposome against receptor binding of the enkephalin unit. The affinity of the immobilized enkephalin conjugate for opioid receptor was dependent on the density of the enkephalin unit on the surface of polymerized liposome, indicating that more than two enkephalin units on a liposome simultaneously bind to receptors in the membrane. IC50 values for the delta- and mu-receptors were 15 and 26 nM, respectively, when the conjugate was immobilized at the molar ratio of [enkephalin]/[phospholipid] of 340. The latter affinity is better than that of Tyr-D-Ala-Gly-Trp-Leu (50 nM). The enkephalin part was located at the membrane surface, as indicated by fluorescence spectroscopy. When 12% cerebroside sulfate was mixed in the polymerized liposome, delta-receptor affinity of the immobilized enkephalin conjugate was improved to 7.4 nM. The environment around the enkephalin part became hydrophilic upon incorporation of cerebroside sulfate into the polymerized liposome, resulting in changes in the receptor affinities.

Animals↗
PubMed1981

Effect of malnutrition and subsequent rehabilitation on the development of mouse brain myelin.

Malnutrition in mice from birth resulted in myelin of brain having higher than normal molar proportions of cholesterol and phospholipids relative to a molar unit of cerebroside + sulphatide. This was found at all ages between 20 and 60 days, and the molar ratio of these lipids in older animals was comparable to that in the younger controls. The phospholipid and the ganglioside patterns were also immature for age. The phospholipid composition was characterized by lower molar proportions of ethanolamine phosphoglyceride (EPG) and sphingomyelin (SPh) and higher proportion of choline phosphoglyceride (CPG), and the ganglioside pattern was characterized by higher molar proportions of the disialogangliosides GD1a and GD1b and markedly lower proportion of the monosialoganglioside GM1. Malnutrition imposed from 30 days of age did not affect the contents of the major lipids (and so their molar ratio), but within the phospholipids there was a small but significant deficit of SPh, which was compensated by a higher content of CPG. The ganglioside pattern was as if the animals were malnourished from birth. Nutritional rehabilitation up to 60 days of age subsequent to malnutrition for the first 30 days fully corrected the ganglioside pattern, but not the molar ratio, of the major lipids (because of persistent deficit in cerebroside + sulphatide) and the composition of the phospholipids (because of small but significant deficit of SPh). The results indicate that malnutrition instituted at any time during the entire programme of myelination can affect one or other aspect of myelin development, and nutritional rehabilitation of animals malnourished in early life cannot fully correct this developmental gap.

Animals↗
PubMedDate not supplied

Endogenous lipids of the earthworm Lumbricus terrestris.

Earthworms (Lumbricus terrestris) were given [1-14C]-labeled palmitic acid by gavage on days 0 and 3, and sacrificed on day 7. The distribution of label among lipid classes indicated that glycerides, sterol esters, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylserine and (or) phosphatidylinositol, phosphatidylcholine, and sphingomyelin turn over in, or are synthesized by, the earthworm. Free fatty acids still had the highest specific radioactivity of any lipid class at the end of the experiment. Incorporation of label into sterol and hydrocarbon fractions was insignificant and there was no detectable label incorporated into gangliosides. Phosphatidylethanolamine apparently turned over quite slowly compared with other lipid classes, while the cerebroside fraction became highly labeled. Elongation of palmitic acid to stearate and oxidation to CO2 occurred extensively, but there was no evidence for desaturation.

Animals↗
PubMed2003

Effects of dietary sphingomyelin on central nervous system myelination in developing rats.

Human milk contains sphingomyelin (SM) as a major component of the phospholipid fraction. Galactosylceramide (cerebroside), a metabolite of sphingolipids, increases along with CNS myelination, and is generally considered a universal marker of myelination in all vertebrates. l-Cycloserine (LCS) is an inhibitor of serine palmitoyltransferase (SPT), a rate-limiting enzyme for sphingolipid biosynthesis that is reported to show increased activity with development of the rat CNS. The present study examined the effects of dietary SM on CNS myelination during development in LCS-treated rats. From 8 d after birth, Wistar rat pups received a daily s.c. injection (100 mg/kg) of LCS. From 17 d after birth, the animals were fed an 810 mg/100g of bovine SM-supplemented diet (SM-LCS group) or a nonsupplemented diet (LCS group). At 28 d after birth, the animals were killed and subjected to biochemical and morphometric analyses. The myelin dry weight, myelin total lipid content, and cerebroside content were significantly lower in the SM-LCS and LCS groups than in a group not treated with LCS (the non-LCS group). However, these levels were significantly higher in the SM-LCS group than in the LCS group. Morphometric analysis of the optic nerve revealed that the axon diameter, nerve fiber diameter, myelin thickness, and g value (used to compare the relative thickness of myelin sheaths around fibers of different diameter) were significantly lower in the LCS group than in the other groups, but were similar in the SM-LCS and non-LCS groups. These findings suggest that dietary SM contributes to CNS myelination in developing rats with experimental inhibition of SPT activity corrected].

Acyltransferases↗
PubMed2005

Cutaneous water loss and lipids of the stratum corneum in house sparrows Passer domesticus from arid and mesic environments.

Birds that live in hot, dry environments must balance water intake with losses in order to maintain water homeostasis. The outer layer of the integument, called the stratum corneum (SC), consists of corneocytes embedded in a matrix of lipids. The SC serves as a barrier to water vapor diffusion through the skin. We measured cutaneous water loss (CWL) in two populations of house sparrow Passer domesticus L., one living in a desert environment in Saudi Arabia, and another living in a mesic environment in Ohio, USA. We found that CWL rates at 30 degrees C were lower in desert individuals (11.9+/-2.2 mg H(2)O cm(-2) day(-1); N=11) than in mesic birds (16.0+/-2.6 mg H(2)O cm(-2) day(-1); N=14). We hypothesized that changes in the lipid composition of the SC could affect CWL. We analyzed four classes of lipids in the SC: ceramides, cerebrosides, cholesterol and free fatty acids, by thin layer chromatography. Compared to mesic sparrows, desert birds had a higher amount of ceramides (49.2+/-10.3 mg g(-1) SC dry mass in Saudi Arabia; 38.2+/-18.0 mg g(-1) SC dry mass in Ohio) and cerebrosides (101.2+/-48.9 mg g(-1) SC dry mass in Saudi Arabia; 56.5+/-34.0 mg g(-1) SC dry mass in Ohio), and a lower percentage of cholesterol (4.1+/-3.6% in Saudi Arabia; 5.4+/-2.5% in Ohio) in their SC. Although CWL was lower in sparrows from Arabia, and lipid composition of their SC differed, we could not detect differences between rates of water loss through non-living skin attached to glass vials (46.0+/-15.7 mg H(2)O cm(-2) day(-1) for sparrows in Saudi Arabia; 45.8+/-27.2 mg H(2)O cm(-2) day(-1) for sparrows in Ohio). These results suggest that biological control mechanisms interact with layers of lipids in the stratum corneum to adjust CWL to the environment.

Animals↗
PubMed2001

Confirmation of minor components of less polar neutral and acidic glycolipids in monkey brain tissue.

Crude glycolipids, prepared without alkali treatment in advance, were separated into neutral and acidic glycolipids by DEAE-Sephadex A-25 (acetate form) column chromatography. Each glycolipid was further fractionated by a Silica gel 60-column chromatography. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS) of the intact glycolipid fractions, the less polar neutral glycolipids were found to contain alkali-labile ester cerebrosides and Galb-1-Diradylglycerols, whereas the less polar acidic glycolipids were found to contain alkali-labile ester sulfatide, HSO(3)-3Gal-1-Diradylglycerols, and novel alkali-stable plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols as minor components of glycolipids in monkey brain tissue. In conclusion, minor components of less polar neutral and acidic glycolipids in monkey brain tissue were confirmed as ester cerebrosides, Galb-1-Diradylglycerols, ester sulfatides, HSO(3)-3Galb-1-Diradylglycerols, and novel plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols by DE MALDI-TOF MS.

Animals↗
PubMedDate not supplied

[Brain lipid biochemistry of the sturgeons Acipenseridae].

Brain lipids of 4 species of sturgeons-Acipenser güldenstädti, A. stellatus, A. ruthenus and Huso huso-have been investigated. In whole brain homogenates the content of the basic phospholipid families, their fatty acid composition as well as the content of glycosphingolipids-gangliosides, cerebrosides and sulfatides were studied. The quantitative relations between different molecular ganglioside fractions and their fatty acid composition, as well as the relative content of hydroxycerebrosides and cerebrosides with normal fatty acids were determined. The brain lipids of Acipenseridae were compared to previously obtained data on the brain of Elasmobranch and Teleost fishes. The brain lipid pattern of the four sturgeon species is very similar, but it differs greatly from that of Elasmobranch and of Teleostean brain, being in some respect more like the type of bony fish brain, in others the kind of brain of sharks and rays, and frequently considerably differing from both these types. The conspicous peculiarity in the content and molecular characteristics of the brain lipids of Acipenseridae compels us to agree with the opinion expressed by the late A.N. Belozerski and his coworkers [26, 27], based on a detailed study of fish DNA nucleotides, that the Ganoid fish are to be regarded as an independent class.

Animals↗
PubMed1975

Dissociated cultures of newborn mouse brain. I. Metabolism of sulfated lipids and mucopolysaccharides.

The metabolism of 35SO4-sulfated lipids and mucopolysaccharides was studied in dissociated brain cell cultures from newborn albino mouse brains. The cultures were maintained under an atmosphere of 40% O2 and 5% CO2 in apparent good health up to 30 days. Early morphological examination of the dissociated cells demonstrated an initial partial reaggregation of the cells, which later settled and became confluent bilayered cultures. Cell proliferation measured by DNA and protein determination, morphological differentiation and biochemical differentiation took place in the dissociated brain cell cultures analogous in some respects to the in vivo situation. A timed increase in the synthesis of a myelin precursor, cerebroside 35SO4, was observed after 6 to 8 days in culture (DIC). A peak of cerebroside sulfate was evident at 17 DIC. No stable sulfatide was observed at any time. Protein-bound macromolecular 35SO4-MPS was synthetized and secreted from the cells into the culture medium. Maximal synthesis and secretion occurred at 8 DIC. This culture system proves to be a useful model for studying some aspects of differentiation of brain cells under controlled external conditions.

Animals↗
PubMed1992

Transgenic mice expressing polyoma virus large T antigen in astrocytes develop severe dysmyelination of the central nervous system.

Transgenic mice were generated using a construct that encodes mouse polyoma virus large T antigen, one of three oncogenic products of the "early region" of the polyoma viral genome. Of 16 transgenic families developed, 1 was characterized by a neurologic disorder consisting of constant tremor and recurrent seizures. Morphologic analysis of the central nervous system (CNS) of affected transgenic mice included: classical light and electron microscopic examination; immunohistochemical assessment of the presence and localization of myelin-specific proteins, of the astrocyte marker glial fibrillary acidic protein, of the oligodendrocyte marker galactosyl cerebroside, and of large T; double immunolabeling of glial fibrillary acidic protein or galactosyl cerebroside and large T to identify the CNS cell type in which large T is expressed; and in situ hybridization to study myelin basic protein gene expression. Our results suggest that polyoma large T is expressed in astrocytes, possibly resulting in altered glial-glial interactions causing impaired oligodendroglial development and secondary dysmyelination. Transgenic oligodendrocytes exhibit features of immaturity, failing to myelinate axons properly and producing morphologic phenotypes of early stages of myelination, such as numerous mesaxonal profiles. Myelin proteins are markedly reduced in transgenic CNS, and myelin basic protein transcripts, while present, are generally decreased. We believe that expression of large T in astrocytes could influence the complex and dynamic interactions between astrocytes and oligodendrocytes, perhaps with regard to the molecular (trophic) signals in the local CNS environment, bringing about arrested oligodendroglial maturation and hypomyelination. This raises intriguing questions concerning the importance of glial-glial interactions in the CNS and the complex levels of control involved in biological expression of genetic information in glial cells.

Animals↗
PubMed1992

Purification and activation of brain sulfotransferase.

Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.

Adenosine Triphosphate↗
PubMed1975

Glycolipids of the fish testis.

Glycolipids were purified from the total lipid extract of the testis or milt of a kind of puffer (Fugu rubripes rubripes) by adsorption column chromatography using silicic acid and magnesium silicate and by preparative silica gel TLC. The glycolipids were identified as glucosylceramide (116 mug/g wet tissue) and galactosylceramide 26.7 mug/g). Seminolipid, a sulfagalactolipid specific to mammalian testis was not detected, but the presence of a small amount of sulfatide (15.2 mug/g) was demonstrated. The long-chain bases of both cerebrosides were mainly C18-sphingenine, but in sulfatide, C20-sphingenine was more abundant than C18-sphingenine. In both cerebrosides and sulfatide, the fatty acid compositions were similar, with nervonic acid as the predominant component. Two species of gangliosides were also obtained and were identified as N-acetylgalactosaminyl(1 leads to 4)[N-acetylneuraminyl(2 leads to 3)]galactosyl(1leads to 4)glucosylceramide (59.8 mug/g) and N-acetylneuraminyl(2 leads to 3)galactosyl(1 leads to 4)N-acetylglucosaminyl(1 leads to 3)galactosyl(1 leads to 4)glucosylceramide (45.0 mug/g). The long-chain bases of the two gangliosides consisted of C18-spingenine and C20-sphingenine, and the major fatty acids were palmitic and stearic acids.

Animals↗
PubMed1979

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.

Animals↗
PubMed1989

Modulation of phosphorylase kinase activity by sphingolipids.

Psychosine (galactosyl sphingosine) potently inhibits the activity of both nonactivated and activated by covalent modification (autophosphorylation and limited proteolysis) rabbit skeletal muscle phosphorylase kinase. Half-maximal inhibition was observed at 44 microM or 66 microM when the kinase activity was assayed at pH 6.8 or 8.2 respectively. Sphingosine was also inhibitory, but only at pH 6.8 (half-maximal inhibition was observed at 130 microM). In this respect, sphingomyelin, cerebroside and cerebroside sulfate were ineffective. On the other hand, a number of gangliosides stimulated the activity of nonactivated phosphorylase kinase at neutral pH. Among the individual gangliosides tested the activation potency was GD1a greater than GT1b greater than GM1, while GM3 was without effect. Most important, GD1a dramatically increases the activity of the kinase at low Ca2+ concentrations. Both psychosine and GD1a increased the rate of kinase autophosphorylation on alpha- subunit only, but although ganglioside-induced stimulation of autophosphorylation was accompanied with an enhancement of the rate of autoactivation at pH 6.8, psychosine completely blocked autoactivation.

Animals↗