Pierre Stoffyn

Identification of amino sugars by paper chromatography

Abstract A method is described which permits separation, identification, and determination of glucosamine and galactosamine by paper chromatography. Amounts as small as 2–5 μg. of one of the amino sugars can be detected in the presence of more than 100 μg. of the other.

PROTEOLIPIDS FROM MEMBRANE SYSTEMS

In 1951 Folch and Lees described the presence in brain tissue of a protein material insoluble in water and aqueous solvents and soluble in chloroform: methanol mixtures. Since all other known proteins were completely insoluble in these solvent mixtures, it was assumed that the proteins involved were combined with a lipid moiety that gave the complex its particular lipidlike solubility. To designate this new type of substances, they coined the term proteolipid to indicate that they were lipoproteins with some of the physical properties of lipids, in sharp contrast to other then known lipoproteins which were soluble in water and aqueous solutions and, not only insoluble in chloroform:methanol, but usually rendered insoluble in water (“denatured”) by exposure to this particular solvent mixture. Subsequent work (which is discussed below) showed that proteolipids are general tissue constituents usually associated with membranous structures and that, in addition to their unusual solubility properties, they are characterized by resistance to most proteolytic enzymes and by a pattern of amino acid composition that may vary according to the source of the proteolipid but retains throughout the group some special characteristics.

On the type of linkage binding fatty acids present in brain white matter proteolipid apoprotein

Summary Fatty acids (FA) which constitute about 2.5% by weight of exhaustively delipidated proteolipid apoprotein (PLA) have been shown to be bound mainly, if not exclusively, by ester linkages. Indeed, they are progressively released by mild alkaline hydrolysis, and reduction of PLA with NABH 4 yields corresponding fatty alcohols. The FA are not constituents of residual contaminating known lipids except possibly in a very small proportion.

BIOSYNTHESIS OF LACTOSYLCERAMIDE BY RAT BRAIN PREPARATIONS AND COMPARISON WITH THE FORMATION OF GANGLIOSIDE GM1 AND PSYCHOSINE DURING DEVELOPMENT

—This report deals with some properties of the enzyme from rat brain, which catalyses in vitro the formation of lactosylceramide, a probable precursor of the gangliosides, from UDP‐galactose and glucosylceramide. The enzyme is present in microsomes, mitochondria and synaptosomes, the latter having the highest specific activity, and appears to be firmly bound to the membranes of these particles. The enzymic activity is optimal at pH 6·8 and requires the presence of Mn2+.

Specificity of Sialyltransferase: Structure of α1‐Acid Glycoprotein Sialylated in vitro

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.

Structure of kidney ceramide dihexoside sulfate

Abstract 1. 1. It has been established by methylation that kidney ceramide dihexoside sulfate is a lactosyi ceramide with the sulfate group at C-3 of the galactose moiety. Thus, it is structurally related on the one hand to kidney neutral glycolipids which possess a lactosyl unit and on the other hand to the simpler galactose-containing cerebroside sulfate esters which have the sulfate group at C-3 of galactose. 2. 2. These results have been obtained by microtechniques using a few mg only of material.

The structure and chemistry of sulfatides

Recent advances in the chemistry of cerebroside sulfates which have led to a precise formulation of their structure and to a detailed knowledge of their composition are reviewed.Qualitative and quantitative analyses of the constituent fatty acids and sphingosine-type bases of cerebrosides and sulfatides have shown a close relationship between the cerebrosides and their sulfate esters.Sulfatides were converted into cerebrosides in high yield by mild desulfation with methanolic HCl at room temperature. The products of partial degradation of cerebrosides—ceramide and psychosine—were found to be identical to the products obtained under similar conditions from desulfated sulfatides.By methylation and periodic acid oxydation, the sulfate group has been located conclusively and apparently only at C-3 of the galactose moiety in the sulfatide molecule.Δ β-configuration of the galactosidic linkage in cerebrosides and in sulfatides has been demonstrated by direct comparison of the products of periodate oxidation of dihydropsychosine obtained from cerebrosides and from sulfatides, with the products of oxidation of 1-D-glycerityl β-D-galactoside of known configuration.Investigations of the composition and structure of sulfatides abnormally accumulated in the brain and kidneys in cases of metachromatic leukodystrophy, a demyelinating disorder, are reported.

A COMPARATIVE STUDY OF BRAIN AND KIDNEY GLYCOLIPIDS IN METACHROMATIC LEUCODYSTROPHY

Sulphatides and cerebrosides from white matter of brains of patients with metachromatic leucodystrophy (MLD) have been isolated and compared in fatty acid composition to those glycolipids found in MLD kidney tissue. A marked difference in glycolipid composition was found between the brain and kidney tissues. The sulphatides accumulated in MLD kidney have the same fatty acid profile as those found in normal kidney tissue and are typical‘kidney sulphatides.’The neutral glycolipids of MLD kidney retain larger amounts of the longer chain acids than do the cerebrosides of MLD brain white matter and thus resemble more closely in fatty acid composition, glycolipids of normal tissue.

Methylation of 2-acetamido-2-deoxy-d-hexoses

Abstract Methyl iodide in the presence of the dimethylsulfinyl carbanion leads to complete O -methylation and N -methylation of 2-acetamido-2-deoxyglycosides. Thus, methylation of methyl 2-acetamido-2-deoxy-α- d -glucopyranoside and -galactopyranoside gave, respectively, methyl 2-deoxy-3,4,6-tri- O -methyl-2-( N -methylacetamido)-α- d -glucopyranoside and -galactopyranoside. Methylation, under these conditions, with methyl- 14 C iodide showed that fully methylated 2-acetamido-2-deoxyglycosides, detectable in small amounts by radioautography, are obtained in quantitative yield. Methylation of methyl 2-acetamido-2-deoxy-6- O -trityl-α- d -hexopyranosides, followed by removal of the trityl group and acetylation gave methyl 6- O -acetyl-2-deoxy-3,4-di- O -methyl-2-( N -methylacetamido)-α- d -hexopyranosides. Separation and identification of these N -methylated 2-acetamido-2-deoxyhexosides was accomplished by g.l.c. and m.s. Acid hydrolysis of these derivatives gave the corresponding hydrochlorides.

Biosynthesis in vitro of mono- and di-sialosylgangliosides from gangliotetraosylceramide by cultured cell lines and young rat brain. Structure of the products, and activity and specificity of sialosyltransferase

Incubations in vitro of GA1, labeled with 3H in the terminal D-galactopyranosyl group, with nonradioactive CMP-NeuNAc in the presence of homogenates of C21 rat brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV 40-transformed 3T3 cells, chick embryo fibroblasts, Rous sarcoma virus-transformed chick embryo fibroblasts, and 9-day old rat brain resulted in all cases in the formation in high yield of GM1b, in which the neuraminidase-labile NeuNAc group is linked at O-3 of the terminal D-galactosyl residue, as shown by permethylation studies. No trace of the naturally occurring neuraminidase-stable GM1a was detected in any case. In addition, with NIE cells, and normal and RSV-transformed chick embryo fibroblasts, a disialosylganglioside (GD1) differing from GD1a and GD1b, and bearing only one substituent at O-3 of the terminal D-galactopyranosyl residue was formed. It was also biosynthesized from GM1b and CMP-NeuNAc by NIE and chick embryo cells but not by C21 cells, or rat brain. However, C21 cells and rat brain were capable of synthesizing GD1a from GM1a. Periodate oxidation degraded both NeuNAc groups in GD1 to a 7-carbon fragm:nt, indicating lack of substitution at O-8. GM1b could not be detected as a natural product in rat brain.