Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid ( N -acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.     

Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase

Abstract: N′-Acetyl-d -[6-³H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.     

THE REGIONAL DISTRIBUTION OF SIALOGLYCOPROTEINS, GANGLIOSIDES AND SIALIDASE IN BOVINE BRAIN

Abstract— Sialoglycoproteins and gangliosides were characterized in various bovine brain regions by determining the amount of sialic acid. Expressed per g dry weight, the gangliosidic sialic acid ranged from 11·20 to 1·93 μmol and the glycoprotein sialic acid from 8·93 to 1·84 μmol in grey and white matter respectively (values not corrected for incomplete release and breakdown during hydrolysis). Both the sialoglycoproteins and the gangliosides occur in highest concentration in areas predominating in neuronal cell bodies (cerebral grey, cerebellar grey, caudate nucleus). The lowest concentrations are found in those areas, consisting largely of myelinated fibre tracts and glial cells (pons, medulla, corpus callosum, cerebral white). Relative to the gangliosides the sialoglycoproteins are somewhat more concentrated in white matter.
The sialidase activity was investigated with endogenous substrate as well as with additional gangliosides or sialoglycopeptides. In all conditions the activity was much greater in grey matter than in white matter. The regional sialidase distribution more or less parallels the distribution of sialic acid in the various regions. At high substrate level the sialoglycopeptides inhibit the sialidase activity. There are indications that gangliosides are a far better substrate for brain sialidase than glycoproteins or glycopeptides. The possible significance of this phenomenon is discussed.     

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.     

THE ISOLATION AND LIPID COMPOSITION OF MYELIN-FREE AXONS FROM RAT CNS1

—Myelin-free axons were isolated from rat CNS using a modification of the method of De Vries et al. (1972). On a dry weight basis, the axons contained 15·2% lipid composed of 19·4% cholesterol, 56·9% phospholipid and 23·7% galactolipid with a weight ratio of cerebroside to sulfatide of 3·6-1. The phospholipid was composed of 11·0% ethanolamine phosphatides (44·4% in the plasmalogen form), 21·0% choline phosphatides (9·3% in the plasmalogen form), 4·5% sphingomyelin, 4·5% phosphatidyl serine, 4·3% phosphatidyl inositol, 3·0% diphosphatidyl glycerol and 8·5% unidentified phospholipid. The rat axons contained 0·18 μg ganglioside NeuNAc/mg dry wt. In addition to the 4 major brain gangliosides, the rat axons contained gangliosides G_D2 and G_D3. The axonal galactolipid could not be accounted for by myelin contamination as revealed by electron microscopy, absence of the characteristic ratio of myelin specific proteins in the axonal protein profile as shown by polyacrylamide gel electrophoresis, and the axonal level of the myelin marker enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The relationship between lipids of axons isolated from rat and bovine CNS, and rat whole brain and CNS myelin is discussed.     

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 GM_1a (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); GM_1a, 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 GM_1a and exogenous DS-fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM₃, while exogenously added GM_1a was converted to GD_1a. 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. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM_1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM₃, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM_1a. This was supported by the observation that exogenously added GM_1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS-fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP-NeuNAc.     

On the mechanism of a calcium-associated defect of oxidative phosphorylation in progessive muscular dystrophy

Partially purified elongation factor 1 preparations from calf brain, sheep brain, calf liver, and rabbit reticulocytes have been compared in their ability to interact with GTP and Phe-tRNA. A nitrocellulose filter assay has been used to study these interactions, and with all the EF1 preparations studied, evidence has been obtained for the formation of a Phe-tRNA·-EF1·-GTP complex. The ternary complex reacts with calf brain ribosomes in the presence of poly(U) resulting in a rapid hydrolysis of GTP and the binding of Phe-tRNA to the ribosome. Indirect evidence indicates that EF1·GDP is a product of this reaction. In the absence of poly(U) the intact complex reacts with the ribosomes without hydrolysis of GTP. The stability of the ternary complex was different with the various EF1 preparations, but the most stable complexes were prepared with calf brain EF1. Sephadex chromatography of the ternary complex shows that it contains a low molecular-weight species of the enzyme.     

THE ACTION OF CALF BRAIN SIALIDASE ON GANGLIOSIDES, SIALOGLYCOPROTEINS AND SIALOGLYCOPEPTIDES

Abstract— In agreement with other investigators it has been shown that endogenous as well as added gangliosides are a substrate for brain sialidase. The release of sialic acid was enhanced in the presence of Triton X-100; this might be due to the action of the detergent on the ganglioside micelles. The sialic acid release from endogenous gangliosides was observed over 48 h and compared with the effect of the sialidase on the endogenous glycoproteins. Though the hydrolysis of sialic acid from gangliosides is much faster in the first hours, after 48 h 40 per cent of the total bound sialic was released from both substrates at pH 4.0 and 37°C.
Sialoglycopeptides obtained from brain glycoproteins are also metabolized by the sialidase. No effect of Triton X-100 on this substrate has been observed. From sialoglycopeptides, fractions can be obtained by DEAE-Sephadex A-50 column chromatography with a sialic acid content from 8 to 26 per cent. The fractions with a high sialic acid content were about equally active towards brain sialidase as gangliosides. The results agree with the similar turnover rate observed for the carbohydrate chains from gangliosides and glycoproteins, but are in contrast to the observations of other investigators who have stated that glycoproteins are a poor substrate for brain sialidase. In our experiments bovine and ovine submaxillary mucins and sialyl-lactoses showed only slight activity compared to gangliosides and selected brain sialoglycopeptides.     

Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates.     

Topographic Distribution of Enzymes Synthesizing Phosphatidylcholine and Phosphatidylethanolamine in Chicken Brain Microsomes

Abstract: The localization of phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes within the transverse plane of chicken brain microsomes was investigated by using proteases (trypsin and pronase) and neuraminidase. Treatment of intact microsomes with the proteases inactivated the phosphocholine transferase completely and the ethanolamine phosphotransferase only slightly. This latter enzyme was, however, completely inactivated when deoxycholate-treated microsomes were exposed to proteases. Treatment of intact microsomes with neuraminidase had no effect on both phosphotransferases, although 65% of the sialic acid of sialoglycoproteins and 37% of that of gangliosides were removed. With deoxycholate-disrupted microsomes nearly all sialic acid from the sialoglycoproteins and about 70% of that of gangliosides were released. In parallel, the phosphoethanolamine transferase was 90% inactivated. It is suggested that phosphocholine transferase is localized on the outer face of the microsomal vesicle, whereas the phosphoethanolamine transferase could be a sialoglycoprotein, possibly situated on the inner face of the vesicle, or perhaps a transmembrane protein.     

STUDIES ON THE BIOSYNTHESIS OF GLYCO-SPHINGOLIPIDS IN CULTURED MOUSE NEUROBLASTOMA CELLS: CHARACTERIZATION AND ACCEPTOR SPECIFICITIES OF N-ACETYLNEURAMINYL- AND N-ACETYLGALACTOSAMINYLTRANSFERASES

Abstract— The pathway of biosynthesis of N -acetylgalactosamine-containing gangliosides in mouse neuroblastoma has been studied using NB41A cells grown in monolayer tissue culture. Cell-free enzyme preparations catalyzed the transfer of NeuNAc from CMP-NeuNAc to lactosylceramide (GL-2a), to form G_M3. Asialo-G_M2 was neither an acceptor nor a competitive inhibitor of the sialyltransferase (CMP-NeuNAc: GL-2a N-acetylneuraminyltransferase, EC 2.4.99.-) under a variety of experimental conditions. Enzyme preparations also contained an N -acetylgalactosaminyltransferase (UDP-GalNAc. G_M3 N -acetylgalactosaminyltransferase, EC 2.4.1.-) which catalyzed the conversion of G_M3 to G_M2. No significant transfer of N -acetylgalactosamine to GL-2a could be demonstrated. The results of the glycosyltransferase assays support the concept that the first NeuNAc of brain gangliosides is introduced into GL-2a. The present data suggests that the occurrence of asialo-G_M2 in NB41A cells under some culture conditions is a consequence of the catabolism of higher gangliosides.     

Cerebral proteinases in the growing rat

—The proteolytic activity of brain homogenates obtained from 1-, 5-, 14-, 60-, 150-, and 300-day-old rats was assayed with urea-denatured haemoglobin and casein, endogenous tissue proteins, Nα-benzoyl-dl-arginine 2-naphtylamide (BANA), N^α-benzoyl-dl-arginine methyl ester (BAME), N^α-toluene p-sulphonyl-dl-arginine methyl ester (TAME), N^α-benzoyl-dl-phenylalanine 2-naphthyl ester (BPANE), and N^α-acetyl-dl-tyrosine ethyl ester (ATEE) as substrates. Several peaks of activity were detected with all these substrates in different pH ranges. Activity was highest with protein substrates at pH 3·0-4·0, with smaller peaks of activity at pH 5·5-6·5 and 8·0-9·0. At pH 3·0 the activity with trypsin substrates, viz. BANA, BAME and TAME, was also relatively high, but much less with chymotrypsin substrates, ATEE or BPANE. With BAME, TAME, BPANE and ATEE the hydrolysis rate was highest at neutral or slightly alkaline pH. During postnatal development the hydrolysis of protein substrates increased three-fold at pH 3·0 and about two-fold at pH 6·5 and 8·5. The rate of hydrolysis of BANA, BAME and TAME generally increased during the first 2 postnatal weeks and thereafter decreased, whereas no marked increase in the rate of hydrolysis of BPANE and ATEE occurred until the age of about 2 weeks. The results were less consistent with synthetic substrates than with protein substrates, indicating the existence of non-uniform alterations during development in the activity of the individual hydrolytic enzymes participating in the breakdown of brain proteins.     

Incorporation of N-Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol-Induced Convulsions on Brain Gangliosides1

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides G_T1b and G_Q1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, G_T1b and G_Q1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.     

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a-similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. G _{d 3} and G _{d 1b} decreased rapidly in per cent; correspondingly, G _{d 1a} increased during embryonic life and became the major ganglioside in place of G _{d 3}. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.     

GLYCOPROTEINS AND GLYCOLIPIDS OF SUBCELLULAR FRACTIONS FROM BOVINE RETINA. INCORPORATION OF MANNOSE AND SIALIC ACID1

Abstract— Endogenous lipids and proteins of bovine retina subcellular fractions were labelled from CMP-[³H]NeuNAc and GDP-[¹⁴C]mannose. The bulk of NeuNAc and mannose transfer activity was in membranes other than those from the rod outer segment (ROS). Lighter and heavier membranes, obtained from ROS free membranes by density gradient centrifugation, were the most active for the incorporation of NeuNAc and mannose, respectively. NeuNAc bound to a lipid indistinguishable from gangliosides, and a lipid that contains mannose (mannolipid-I) were found in the fraction extractable with chloroform-methanol (2:1, v/v). Mannose was also incorporated into a lipid fraction extractable with chloroform-methanol-water (1:1:0.3, by vol) (mannolipid-II). Mannolipid-I and mannolipid-II were labile to mild acid hydrolysis. In the presence of ROS free membranes, radioactivity of mannoli-pid-I was transferred to mannolipid-II and from this to proteins. Analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the proteins labelled from GDP-mannose migrated as a broad peak covering the range of molecular weights 20,000–30,000 and including the zone of rhodopsin migration. The proteins labelled from CMP-NeuNAc showed four radioactive peaks that were coincident with three out of four periodic acid-Schiff (PAS) positive bands.     

Intestinal neuraminidase activity of suckling rats and other mammals. Relationship to the sialic acid content of milk.

1. The neuraminidase activity of homogenates of the mucosa of the middle and distal thirds of the small intestine of rats increased about 5-fold between birth and 4 to 8 days of age, and then gradually declined to the much lower adult activity by 24 days. No comparable changes occurred in the proximal third. 2. In 8-day-old rats, the neuraminidase activity of the middle and distal thirds of the small intestine was about 10 times greater than that of the proximal third, 20 times greater than that of the colon and at least 100 times greater than that of the liver, brain, gastric mucosa or pancreas. 3. In all other species investigated (mice, rabbits, cats and guinea pigs), the neuraminidase activity of the middle and distal thirds of the small intestine was greater in suckling animals than in adults. 4. The sialic acid content of rat milk increased about 2-fold between birth and 8 days post partum and then declined. 5. There was a highly significant positive correlation between the intestinal neuraminidase activity of suckling animals of various species and ages and the sialic acid content of milk obtained from the corresponding species and stage of lactation. 6. It is suggested that the intestinal neuraminidase of suckling mammals functions primarily to remove sialic acid from various components of milk, thus providing sialic acid for the synthesis of sialoglycoproteins and gangliosides by the young.     

N-Acetylneuraminic acid molecules as possible serotonin binding sites.

5-Hydroxytryptamine (5-HT), but not acetylcholine, carbamylcholine or L-D-noradrenaline, binds to ox brain ganglioside micelles, to phosphatidylcholine smectic mesophases (liposomes) containing gangliosides and to the glycoprotein fetuin, through the negatively charged N-acctylneuraminic acid (NeuNAc) residues. The 5-HT binding to NeuNAc is reversible, saturable, prodeeds in a 1: 1 fashion and can be specifically blocked by 7-methyltryptamine. Thc affinity constant at equilibrium for the reaction is of the order of 10² 1. mol-¹. No special ganglioside was identified as specifically associating with the amine. A terminal NeuNAc in the gangliosides is not a prerequisite for binding, although it seems important for binding 5-HT in entire mcmbrane preparations (MARCHBANKS, 1966) or for bringing about the 5-HT induced contraction of smooth muscle cells (WOOLLCY & GOMML 1964a). It is proposed that in 5-HT target cells, NeuNAc residues, probably attached to membrane surface glycoprotein(s) are involved in thc mechanism of action of the drug.     

Changes in Rabbit Brain Cytosolic and Membrane-Bound Gangliosides During Prenatal Life

The present study deals with the developmental profile of cytosolic and membrane-bound gangliosides in rabbit whole brain from the 21st day of pregnancy, the time at which brain could be macroscopically recognized and handled, till birth. In this period of prenatal life the content of membrane-bound gangliosides showed a 2.5-fold increase, referred to fresh and dry brain weight and to membrane-bound protein; the content of cytosolic gangliosides reached a maximum at 21-22 days of pregnancy, and then underwent to birth a threefold diminution. The qualitative pattern of membrane-bound gangliosides, in the same period of life, was characterized by an increase of G_D1a and G_M1 (more marked for G_D1a), a decrease of G_T1a, G_T1b and G_Q1b and a constant level of G_D3 and G_D1b. At 21 days of pregnancy the most abundant gangliosides were G_T1b, and G_Q1b, followed by G_D1a and G_D1b; at birth it was G_D1a followed by G_T1b G_D1b, and G_M1 The qualitative pattern of cytosolic gangliosides closely resembled, during the entire period of prenatal life examined, that of membrane-bound gangliosides.     

Influence of gangliosides or LPS-like gangliosides on the tumoricidal activity of adherent leukocytes

We previously showed that highly metastatic clones derived from the poorly metastatic human melanoma cell line M4Be are very radiosensitive provided that they are deficient in complex gangliosides. Here, we report that the highly metastatic clone 4 appears more sensitive to activated adherent leukocytes than M4Be via a transmembrane TNF-alpha-dependent mechanism. Adherent leukocytes (AL) were freshly isolated from different blood donors and were activated with Esherichia coli lipopolysaccharide (LPS). These AL contain 80% (73-93%) monocytes, 15% (6-20%) B lymphocytes and 5% (1-8%) T lymphocytes. The tumour cell survival following contact with AL was estimated with a clonogenic assay where isolated tumour cells were plated for 14 days with AL. We show on the one hand that either exogenous bovine brain GM1 gangliosides or Campylobacter jejuni LPS with GM1-like structure (LPS-like GM1) significantly decrease the hypersensitivity of clone 4 to AL. On the other hand, the cleaving with neuraminidase of more than 50% of the sialic residues bound to endogenous gangliosides in resistant M4Be cells significantly increases their sensitivity to AL. Thus, our highly metastatic cells appear both very sensitive to activated AL when they are deficient in complex gangliosides and resistant to AL when they are transiently exposed to exogenous gangliosides or LPS-like gangliosides. These in vitro data may reflect the paradoxidal behaviour of highly metastatic cells in vivo which appear both very sensitive to physiological stresses and able to survive to form secondary tumours.     

ON THE ASSOCIATION OF NON-SPECIFIC ESTERASE ACTIVITY WITH CENTRAL NERVE MYELIN PREPARATIONS

Abstract— Isolated bovine central nerve myelin sheath preparations showed non-specific esterase activity towards naphthyl ester substrates of increasing chain length from acetate to palmitate. Short chain esters were hydrolysed much faster than long chain substrates by myelin, the specific activity for the hydrolysis of β-naphthyl acetate being the highest. Micro-somal fractions from brain white matter were much higher in esterase activity to all naphthyl ester substrates. NADPH-cytochrome c reductase activity was absent from isolated myelin samples. Distilled water and salt and buffer solutions of different ionic strengths and pH were ineffective in releasing non-specific esterase activity from myelin although tri-potassium citrate caused marked inhibition of the membrane-bound esterase activity. The detergent Triton X-100 released esterase activity from the myelin preparations but at a concentration of 0.1 per cent was also inhibitory.