Saul Roseman

The Svennerholm resorcinol method for the quantitative determination of the sialic acids was modified by introducing a periodate oxidation step prior to heating with the resorcinol reagent. Sialic acid glycosides could be determined without acid hydrolysis. The glycosides gave chromogens stable to periodate oxidation at 37°, whereas free sialic acid gave chromogens that were destroyed at this temperature, but were stable at 0°. Using these properties, the periodate-resorcinol method was applied to the determination of total, free, or bound sialic acid. The periodate-resorcinol method was substantially more sensitive than the resorcinol procedure, was not affected by lipids, amino acids, or sugars, and could be used to detect free or glycosidically bound sialic acids on paper chromatograms.

Abstract The following trisaccharide unit terminates the oligosaccharide chains of several plasma glycoproteins: sialic acid → galactose → N-acetylglucosamine → oligosaccharide → protein. Previous studies showed that this trisaccharide unit was synthesized by the sequential action of three glycosyl-transferases isolated from goat colostrum; each glycosyltransferase catalyzed the transfer of a monosaccharide residue (N-acetylglucosamine, galactose, or sialic acid) from its nucleotide derivative to the appropriate glycoprotein acceptor. The present studies are concerned with the intracellular location of these glycosyltransferases in rat liver. Kinetic studies were first performed to establish optimum conditions for determining quantitatively the particle-bound rat liver glycosyltransferases; these enzymes exhibited similar properties to the soluble, partially purified glycosyltransferases previously obtained from goat colostrum. The subcellular localization of the enzymes was then investigated by two techniques, differential centrifugation and discontinuous sucrose density gradient centrifugation. In these studies, the following were used as markers for various subcellular organelles: DNA, RNA, glucose 6-phosphatase, NADPH-cytochrome c reductase, acid phosphatase, 5'-nucleotidase, and glutamic dehydrogenase. The results indicated that the glycosyltransferases were all located in the same membranous subcellular component and that this component was different from organelles containing the above marker substances; the active subcellular particle was characterized by electron microscopy as being rich in Golgi apparatus. The apparent location of the glycosyltransferases in the Golgi apparatus suggests that these enzymes may be involved in terminating the synthesis of plasma glycoproteins by the liver during secretion, and may possibly be required for secretion of these proteins.

Mammalian tissues contain a kinase involved in the intermediary metabolism of the sialic acids.1' 2 This enzyme has been extensively purified,3 studied in detail, and catalyzes the following reaction: N-Acyl-D-mannosamine + ATP Mg++ N-Acyl-D-mannosamine-6-P + ADP.To determine whether this kinase occurred in bacteria, such as Aerobacter cloacae and Escherichia coli K235,4 that metabolize N-acetyl-D-mannosamine, extracts of these organisms were examined and found to contain a novel phospho-transferase system.The system obtained from E. coli K235 consisted of two enzymes, I and II, and a histidine-containing, heat- stable protein (HPr).The sequence of reactions is: I Phosphoenolypyruvate (PEP) + HPr Mg+ Phospho-histidine- protein (P-HPr) + Pyruvate (A) P-HPr + Hexose -., -Hexose-6-P + HPr (B)PEP + Hexose M Hexose-6-P + Pyruvate (A+B)The intermediate in the system, P-HPr, is protein-bound phosphohistidine.Materials and Methods.-Unlessotherwise specified, all materials were obtained from comi- mercial sources.Previously published methods', 6 were used for the preparation, separation, and characterization of C"1-and C'4-hexosamines, N-acylhexosamines, the corresponding 6-phosphate esters, and for the periodate oxidation of the esters and the characterization of glycolaldehyde- phosphate.The following compounds were prepared as described: P-histidine,6 N-phospho- glycine,7 phosphoramidate,8 and PEP."An essential substrate for these experiments, P32-PEP was prepared enzymatically by a published procedure'0 and with the invaluable help of Dr. M. F. Utter and Mr. Douglas Kerr,-to whom we are most grateful."The P32-PEP (5-10 ,umoles per experiment) contained 200-400 c of P8a per ,umole and was purified by ion-exchange chromatog- raphy; paper chromatography and electrophoresis indicated that it was homogeneous.It was diluted with unlabeled PEP prior to use.. Purification of enzymes I, and II, and HPr: The organism, E. coli K235, was grown to the sta- cionary phase in Todd-Hewitt (Difco) broth supplemented with 1.5% glucose in a New Brunswick fermentor.Maximum yields of the phospho-transferase system were obtained when the culture was stirred during growth but without passage of air through the sparger.After washing with 1% KCl solution, the wet cell paste was stored at -18°.The cells were ruptured by sonic oscilla- tion following suspension in 0.025 M phosphate buffer, pH 7.6 (containing 0.1% 2-mercapto- ethanol and 10-3 M EDTA when enzymes I and II were desired).After centrifugation, the supernatant fluid (crude extract) was treated with charcoal to remove HPr and fractionated for I and II as outlined in Table 1.The critical step was the Qy alumina gel treatment since I was adsorbed while II was not; after washing the gel with 0.01 and 0.05 M phosphate buffers, pH 7.6, I was eluted with 0.10 M buffer.These data suggest that both enzymes were purified approximately 300-fold.Since we have not yet determined which enzyme, I or II, was present at rate-linmiting concentrations prior to their separation, the purification factor is cor- rect for only one of these enzymes, and is not known for the other.However, the availability of the purified enzymes I and II will now permit accurate analysis for each enzyme.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.