Joel A. Dain

The enzymic synthesis of ganglioside: IV. UDP-N-acetylgalactosamine:(N-acetylneuraminyl)-galactosylglucosyl ceramide N-acetylgalactosaminyl-transferase in rat brain

Abstract This paper describes the subcellular distribution and general characteristics of UDP-N-acetylgalactosamine:(N-acetylneuraminyl)galactosylglucosyl ceramide N-acetylgalactosaminyltransferase (UDP-N-acetylgalactosamine: GM3N-acetylgalactosaminyltransferase) in the rat brain. 1. 1. The subcellular distribution of the enzyme in 7 day-old rat brain found 44% of the activity in the microsomal and 36% in the mitochondrial particles. Subfractionation of the mitochondrial particles showed the enzyme to be primarily associated with the synaptic membranes within this fraction. 2. 2. The enzyme had greatest specificity for Gm3 (N-acetylneuraminyl) and required Mn2+ for optimal activity. This Mn2+ requirement could not be satisfied by Mg2+, K+, Cu2+, Ni2+, or Al3+. A broad pH optimum was observed between pH 6.8 and 7.2, and the Km values were 16.6 μM for GM3 and for UDP-N-acetylgalactosamine, 57 μM. 3. 3. The enzyme showed highest activity in brain tissue and the specific activity of the enzyme decreased with increasing age of the rats.

Sialyltransferases in rat brain: reaction kinetics, product analyses, and multiplicities of enzyme species.

Abstract— The incorporation of NeuNAc from CMP‐NeuNAc into endogenous glycolipids and glyco‐proteins, and exogenously added GM1a (monosialoganglioside) and desialylated fetuin (DS‐fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP‐NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM1a, 0.20 mm; DS‐fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM1a and exogenous DS‐fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM3, while exogenously added GM1a was converted to GD1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS‐polyacrylamide gel electrophoresis. Neura‐minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species.

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP‐N‐acetylgalactosamine: GM3 N‐acetyl‐galactosaminyl transferase and UDP‐galactose: GM2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7‐day‐old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the N‐acetylgalactosaminyl transferase involved in the formation of GM2 from GM3 is different from the N‐acetylgalactosaminyl transferase involved in the formation of GalNAoGal‐Glc‐ceramide from Gal‐Glc‐ceramide, whereas in contrast, both the formation of GM1 from GM2 and of Gal‐GalNAc‐Gal‐Glcceramide from GalNAc‐Gal‐Glc‐ceramide appear to be catalysed by the same galactosyl transferase.

The enzymic synthesis of ganglioside: I. Brain uridine diphopphate D-galactose: N-acetyl-galactosaminyl-galactosyl-glucosyl-ceramide galactosyl transferase

An enzyme which catalyzes the transfer of galactose from UDP-galactose to galNAc-gal-glc-ceramide is described. The enzyme is found mainly in the nervous tissue of tadpole (Taylor and Kollros stage 17), adult frog, adult and 8 day old rat. The enzymic activity is localized in the 11,500 xg, 20,000 xg and 100,000 xg particles. The UDP-galactose: galNAc-gal-glc-ceramide from the particles by treatment with sodium desoxylcholate and Triton X-100. The pH optimum for the solubilized enyme is between 6.8 and 7.0 in cacodylate buffer, and the Km is 4.25 ×10−5 M. The enzymic reaction is proportional to time for 4 hr and to the amount of protein added. The product of the transferase reaction, using galNAc-gal-glc-ceramide. A pathway for the biosynthesis of brain gangliosides requiring UDP-galactose: galNAc-gal-glc-ceramide galactosyl transferase is proposed.

The enzymic synthesis of ganglioside. II. UDP-galactose: N-acetylgalactosaminyl-(N-acetyl-neuraminyl)galactosyl-glucosyl-ceramide galactosyltransferase in rat brain

Abstract This paper describes the developmental pattern, subcellular distribution and general properties of UDP-galactose: N -acetylgalactosaminyl-( N -acetylneuraminyl)-galactosyl-glycosyl-ceramide galactosyltransferase (UDP-galactose: G M 2 ganglioside galactosyltransferase) in rat brain. 1. 1. The enzyme was detected in fetal rat brain, and the specific activity increased linearly up to 5 days post partum . A sharp peak of enzyme activity occurred during the period of active myelination. Significant enzymic activity was detected in adult rat brain. 2. 2. The enzyme from fetal rat brain was localized with the mitochondrial and microsomal particles. The percentage of enzyme activity in the mitochondrial fraction increased after birth, and was found to be localized with this fraction at 10 days of age. Appreciable enzymic activity in the nuclear fraction was found only in the adult brain. 3. 3. General properties of the enzyme: (a) The enzyme was specific for G M 2 ganglioside and UDP-galactose. (b) EDTA inhibited the enzyme which also required Mn 2+ for maximum activity. (c) The K m value for G M 2 ganglioside was 94 μM and 12 μM for UDP-galactose. G M 2 ganglioside was inhibitory at a concentration equal to or greater than 0.35 mM. (d) The pH optimum ranged between 6.5 and 7.15.

The Natural Occurrence of Sialic Acids

The sialic acids are widely distributed in nature, either free or as components of homo- and heterosaccharides, glycoproteins, and glycolipids (Tuppy and Gottschalk, 1972). Although a considerable literature has accumulated on sialic acids and the sialo compounds, a unified concept about the biological roles of sialic acids has not yet developed due to both their heterogeneous occurrence and their possible involvement in diverse cellular functions (Faillard and Schauer, 1972; Gottschalk, 1972; Marshall, 1972; Mehrishi, 1972; Curtis, 1973; Hughes, 1973; Schauer, 1973; Weiss, 1973). This chapter will approach a better understanding of the biological roles of sialic acids by examining their occurrence in nature. A possible correlation between the evolution and biological roles of sialic acids is considered.

Sialyltransferases in rat brain: intracellular localization and some membrane properties.

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.

The appearance of ganglioside during embryological development of the frog.

Embryological stages after fertilization were examined for the appearance of gangliosides. In Rana pipiens, gangliosides were found to appear first at the late gastrula, a stage well before any morphological appearance of a nervous system. Furthermore, at this stage and at a later stage, hatching, only two of the four major gangliosides were detected, namely, monosialoganglioside GM1 and disialoganglioside GD1a.

Colorimetric assay for free and bound L-fucose.

A novel, rapid, and reliable colorimetric method for measuring L-fucose has been developed. This method utilizes NADH formed from the interaction of L-fucose with fucose dehydrogenase and NAD to generate color in a reaction involving CuSO4 and neocuproine. NADH reduces Cu2+ to Cu1+ and the latter interacts with neocuproine to yield a complex with a maximal absorption at 455 nm. The reaction of NADH with copper-neocuproine is immediate and under the conditions of the assay the color formed remains stable for at least 2 h. When the assay is used to determine levels of L-fucose, the absorbance is found to be linearly proportional to exogenously added fucose concentrations from 16 to 179 nmol with resulting molar extinction coefficient of 13,660. Using this procedure, L-fucose released by acid hydrolysis from porcine submaxillary mucin, and by alpha-L-fucosidase from p-nitrophenyl-alpha-L-fucopyranoside, was quantitated.

Glycation of human erythrocyte glutathione peroxidase: Effect on the physical and kinetic properties

BACKGROUND Glutathione peroxidase (GPx) is a significant antioxidant enzyme that plays a key role in protecting the body from reactive oxygen species (ROS) and their toxicity. As a biocatalyst, the enzyme has been shown to reduce hydrogen peroxide to water and lipid hydroperoxides to their respective alcohols. The increased levels of ROS in patients with diabetes have been speculated to arise, in part, from alterations in the activity of glutathione antioxidant enzymes, perhaps, by mechanisms such as the glycation of the protein, in vivo. METHODS Under physiological conditions of temperature and pH, we investigated the susceptibility of human glutathione peroxidase to glycation, determined the effects of glycation on the physical and kinetic properties of the enzyme, and identified the proteins vulnerable amino acid sites of glycation. RESULTS Circular dichroism, UV and mass spectrometry studies revealed that methylglyoxal and DL-glyceraldehyde are potent glycators of glutathione peroxidase; destabilizing its structure, altering its pH activity and stability profiles and increasing its Km value. CONCLUSIONS In comparison to DL-glyceraldehyde, methylglyxol was a more potent glycator of the enzyme and was found to nonenzymatically condense with Arg-177, located near the glutathione binding site of GPx.