Anne Stoffyn

The estimation of urinary metabolites of administered estradiol-17 β-16-C14

Abstract A procedure for the quantitative estimation of five identified urinary metabolites and two groups of urinary metabolites of estradiol-C 14 is described. It is based upon ( a ) hydrolysis of glucosiduronates with bacterial β-glucuronidase, ( b ) separation of neutral from phenolic substances with an anion-exchange resin, and ( c ) the separation by gradientelution partition chromatography of 2-methoxyestrone, estrone, estradiol-17β, 16-epiestriol, and estriol. In addition, a group of metabolites eluted between estradiol-17β and 16-epiestriol and a group of metabolites more polar than estriol may be separated and estimated.

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.

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.

Structure of trihexosylceramide biosynthesized in vitro by rat kidney galactosyltransferase

Abstract Trihexosylceramide biosynthesized in vitro from lactosylceramide and UDPgalactose in the presence of galactosyltransferase from rat kidney microsomes was shown by permethylation to consist of two positional isomers in which the galactopyranosyl units are linked 1 → 3 and 1→ 4, respectively.

A method for the determination of the chemical structure of glycolipids biosynthesized in vitro and the specificity and activity of glycosyltransferases

A very sensitive method for the qualitative and quantitative investigation of the biosynthesis of the oligosaccharide chains of glycolipids in such tissues as cultured cells, biopsies, or cell organelles, is described. It permits the determination, on an ultramicro-scale, of the specificity of glycosyltransferases in terms of the exact chemical structure of the products that are synthesized from known labeled glycolipid precursors, and defines without ambiguity the precursor-to-product relationship in biosynthetic incubations in vitro.

Structure of trihexosylceramide isolated from rat spleen

Abstract Trihexosylceramide isolated from rat spleen was shown by methylation to consist mainly of a molecular species in which two galactosyl residues are linked 1→ 3. Thus a compound of this structure occurs normally in rat spleen. This fact is consistent with the previously reported biosynthesis in vitro of galactopyranosyl-α-(1→3)-galactopyranosyl-β-(1→4)-glucopyranosyl-β-(1→1)-ceramide in the presence of galactosyltransferases from rat spleen and bone marrow.

STRUCTURE OF THE OLIGOSACCHARIDE MOIETY OF GLYCOSPHINGOLIPIDS BIOSYNTHESIZED IN VITRO. ACTIVITY AND SPECIFICITY OF GLYCOSYL TRANSFERASES

Abstract A methodology is described which permits the determination on a ultramicro-scale, of the activity and specificity of glycosyltransferases in terms of the chemical structure of the products synthesized from known labeled substrates. Its potential and limitations are discussed taking the biosynthesis of sialosylgalactosylceramide (G M4 ) and sialosyllactosylceramide (G M3 ) as examples.