Major Subfractions Research Articles

A Study of Tobacco Carcinogenesis. XX. Role of Catechol as a Major Cocarcinogen in the Weakly Acidic Fraction of Smoke Condensate<xref ref-type="fn" rid="fn2">2</xref>, <xref ref-type="fn" rid="fn3">3</xref>, <xref ref-type="fn" rid="fn4">4</xref>

The weakly acidic fraction of cigarette smoke condensate was fractionated by preparative high-pressure liquid chromatography into major subfractions I-IV. Major subfractions II and III were fractionated further into subfractions A-J. Subfractions A-J were tested for cocarcinogenicity on the skin of noninbred Ha:ICR Swiss albino mice by application with 0.003% benzo[a]pyrene. Subfractions A-C and F-J showed significant cocarcinogenic activity; subfractions A, F, and H were the most active. Catechol was a major component of subfraction A and was also detected in subfractions B-D and F. Major components of the other subfractions included hydroquinone (B), coniferyl alcohol (C and H), hydroxyphenyl alcohols (D), alkyl-2-hydroxy-2-cyclopenten-1-ones (C, D, and F), hydroxyacetophenones (F), phenolic cyano compounds (F), and fatty acids (F). The results demonstrate the importance of catechol as a cocarcinogen in the weakly acidic fraction of cigarette smoke condensate and indicate the presence of other cocarcinogens.

Thermally induced heterogeneity in microsomal membranes of fatty acid-supplemented Tetrahymena: lipid composition, fluidity and enzyme activity.

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [ 3H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.

Characterization of lens proteins. III. A group of labile rat lens proteins involved in cataracts

In the fractionation of soluble rat lens proteins by Sephadex G-200, superfine, a group of labile proteins, designated F-II, has been obtained. The soluble protein of normal rat lens contains 17% F-II which is not found in that of severe cataract (5+) lens induced by galactose feeding. F-II is prone to irreversible precipitation by a variety of laboratory techniques, such as concentration, lyophilization and freeze-thaw. The apparent molecular weight of the F-II has been estimated to be 240 000 daltons from the position of its elution from the Sephadex G-200, superfine, column. The amino acid composition of the F-II has been found similar but not identical to the β 1 fraction of the β-crystallin. F-II consists of a number of polypeptides with slight variations in surface charge having pI in the range pH 6·3–7·1, distinct from other crystallins. The extinction coefficient of F-II was E 280 nm 1% = 19·4. The amino terminals of the fraction appeared to be blocked, but analyses with carboxypeptidase Y indicated the presence of a carboxyl terminal sequence, -(Val, Ile)-Tyr-Phe-Leu-Ser(OH). When the F-II was further fraction-ated on DEAE-cellulose, the first four major subfractions were found in regions where rat β-crystallin fractions were eluted. The last major subfraction was eluted in the α-crystallin region. All fractions appear to be closely related in structure as they showed nearly identical amino acid composition and identical carboxyl terminal. By a freeze-thaw process, F-II and β-crystallin fractions have shown various degrees of precipitation but the α- and γ-crystallin fractions are unaffected. Attempts are in progress to determine the relationship of this group of labile proteins to the “cold precipitable protein” for the purpose of understanding the cold cataract phenomenon.

Maize histone H1: a partial structural characterization

The first H1 histone from a plant, Zea mays, has been characterized and partially sequenced. The maize H1 molecule shares many sequence features with rabbit and sea urchin H1 and chicken H5. The central hydrophobic region of the protein exhibits sequence microheterogeneity, indicating the presence of multiple H1 proteins in maize. The cause of the genetically controlled electrophoretic variation in the major H1 subfraction of maize has been localized to the carboxy-terminal region of the molecule.

THE HIGH OCCURRENCE OF LOW DENSITY LIPOPROTEIN SUBFRACTIONS IN CORONARY HEART DISEASE

This study was made to determine whether patients with coronary artery disease have any specific abnormality in their serum lipoprotein pattern which might be a factor in the etiology of the disease. Serum lipoproteins (Lps) of 18 patients with myocardial infarction (group A), 28 patients with other forms of coronary artery disease (group B), and 22 healthy control subjects (group C) were studied using ultracentrifugal and electrophoretic techniques. All subjects were Japanese living in Japan. Acrylamide gel electrophoresis of the patients' sera showed, in addition to bands of α- and β-globulin and pre-β-lipoprotein mobility, one of mobility between that of β and pre-β-mobility, mid-band. It was present in 100% of sera of group A, 93% of group B but only 50% of group C. The midband is a subfraction of the LDL fraction isolated by ultracentrifugation at d 1.006 to 1.063. The midband may be resolved as a single, double or multiple band fraction on acrylamide gel. By ultracentrifugation subfractions of LDL were detected in 16 of 18 patients in group A (89%), 10 of 13 patients in group B (77%) and 3 of 11 in group C (27%), respectively. Of LDL subfractions Sf 10 LDL was most frequent; in 77% of group A's sera, and 89% of group B's, Sf 10 was the sole or major subfraction. Serum HDL, determined ultracentrifugally was significantly lower (p 0.01) in group A and B (179.3 14.3, and 219.1 20.3, mean SE) than in group C persons (319.9 16.7). The above findings indicate that accumulation of Sf 10 LDL and other LDL subfractions may occur in association with a decrease of HDL in atherosclerotic coronary artery disease. It is suggested that those abnormalities may contribute to the genesis of atherosclerotic coronary artery disease.

In-vitro evaluation in man of immunostimulation by subfractions of Actinomyces viscosus

Actinomyces viscosus was fractionated into cell wall (PEL) and intracellular supernatant (SUP) fractions following ultrasonication. In vitro lymphocyte transformation induced by each fraction was assessed using the lymphocytes obtained from subjects with minimal periodontal disease. In kinetic experiments, a detectable blastogenic response to both fractions was measured by the fourth day, with a peak at day 7. Nuclease treatment enhanced the immunostimulatory activity of the PEL fraction 5.5-fold over untreated PEL; it had no effect on the activity of SUP. Although pronase treatment had no effect on the PEL fractions, it abrogated the ability of SUP to activate lymphocytes. Fractions of nuclease-treated and untreated SUP were batch-eluted from DEAE-sepharose columns with increasing concentrations of NaCl, producing 3 major stimulatory subfractions which accounted for 75 per cent of the activity of unfractionated SUP. Comparisons of lymphocyte responses to the DEAE-fractionated SUP indicated that not all individuals responded equally to each subfraction. As there was a differential responsiveness to the various antigenic components, detailed evaluation of the antigens of A. viscosus is warranted to define differences in antigen responsiveness in individuals with differing severities of periodontal disease.

Chemical nature of human urinary glycosaminoglycans.

To elucidate precise chemical nature of urinary glycosaminoglycans (GAG), GAG in the seven major acidic subfractions in a previous paper (Endo et al. 1980a) were analyzed for the chemical compositions and studied on the linkage between carbohydrate and peptide. All the subfractions examined contained galactosamine, hexuronic acid as the major constituent sugars, and xylose, galactose, L-fucose as the minor ones. Some of the subfractions contained additional glucosamine. Sulfate was also found in all the subfractions as their major constituent. The contents of total amino acids ranged from 1.20 to 17.9%. Serine, threonine, aspartic acid, glutamic acid, glycine, alanine and leucine were found as major amino acids. Pronase digestion did not significantly change their molecular size. The presence of O-xylosyl-serine linkage between carbohydrate chains and peptide moieties ws verified byan alkali treatment. Nevertheless, the presence of small portions of galactose and xylose residues at the reducing ends of the carbohydrate chains suggested a possibility of exertion of endo-beta-galactosidase and endo-beta-xylosidase activities for the linkage regions.

Heterogeneity of Human Urinay Glycosaminoglycans

Crude urinary glycosaminoglycans (GAG) obtained from normal adult men were fractionated by ethanol-fractionation, followed by Dowex 1 column chromatography. The resulting acidic subfractions were qualitatively analyzed for monosaccharide composition and examined by electrophoresis on cellulose acetate membrane and paper chromatography before and after mucopolysaccharidase digestions. Some of the major subfractions were also examined by gel filtration and infrared spectral analysis. The result showed that urinary GAG were extremely heterogeneous in the molecular size, negative charge and the chemical composition. Hyaluronic acid, heparan sulfate, chondroitin sulfates A and C, chondroitin, dermatan sulfate and keratan sulfate were found in wide distribution in these acidic subfractions. Of these GAG, chondroitin sulfate isomers were distributed into almost all acidic subfractions, indicating to be the most heterogeneous.

Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver.

The hepatic concentration of a low molecular weight, metal-binding protein in 1- to 4-day-old rats is 20 times that in 70-day-old adults. Biochemical purifications were performed to characterize this metal-binding protein in the liver of 1- to 4-day-old rats. Gel filtration of hepatic cytosol on Sephadex G-75 demonstrated that the major Zn-containing fraction of newborns had a relative elution volume and low absorbance at 280 nm, similar to the metallothionein fraction of Zn-treated adult rats. These fractions were further purified by DEAE-Sephadex A-25 chromatography and two major subfractions (MT-A and MT-B) were obtained from both the newborns and Zn-treated adults, with the corresponding subfractions eluted in buffer of similar conductivity. MT-A and MT-B of newborns also exhibited mobilities on polyacrylamide gel electrophoresis similar to the corresponding subfractions from Zn-treated adults. Both subfractions from the newborns had an abundance of half-cystine and a deficiency in most aromatic amino acids which are characteristics of metallothionein. Therefore, according to all of the criteria commonly used for the indentification of metallothionein, these data demonstrate that the metal-binding protein which is highly concentrated in newborn liver is metallothionein. The variation of the concentration of hepatic and renal metallothionein with age was also determined.The hepatic concentrationmore » peaks at 1 to 4 days of age and falls rapidly after that, reaching the adult level at 28 days of age. However, the renal concentration showed no variation with age.« less

THE TURNOVER AND SUBCELLULAR LOCALIZATION OF THE CHICK FOREBRAIN SMALL NUCLEAR RNAs

AbstractAnalysis of 2 day old chick forebrain RNA on polyacrylamide gels revealed the presence of the 29S, 18S, 5.5s and 5s rRNAs, 4S tRNA and the small mol wt nuclear RNA (snRNA) species K, L, C, DD and G. The turnover of these molecules was studied following the intracerebral injection of [3H]uridine into 2 day old animals. The specific activities of these molecules declined over a 23 day time course and all displayed first‐order decay kinetics. Apparent half‐lives (in days) for 29S (7). 18S (S), L (24), C (6.5), DD′ (8.5), 5.5S (7), 5S (13), G (6) and 4S (7) were obtained.The subcellular localization of the snRNAs was studied in 5 day old chick forebrain. The snRNAs accounted for almost 17% of nuclear RNA, in contrast to their low levels in whole forebrain (1.2%). This was due to an approx 20‐fold nuclear enrichment of C, DD′, G′ and H. Relatively low levels of K and L were present within the nuclear fraction. However, K and L, and to a lesser extent C and DD, were readily detected within the post‐microsomal supernatant. Production of a pH 5 enzyme fraction from this supernatant resulted in a 2‐3‐fold enrichment of K, L, C and DD′ with respect to tRNA. The polysome containing fractions also displayed significant levels of L with about 1 mol of L per 30 of 5S rRNA.A new method for the determination of the subcellular distribution of the snRNAs is described. It depends upon the purity of the nuclear, microsomal and cell sap subfractions rather than upon total recoveries from such fractions.Utilizing the relative amounts of these RNA species within whole forebrain and the major subfractions, distributions (as a percentage within each fraction) were obtained. The observation of significant levels of K, L, C and DD within the cytoplasmic fractions raises some doubts about previous suggestions of an exclusively nuclear role for these molecules. It is proposed that the snRNAs may be involved in a multistage interaction within the process of eukaryotic gene expression.

Intrinsic ADP-Ribose Transferase Activity versus Levels of Mono(ADP-Ribose)Protein Conjugates in Proliferating Ehrlich Ascites Tumor Cells

Transition of proliferating Ehrlich ascites tumor cells (3 days after transplantation) to the non-proliferating status (8--14 days after transplantation) was associated with an increase in total mono (ADP-ribose) protein conjugates. This increase was largely confined to the NH2OH-resistant subfraction. When the amounts of mono-(ADP-ribose) conjugates from 20% trichloroacetic acid precipitates were compared with those from 5% perchloric acid precipitates, no significant differences were seen. This fact excludes histone H1 as a major mono (ADP-ribose) acceptor in vivo in these cells. Transition to the resting state was also associated with a small decrease in NAD levels, and with no significant changes of total ADP-ribose transferase activity. However intrinsic ADP-ribose transferase activity as expressed in permeabilized cells was increased, being correlated with the changes in the level of the NH2OH-resistant mono (ADP-ribose) protein conjugates. This shows that alterations in intrinsic transferase activity may, in general, indicate similar alterations in major subfractions of ADP-ribose conjugates. Intrinsic ADP-ribose transferase activity exhibited an inverse relationship to ornithine decarboxylase activity.

Interaction of plasma high density lipoprotein HDL2b (d 1.063–1.100 g/ml) with single‐bilayer liposomes of dimyristoylphosphatidylcholine

AbstractIncubation of a major subfraction, HDL2b (d 1.063–1.100 g/ml), of human plasma high density lipoproteins, HDL (d 1.063–1.21 g/ml), with single‐bilayer liposomes of dimyristoylphosphatidylcholine (DMPC) resulted in uptake of DMPC by the HDL2b and dissociation of lipid‐free apolipoprotein A‐I (apoA‐I). In the presence of excess DMPC, the dissociated apoA‐I was also incorporated with DMPC into discoidal complexes. Preliminary studies with model apoA‐I‐DMPC complexes indicated that they also can interact with native HDL2b with the resultant transfer of their DMPC to HDL2b and the concomitant release of their apoA‐I. After interaction of HDL2b with DMPC liposomes, the DMPC‐enriched HDL2b product showed a lower hydrated density and a larger particle size than the control HDL2b. The molecular properties of the lipoprotein product suggest that stabilization of the apoA‐I‐depleted HDL2b probably occurred via substitution of DMPC for the apoA‐I at the HDL2b surface rather than by fusion of the apoA‐I‐depleted HDL2b. The above interactions of HDL2b with single‐bilayer liposomes and discoidal complexes indicate pathways of phospholipid transfer relevant to the possible role of HDL in the metabolism of lipoprotein surface components in vivo.

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.

A study of tobacco carcinogenesis. XIII. Tumor-promoting subfractions of the weakly acidic fraction.

The weakly acidic fraction (WAF) of cigarette smoke particulate matter was fractioned by silica get chromatography. We assayed the various primary subfractions for potential tumor-promoting activity by measuring the incorporation of tritiated thymidine into mouse epidermal DNA as induced by these subfractions. Based on these results and on chemical composition, the primary subfractions, were then combined into four major subfractions and tested on initiated mouse skin for tumor-promoting activity by long-term application. Two of these subfractions (40% of WAF) were inactive, whereas the other two (18 and 35% of WAF) showed tumor-promoting activity. The two active portions were then further chromatographed and tested by the short-term bioassay. Some major components of the resulting active fractions included alkyl-2-cyclopenten-2-ol-1-ones, catechols, hydroquinone, fatty acids, and 3-hydroxypyridines. Among these components, catechol, hydroquinone, 3-hydroxypyridine, 6-methyl-3-hydroxypyridine, linolenic acid, and linoleic acid were inactive as tumor promoters in the experimental animal. The activity of the alkyl-2-cyclopenten-2-ol-1-ones is unknown. Other components remain to be identified.

Lipid Metabolism in Pregnancy. III. Altered Lipid Composition in Intermediate, Very Low, Low, and High-Density Lipoprotein Fractions

The hyperlipidemia of pregnancy consists primarily of an increase in triglyceride with lesser rises in cholesterol and phospholipid. As a further characterization, we have analyzed all lipids in the major lipoprotein subfractions in fasting pregnant and non-pregnant women. An elevated triglyceride in the major lipoprotein fractions in pregnancy is confirmed. The triglyceride rises in VLDL and IDL (density 1.006-1.019 lipoprotein) are associated with proportional rises in cholesterol and phospholipid. The result is a 3-4-fold increase of compositionally unchanged lipoprotein lipid. Contrasting changes are seen in LDL, density 1.019-1.063 lipoprotein, and HDL. In these fractions, triglyceride rises more than cholesterol and phospholipid. As a result, an increase in triglyceride on a percentage basis tends to reduce the contribution of the other two lipids. Nonetheless, on an absolute basis HDL cholesterol is not significantly reduced. The proportional increases in all lipids of VLDL and IDL fractions are consistent with increased VLDL production in pregnancy as suggested by data from animal systems. However, alterations in removal are not rules out. Maintenance of the HDL cholesterol level distinguishes pregnancy from other endogenous hypertriglyceridemias where HDL cholesterol is reduced. One may speculate that these physiological adaptations in material lipid transport can serve the increased energy needs of the mother, supply steroid hormone precursors for the placenta, and provide cholesterol and essential fatty acids for the fetus.

Investigation of possible intestinal peptidase deficiency in coeliac disease

The possibility that patients with coeliac disease may lack an enzyme normally involved in the later stages of digestion of certain components of gliadin was explored further. Gliadin, from wheat gluten, was digested with pepsin, trypsin and pancreatin and the resultant peptides separated into 13 fractions by ion exchange chromatography. Components present in Fraction 9 appeared to be digested to a lesser extent by duodenal mucosa from coeliac children in remission than by normal mucosa, whereas all other fractions were equally well digested. The major amino acids remaining after digestion of Fraction 9 with coeliac mucosa were glutamine/glutamic acid, proline and serine, suggesting that a peptidase or peptidases capable of hydrolysing this combination of amino acids is lacking in patients with coeliac disease. Re-chromatography of Fraction 9 yielded two major sub-fractions each containing peptides having leucine and glutamine as the major N-terminal amino acids.

Genes coding for polysomal 9S RNA of sea urchins: conservation and divergence.

The DNA sequences coding for the individual major subfractions of the putative histone mRNAs are all 1,200-fold reiterated and are contained in a “cryptic” density satellite in Psam-mechinus DNA. While random mutational drift cannot be detected in the 9S mRNA coding sequences within the species, it is very much in evidence during the evolution of two related sea urchin species.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: physical and chemical studies of low and high density lipoproteins.

LOW DENSITY LIPOPROTEINS (LDL) AND HIGH DENSITY LIPOPROTEINS (HDL) FROM THE PLASMA OF PATIENTS WITH FAMILIAL LECITHIN: cholesterol acyltransferase (LCAT) deficiency have been characterized by gel filtration, analytical ultracentrifugation, and gel electrophoresis, and their relative content of lipid and protein has been determined. The LDL of d 1.019-1.063 g/ml show marked heterogeneity. A subfraction of the LDL emerges from columns of 2% agarose gel with the void volume, has corrected flotation rates (S(f) degrees ) in the range of 20-400, and contains 4-10 times as much unesterified cholesterol, phosphatidylcholine, and triglyceride per mg protein as normal LDL. A major subfraction of the LDL emerges from the gel in the same general position as normal LDL, but exhibits somewhat higher flotation rates and contains 1.5-3 times as much unesterified cholesterol and phosphatidylcholine and 13 times as much triglyceride per mg protein. The HDL, shown to be heterogeneous in earlier studies, are mainly comprised of molecules which have flotation rates of F(1.20) 3-20, migrate in the alpha(1)-alpha(2) region on electrophoresis, and contain about 12 times as much unesterified cholesterol and 5 times as much phosphatidylcholine per mg protein as normal HDL. Smaller molecules are also detected, which have flotation rates of F(1.20) 0-3, migrate in the prealbumin region on electrophoresis, and contain only slightly more unesterified cholesterol and phosphatidylcholine per mg protein than normal HDL.

A comparative study of gangliosides from brains of various species.

AbstractA comparison is made of the sialic acid and hexose content of crude gangliosides from the brains of 13 species, including mammals, a bird, and amphibian, a reptile and fish. The sialic acid content is relatively constant from species to species. Gangliosides are less abundant in tissues other than the brain. Four species (cat, dog, pig and sheep) were selected for the determination of their major ganglioside subfractions. The ganglioside subfractions were isolated (after extraction and partition dialysis) using descending thin layer chromatography. The population of ganglioside molecules varied from species to species. From dog and sheep a mono‐, a di‐ and a trisialoganglioside were obtained; from cat a mono‐, a di‐ and two trisialogangliosides; and from pig a mono‐ and two disialogangliosides. Each ganglioside subfraction was found to contain glucose, galactose and galactosamine in the ratio of approximately 1∶2∶1. The fatty acid moieties consisted of more than 80% stearic acid with lesser amounts of arachidic, palmitic and behenic acid. Sphingosine analyses indicated ratios of sphingosine to icosisphingosine of 7∶3 for the monosialo‐, 1∶1 for the disialo‐ and 3∶7 for the trisialogangliosides.