Isolation and characterization of glycosphingolipids with J blood group activity from bovine spleen1,2

Two glycosphingolipids with J blood group activity were found in J-positive bovine spleen. They were tentatively identified as ceramide deca- and dodecahexosides containing galactose, glucose, N -acetylgalactosamine and N -acetylglucosamine in a molar ratio of 5:3:1:1 and 6:3:2:1, respectively. Fucose was not present. Ceramide decahexosides without J activity were also found in J-negative bovine spleen. The principal component fatty acids of the J-active glycosphingolipids were saturated even-numbered long-chain acids with 16 to 24 C atoms. Their principal long-chain bases were sphingosine and dihydrosphingosine with smaller amounts of phy-tosphingosine.
Both J-active glycosphingolipids were readily water-soluble and showed strong activity in the bovine J and in the porcine A blood group system. They exhibited no cross-reactivity in the human A system. However, a J-negatiye glycosphingolipid fraction - also from J-negative spleen - with shorter carbohydrate chain-length showed strong activity in the human A system.     

Occurrence of J blood group activities in lipid and non-lipid fractions of organs and body fluids of cattle

Total lipids and protein-containing residues obtained after lipid extraction from various organs and body fluids of J-positive cattle were tested for J activity in the bovine J blood group system. Polar lipids prepared by column chromatography of total lipids, which contain predominantly neutral lipids, were also tested. Total lipids (or polar lipids, respectively) were analysed for lipid phosphorus, lipid sugar, and hexosamine. Both lipids and non-lipid fractions of brain, myocardium, skeletal muscle, and adipose tissue show no J activity. The lipids of urinary bladder epithelium, spleen, liver, and kidney are J-positive, whereas their non-lipid fractions are J-negative. Both the lipids and the non-lipid fractions of seminal plasma, spermatozoa, and faeces are J-active. The lipids extracted from hair show no J activity, while those of cornea and eyelens are J-active. The high amount of glycolipids from seminal plasma, spermatozoa and spleen stimulates further studies of these lipids.     

Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH4, LiBH4), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing anunusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactosamine unit as suggested previously.     

Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH₄, LiBH₄), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing an unusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactos-amine unit as suggested previously.     

The transfer of bovine J blood group activity to erythrocytes: chemical nature of transferable and of non-transferable J1

The bovine J blood group substance exists as a glycosphingolipid (ceramide deca-hexoside as well as ceramide dodecahexoside) and as a glycoprotein. The lipidic form occurs in erythrocyte membranes, both forms are found in serum. The lipidic J substances were isolated from erythrocytes and from serum, and identified by thin-layer chromatography with lipidic J substances isolated from spleen. The glycoprotein nature of the non-lipidic J of serum was evident by pronase-catalysed hydrolysis yielding J-active glycopeptides of lower molecular weights. The lipidic J was completely extracted from lyophilized stroma with chloroform/methanol. From lyophilized serum, however. it was completely extracted only in the presence of water, indicating different binding partners in serum and in erythrocyte membranes. The J lipid was incorporated as intact molecule into the erythrocyte membrane by a simple incubation technique. The incorporation was inhibited by various glyc-erophospholipids (called blockers). The J glycoprotein could not be transferred to the erythrocyte membrane. Three methods are descrjbed which are suitable for the preparation of a blocker-free fraction enriched with J lipids from J-positive serum.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Structural characterization of blood group A glycosphingolipids recognized by the antibody 3G9-A

In this study, the antibody 3G9-A was assayed for activity against human erythrocyte glycosphingolipids. The antibody was found to recognize glycosphingolipid components from blood group A erythrocytes but not glycosphingolipids from blood group B or O erythrocytes. Subsequent investigation revealed that the glycosphingolipid components recognized by the antibody were also recognized by a blood group A specific monoclonal antibody. The structures of two of the isolated active glycosphingolipid components were structurally characterized using proteon nuclear magnetic resonance (¹H NMR) and gas chromatography-mass spectrometry (GC-MS) techniques and were found to consist of two blood group A glycosphingolipids; the type 2 chain A^b and type 3 chain A^a glycosphingolipids. Subsequent analysis of the remaining active components by GC-MS and immunostaining techniques revealed that all of the active components were blood group A glycosphingolipids. Furthermore, structural studies of the active components suggested that the epitope of the antibody consisted of the group A trisaccharide, GalNAc1,3(Fuc1,2)Gal.Abbreviations GC-MS gas chromatography-mass spectrometry - ¹H NMR proton nuclear magnetic resonance - Gal d-galactose - Glc d-glucose - Fuc l-fucose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Cer ceramide - mAb monoclonal antibody - BSA bovine serum albumin - PBS phosphate buffered saline - FID free induction decay - PMAA partially methylated alditol acetates     

Transfer of bovine J-blood-group determinant onto erythrocytes: isolation and identification of a blocker

The bovine J-blood-group determinant is transferred from a serum glycoprotein to an erythrocyte membrane lipid by incubation in vitro. This transfer is inhibited by a lipid (called 'blocker') occurring in bovine and human serum, in other bovine and human tissues, yeast and plant tissues. The blocker was isolated from bovine spleen and identified as phosphatidylserine. Moreover, phosphatidylinositol acts as a blocker, while a variety of other phospholipids, glycosphingolipids and neutral lipids have no function as blockers. Mild alkaline deacylation deletes the blocker activity of both phosphatidylserine and phosphatidylinositol. Methyl esters of these phospholipids, or exchange of the amino group for a hydroxyl group in phosphatidylserine or N-benzoylation of phosphatidylserine, do not affect the blocker function. The blocker function of phosphatidylinositol is lost after periodate oxidation. The blocker reacts with the J-containing serum protein, not with the erythrocyte membrane. After preincubation of the J-positive serum protein with the blocker and reextraction of excess blocker, the serum protein remains J-positive, but is then unable to transfer the J determinant to the erythrocyte membrane.     

Immunohistochemical characterization of purple acid phosphatase-containing leucocytes in the human placenta

Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.     

Human blood group glycosphingolipids of porcine erythrocytes.

Two glycosphingolipids with human blood group A and H antigenicity were isolated from porcine erythrocyte membranes which were obtained from the pooled blood. The yield of the A- and H-antigenic glycolipids was approximately 0.2 and 0.1% of total neutral glycolipids, respectively. No B antigen was detected. Through several methods the porcine erythrocyte antigens were all found to belong to lactoseries (type 1 chain), IV2Fuc alpha, IV3GalNAc alpha Lc4Cer for type A and IV2-Fuc alpha Lc4Cer for type H, in contrast to the antigenic glycolipids in human erythrocytes, which mostly belong to neolactoseries (type 2 chain). The constituent fatty acids of the A antigen were 75% normal acids and 25% 2-hydroxy acids, and the long chain base was 95% sphingenine. This is the first demonstration of the A- and H-antigenic glycolipids on erythrocytes of pig in whose gastric mucin the human blood group A and H substances have been demonstrated.     

Non-acid glycosphingolipid expression in plasma of an A1 Le(a-b+) secretor human individual: identification of an ALeb heptaglycosylceramide as major blood group component.

Total non-acid glycosphingolipids were isolated from plasma of an A1 Le(a-b+) secretor individual with Refsum's disease (phytanic acid storage disease). The glycolipids were separated into 11 fractions by open column chromatography and by HPLC. The fractions were analyzed by thin-layer chromatography and tested for different blood group A activities as well as blood group Le(a )and Leb activity. The fractions were structurally characterized by proton NMR spectroscopy and FAB mass spectrometry and in selected cases by EI mass spectrometry of the permethylated and permethylated-reduced derivatives. Degradation analysis was performed on partially permethylated or permethylated-reduced alditol acetates. The dominating blood group compound was found to be a blood group A active type 1 chain difucosylheptaglycosylceramide. Other blood group compounds were identified as a blood group A active type 1 chain monofucosylhexaglycosylceramide, a blood group Leb hexaglycosylceramide, a blood group H active type 1 chain pentaglycosylceramide, and a globotetraosylceramide (the P-antigen). The presence of a Le(a) glycosphingolipid and blood group A type 3/4 chain structures were also found by immunostaining. Glucosyl-, lactosyl-, and globotriaosylceramides were the dominating short chain compounds. The amount of phytanic acid incorporated into the monoglycosylceramide fraction was found to be less than 5% of the fatty acids.     

Screening for the presence of polyglycosylceramides in various tissues: partial characterization of blood group-active complex glycosphingolipids of rabbit and dog small intestines

Twenty different human and animal tissues were investigated for the presence of polyglycosylceramides. The glycolipids were isolated by peracetylation of dry tissue residues left after conventional lipid extraction, followed by extraction with chloroform and subsequent Sephadex LH-20, Sephadex LH-60 and silica gel chromatography. In most of the cases only trace amounts of complex glycolipids were found. Distinct bands of glycosphingolipids migrating on TLC plates in a region of brain gangliosides and below were observed in bovine erythrocytes, human leukocytes and human colon mucosa. Definite fractions of polyglycosylceramides were isolated from rabbit small intestine, dog small intestine, human placenta and human leukocytes. The polyglycosylceramides of dog and rabbit intestine were characterized by colorimetric analysis, methylation analysis, mass spectrometry and immunological assays. The dog material contained branched carbohydrate chains with repeated fucosylated N-acetyllactosamine units. Rabbit intestine polyglycosylceramides resembled rabbit erythrocyte polyglycosylceramides with Hex-Hex- terminal determinants but were more complex in respect of sugar composition and structure. The material isolated from dog intestine showed A, H, Lex and Ley blood group activities. Polyglycosylceramides of human erythrocytes, placenta and leukocytes showed strong binding affinity for Helicobacter pylori, while polyglycosylceramide fractions from rabbit and dog intestine were receptor-inactive for this bacterium or displayed only weak and poorly reproducible binding. Abbreviations: C, chloroform; M, methanol; Hex, hexose; HexNAc, N-acetylhexosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; TLC-thin layer chromatography; FAB/MS, fast atom bombardment mass spectrometry; GC/MS, gas chromatography-mass spectrometry; PGCs, polyglycosylceramides; EI/MS, electron impact ionization mass spectrometry; PBS, phosphate-buffered saline; BSA, bovine serum albumin. The carbohydrate and glycosphingolipid nomenclatures are according to recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (Lipids (1977) 12:455–68; J Biol Chem (1982) 257:3347–51; and J Biol Chem (1987) 262:13–18) This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of B and H blood-group active glycosphingolipids from human B erythrocyte membranes

Two blood group B active glycosphingolipids (B-I and B-II) previously isolated and highly purified from human B erythrocytes [21] were analysed first by degradation with α-D-galactosidase from coffee beans, α-L-fucosidase from bovine kidney and with 0,1 N trichloracetic acid; the native B-glycolipids as well as their degradation products were then investigated by methylation analysis with combined gas chromatography-mass spectrometry, by thin layer chromatography, twodimensional immunodiffusion and by the hemagglutination inhibition technique. Together with the results obtained by mass spectrometry of permethylated glycolipids [26] the following structures were elucidated: α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-I glycosphingolipid and α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-II glycosphingolipid. A H active glycolipid fraction from B erythrocytes further purified by thin layer chromatography was also investigated by methylation analysis. The pattern of its partially methylated alditol acetates was essentially the same as that of the α-galactosidase treated and permethylated B-I glycolipid. It also exhibited strongly precipitating and hemagglutination inhibiting H properties as well as the two α-galactosidase treated B-I and B-II glycosphingolipids. Based upon these data the following tentative structure was proposed: α-L-fucopyranosyl-(1 → 2)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide. Gas chromatographic analysis revealed sphingosine and lignoceric, nervonic and behenic acids to be the main components of the ceramide residues of the three glycosphingolipids. From the data presented the H active substance very probably can be regarded as the immediate precursor of the B-I glycosphingolipid from human B erythrocyte membranes.     

Blood group A glycosphingolipid accumulation in the hair of patients with alpha-N-acetylgalactosaminidase deficiency

In the hair of individuals with blood group AB, the level of blood group A glycosphingolipids is much lower than that of blood group B. We hypothesized that in hair, blood group A determinants are converted by alpha-N-acetylgalactosaminidase (alpha-NAGA, E.C.3.2.1.49) to H determinants. To address our hypothesis, the relative amount of ABH glycosphingolipids in hairs and nails of normal subjects, patients with Kanzaki disease, and heterozygous carriers of alpha-NAGA deficiency were analyzed by dot-blotting and enzyme-linked immunosorbent assay. In hair from normal subjects with blood group B, ABH glycosphingolipids consisted of 88% blood group B- and 12% blood group H glycosphingolipids. In blood group A subjects, 14% were group A- and 86% were group H glycosphingolipids. In Kanzaki patients, 81% were blood group A- and 19% were blood group H glycosphingolipids. In 2 alpha-NAGA deficiency carriers, the ABH glycosphingolipids consisted of 67% blood group A- and 33% blood group H glycosphingolipids. These results indicate that blood group A glycosphingolipids are catabolized to H glycosphingolipids by alpha-NAGA, resulting in lower levels of blood group A glycosphingolipids in the hair of normal subjects, and alpha-NAGA deficiency causes accumulation of blood group A glycosphingolipids in the hair of Kanzaki patients. This finding is of clinical relevance because it suggests that hair may be used to diagnose and assess the alpha-NAGA status of individuals.     

Mass spectrometric analysis of permethylated glycosphingolipids I. Sequence analysis of two blood-group B active glycosphingolipids from human B erythrocyte membranes.

Two blood group B active glycosphingolipids (B-I and B-II) formerly isolated and purified from human B erythrocytes (16) were investigated by mass spectrometry after permethylation. B-I yielded fragments up to m/e 1266 and B-II up to m/e 1495, showing the sequence of six and seven carbohydrate residues respectively. In combination with additional experimental evidence (18) the glycosphingolipids are demonstrated to be a gal-[ fuc ]-gal-glcNAc-gal-glc-ceramide (B-I) and a gal-[ fuc ]-gal-glcNAc-gal-glcNAc-gal-glc-ceramide (B-II). Mass spectrometric evidence for the ceramide residues are also obtained indicating besides spingosine C24-,C24:1-, and C22-fatty acids as main constituents.     

Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13

The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Glycosphingolipids from rabbit aorta, plasma, and red blood cells: effects of high cholesterol-high fat diets on fatty acid distribution and quantity of glycosphingolipids

Four glycosphingolipids were isolated from rabbit aorta, plasma, and red blood cells. They were identified, by thin-layer chromatography and by quantitative analysis of hexose and fatty acid, as cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The rabbits had been maintained on a normal diet or on one of three high cholesterol diets for 180 days. The quantities of the glycosphingolipids and their fatty acid distributions were determined, and comparisons were made between the control and experimental animals. Aorta and plasma glycosphingolipids were more affected by the high cholesterol diets than were those from red blood cells. The effects on aorta and plasma glycosphingolipids were similar. The amount of cerebroside was increased in aorta and plasma in all animals in the experimental groups. The amount was also increased in red blood cells in rabbits from two of the experimental groups. The average fatty acid chain length was greater in the lipids from the experimental animals than in those from the control animals for all measured glycosphingolipids from aorta. The average chain length was also greater in cerebrosides from the experimental animals from all three tissues. Probably the most notable differences in the experimental animals were the increased 24:1/24:0 ratios and the increased concentrations of 24:2. These increases occurred in nearly all samples from plasma and aorta, but not in red blood cells. There was also an increase of total unsaturated fatty acids in aorta cerebrosides from the experimental animals. Except for the increase in 24:2, lard generally caused more deviation from normal than did cottonseed oil when the level of cholesterol in the diet was 1%.     

Isolation of Relatively Large Amounts of Endomorphin-1 and Endomorphin-2 From Human Brain Cortex

Hackler, L., J. E. Zadina, L-J. Ge and A. J. Kastin. Isolation of relatively large amounts of endomorphin-1 and endomorphin-2 from human brain cortex. Peptides 18(10) 1635–1639, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endo-morphin-2 (Tyr-Pro-Phe-Phe-NH₂) were previously isolated from bovine brain. Both peptides showed the greatest selectivity and affinity for the mu opiate receptor of any endogenous substance found to date and may serve as natural ligands for the mu-opiate receptor. We have purified them from the fronto-parietal cortex of human brain tissue by solid phase extraction and high performance liquid chromatography. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against endomorphin-1. The isolated endomorphins showed full biological activity. The tetrapeptides were found in human brain in much higher amounts than in bovine frontal cortex.