Characterization of three new fucolipids from hog gastric mucosa.

Three new fucolioids have been isolated from the water-soluble glycolipid fraction of hog gastric mucosa. Fucolipid A and C exhibited blood-group-A activity, whereas Fucolipid B was not active in A-anti-A, B-anti-B and H-anti-H systems. The structures of these glycolipids were identified by partial acid hydrolysis and methylation analysis, as: (see article)     

Complex fucolipids of hog gastric mucosa. Structure of the ceramide eikosahexoside

The structure of a complex fucolipid from hog gastric mucosa containing twenty sugar residues and exhibiting blood-group (A + H) activity has been investigated. Based on the results of immunological assays, partial acid hydrolysis, sequential degradation with specific exoglycosidases, oxidation with periodate and chromium trioxide, and permethylation analysis, we suggest that the carbohydrate chain of this fucolipid contains four termini. One of the termini bears beta Gal1 leads to 4 beta GlcNAc disaccharide, two bear blood-group A determinant and one bears H determinant. Two of the branches, terminated by beta Gal1 leads to 4 beta GlcNAc and blood-group A determinant, and two terminated by blood-group A and H determinants, are linked through beta Gal1 leads to 4 beta GlcNAc1 leads to 3/6 and beta Gal1 leads to 4 beta GlcNAc1 leads to 4 beta GlcNAc1 leads to 3/6 to the galactose residue adjacent to glucosylceramide core.     

Characterization of dog small intestinal fucolipids with human blood group A activity. Differences in dog and human A-active fucolipids.

Glycolipids containing fucose (fucolipids) which carried human blood group A activity were isolated from a number of dog small intestines and analyzed. On the basis of sugar analysis, methylation, periodate oxidation, enzyme degradation, mass spectrometry, and immunologic studies, a structure is proposed for these substances. The ceramides of the dog fucolipids containing only hydroxylated fatty acids with 85% saturated and 15% monoenoic acids ranging from 16 to 25 carbon atoms. Sphingosine and phytosphingosine comprised 48% each of the long chain bases. An A-active fraction isolated from human small intestine was shown to have two components, one of which was immunologically distinct and the other identical with the dog intestinal fucolipids. The human fraction differed from the dog fucolipids in migration on thin-layer chromatography and contained two types of amino sugar substitution. It is proposed that the human fraction was composed of two fucolipids with incomplete structures.     

Partial characterization of the highly complex fucolipids from gastric mucosa.

Six highly complex fucolipids containing 18, 20–21, 24, 28, 32, and 35–36 sugar residues, respectively, have been isolated from hog gastric mucosa. All six compounds exhibited blood-group (A+H) activities, and were different from each other with respect to the number of fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine residues. Based on the results of chemical, immunological and enzymatic analyses, we suggest that the carbohydrate chains of these glycolipids are highly branched. The branches, number of which is proportional to the degree of molecular complexity, are terminated by GalNAcα1→3(Fucα1→2)Gal, Fucα1→2Gal, Galβ→GlcNAc and βGlcNAc.     

The presence of blood group A-active glycolipids in cancer tissues from blood group O patients

Glycolipids were isolated from human gastric cancer tissues and normal mucosae. Sulfogalactosylceramide, ganglioside and neutral glycolipid fractions were separated by DEAE-Sephadex and silica gel column chromatography. Sulfogalactosylceramide contents were higher in the cancer tissues than in the normal mucosae. Ganglioside contents showed considerable variations but in the cancer tissues in mole percentage of ganglioside GM3 was higher than in the normal mucosae. The cancer tissues contained more neutral glycolipids than normal tissues. Glycolipids of lacto-series, including fucolipids, were markedly increased in the cancer tissues. Blood group A-active glycolipids were found in the cancer tissues from two patients with blood group O but not found in the uninvolved tissue associated with the cancer tissue.     

Characterization of dog small intestinal fucolipids with human blood group H activity.

Fucolipids with human blood group H activity were isolated from several dog small intestines. On the basis of mass spectrometry, periodate oxidation, enzyme degradation, methylation, and immunologic studies the following structure is proposed: Fucalpha(1 yields2)Galbeta(1 yields 4)Glc-NAcbeta(1 yields 3)Galbeta(1 yields 4)Glc-ceramide. The ceramide was shown by mass spectrometry to contain hydroxyhexadecanoic acid and phytosphingosine as major consitutuents.     

Structural studies on branched fucosphingolipids of hog gastric mucosa

A structural studies have been performed on new complex glycolipids extracted from hog gastric mucosa by 0.4 M sodium acetate in methanol-chloroform-water, and which were purified to homogeneity by DEAE-Sephadex and Florisil column chromatography and by preparative thin-layer chromatography in three solvent systems. Five branched fucolipids have been purified from this extract, three of which have been characterized previously [1] and remaining two were subject of this investigations. Based on the results of partial acid hydrolases, oxidation with periodate and chromium trioxide, permethylation and serological activities following structures were proposed:Fucolipid IVa

     

Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa.

Partial reactions of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa were studied by means of a rapid-mixing apparatus. At 21 degrees C, in the presence of 2 mM MgCl2 and 5 microM [gamma-32P]ATP there was a rapid phosphorylation of the enzyme with a pseudofirst order rate constant of 1400 min-1. Addition of the ATP about 120 ms before the MgCl2 increased this rate constant to 4400 min-1. In the absence of MgCl2 there was no phosphorylation. Addition of 4 or 10 mM KCl to the phosphoenzyme which had been formed in the absence of KCl produced a rapid initial rate of dephosphorylation (k = 2600 and 3200 min-1 respectively). An additional slow component of dephosphorylation was observed when unlabeled ATP was added together with the KCl (k = 700 to 900 min-1). At a 4 mM concentration, KCl stimulated the ATPase activity about 9-fold. At higher concentrations, the activity was reduced in parallel with a reduction of the steady state level of phosphoenzyme. Addition of KCl to the enzyme before the addition of ATP plus MgCl2 resulted in a low rate and extent of phosphorylation. KCl appeared to inhibit the phosphorylation at a level preceeding the E.ATP complex.     

Purification and properties of blood group A-active glycoprotein from oyster viscera

A blood group A active substance was isolated from an acetone-dried powder of oyster viscera by extraction with 0.1 M NaCl after heating a homogenate with extraction medium, in boiling water. After the removal of the acidic fraction with cetylpyridinium chloride, the separated neutral fraction was digested successively with α-amylase and amyloglucosidase to remove glycogen. The blood group A-active portion was eluted from a Sepharosé 4B column and purified by DEAE-Sephadex column chromatography. The purified active substance was homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated as 100 000 by sedimentation equilibrium.The sugar content of the purified active substance, expressed in percentage of dry weight, was galactosamine, 16.6; galactose, 12.8; fucose, 9.9; glucosamine, 4.6; and glucose, 3.3. Sialic acid was not detected. Total amino acid content was 23.0% and the main constituents were threonine, proline and serine. The ORD spectrum indicated that the hexosamines were N-acetylated. Absence of glycolipid was confirmed by the analysis of fatty acid and sphingosine base.This active substance had a strong blood group A activity (0.04 μg/ml) but neither B nor H activity; it interacted with lima bean lectin but not with concanavalian. A.