Adriana Lombardo

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide as acceptor substrate.

Parallelism of subcellular location of major particulate neuraminidase and gangliosides in rabbit brain cortex

Abstract The subcellular distribution of major particulate neuraminidase and gangliosides in the rabbit cortex was determined. The neuraminidase and gangliosides were found to be present in: (a) the nerve endings; (b) the light membranes of microsomal origin ; (c) the myelin-rich preparation obtained from the crude mitochondrial fraction. Their distribution patterns were very similar also from the quantitative point of view; in fact 40 % of the neuraminidase activity and 43.5 % of the gangliosides were recovered in the nerve endings; 50% of the enzyme and 45% of the gangliosides in the light microsomal membranes; 8 % of the neuraminidase and 11 % of the gangliosides in the myelin-rich preparation. Conversely, the preparations enriched in nuclei, mitochondria and lysosomes were practically devoid of both neuraminidase and gangliosides. The ganglioside patterns of the different subcellular fractions were similar, except for the myelin-rich subfraction which contained higher amounts of monosialoganglioside GM1. The microsomal light membranes had a slightly lower content of trisialoganglioside GTIb and tetrasialoganglioside GQ1 than the nerve endings. These results may be considered consistent with the hypothesis that neuraminidase and gangliosides are fundamental components of the neuronal plasma membrane, thus following the distribution of the different neuronal fragments after homogenization and fractionation.

Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures

Automated fluorimetric procedures for the assay of five lysosomal glycohydrolases-beta-N-acetylglucosaminidase; beta-galactosidase; beta-glucuronidase; alpha-mannosidase; alpha-fucosidase-in human plasma were set up. A Carlo Erba autoanalyser CLA 1500, provided with a sampler refrigerating unit and connected with a recording Turner Mod 111 fluorimeter was employed. The automated procedures, under the established optimal conditions, proved to be highly accurate and reproducible. Using the automated assay procedures the effect of sex and age on the plasma levels of the same enzymes was studied. 1273 randomly selected health subjects were studied. No sex differences were observed for all the enzymes studied with the exception of beta-glucuronidase which displayed higher values (about 30%) in males from 25 to 60 years. The developmental profiles of all enzymes in females and males were similar and characterised by: (a) absolute maximum level in the umbilical cord blood; (b) absolute minimum level at 10-14 years; (c) decrease to a second minimum occurring around 35 years (not displayed by beta-galactosidase and by beta-glucuronidase in males); (e) slow further increase up to the elderly level which was then maintained till the oldest age examined, 74 years.

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane‐bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane‐bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X‐100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates.

Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates

A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).

Behaviour of some lysosomal enzymes in the plasma of insulin dependent diabetic patients during artificial pancreas treatment

The plasma levels of three lysosomal enzymes, beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, and alpha-L-fucosidase, were fluorimetrically determined in seven insulin-dependent diabetic patients one day before, one day after, and during a two-day treatment with the artificial pancreas, at 4 to 5 h intervals. A statistically significant decrease of the plasma level of each enzyme was observed during artificial pancreas treatment. The extent of decrease was 30 to 35% for beta-N-acetylglucosaminidase, 35 to 40% for beta-D-glucuronidase, and 20 to 25% for alpha-L-fucosidase. The decrease occurred earlier (at the first day of treatment) for beta-D-N-acetylglucosaminidase, and later (at the second day of treatment, and lasting to the first day after treatment) for the other two enzymes. These results suggest a direct connection between the lysosomal apparatus and insulin-controlled metabolic pathways, and a potential role for lysosomal enzymes as indicators of the metabolic compensation in diabetes.

Patterns of some lysosomal enzymes in the plasma and of proteins in urine of workers exposed to inorganic mercury

SummaryIn a population of workers (81 subjects) exposed to inorganic mercury vapors in a chlorine alkali plant (airborne mercury concentration between 0.06 and 0.3 mg/m3) and in a group (104 subjects) of people never exposed to mercury the plasma level of β-galactosidase, β-glucuronidase, β-N-acetylglucosaminidase, and β-glucosidase was determined.The results are as follows:1.The plasma level of the four acid lysosomal hydrolases are higher in the group of workers exposed to mercury than in the control group. The difference is significant at the P < 0.001 level for all studied enzymatic activities.2.There is a significant correlation between the increase of the plasma levels of these enzymatic activities and the degree of exposure. These results suggest the use of this procedure for detecting an undue mercury absorption in workers exposed to the metal before any clinical signs.RBC and plasma cholinesterase activities were not affected. The results of the study of urinary proteins were not useful for detecting an early impairment of the kidney function. However, in some cases we observed a marked glomerular proteinuria without any clinical explanation: this fact makes one very cautious about denying the existence of a kidney impairment in workers exposed to the metal before any clinical sign is present.

Serum enzymes of lysosomal origin as indicators of the metabolic control in diabetes: comparison with glycated hemoglobin and albumin.

SummarySeveral lysosomal enzymes (β-N-D-acetylglucosaminidase, β-D-glucoronidase, α-D-galactosidase, β-D-galactosidase, α-L-fucosidase, α-D-glucosidase, α-D-mannosidase, β-D-glucosidase), glycated albumin and glycated hemoglobin (HbA1c) were determined in the serum of 81 insulin-dependent diabetics with different degrees of metabolic control (optimal, 21 patients; good, 39 patients; poor, 21 patients) and without signs of complications, and in 42 control subjects. All parameters examined increased in serum in inverse proportion to the degree of metabolic control. A highly significant correlation (p<0.01) was found between lysosomal enzymes and both glycated albumin and HbA1c. All parameters correlated with hyperglycemia, glycated albumin having the highest γ-value (0.586) and lysosomal enzymes the lowest one. Unlike glycated albumin and HbA1c, serum levels of lysosomal enzymes in patients with optimal metabolic control were undistinguishable or even lower than those of controls. A 2-month longitudinal monitoring of a patient who was hospitalized in conditions of poor metabolic control and adequately treated, proved that lysosomal enzymes diminished in serum parallel to glycated albumin and HbA1c in relation to improvement of the metabolic situation. The conclusion is drawn that serum lysosomal enzymes are good indicators of the metabolic control of diabetic patients probably reflecting the overall metabolic state connected with insulin action rather than hyperglycemia.

Behaviour of several enzymes of lysosomal origin in human plasma during pregnancy

The following enzymes of lysosomal origin were fluorimetrically determined in maternal plasma from the second to the ninth month of pregnancy at 1-mth intervals: beta-D-N-acetylglucosaminidase (EC 3.2.1.30), beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1.51) and alpha-D-mannosidase (EC 3.2.1.24) (pH 4.0). As reference microsomal alpha-D-mannosidase (pH 5.7) was also studied. Thirty-eight healthy women, aged 18-37 yr, who had a normal pregnancy followed by normal parturition, were studied. All enzymes, with the only exception of beta-D-galactosidase, showed a progressive and statistically significant increase of activity throughout pregnancy. At the end of pregnancy, the increase ranged from a maximum of 5.6-fold for beta-D-N-acetylglucosaminidase to a minimum of 0.55-fold for alpha-D-mannosidase, pH 5.7. In the case of beta-D-N-acetylglucosaminidase, the level at the fifth month of pregnancy was significantly higher than that at the third month, and from the sixth to the ninth month each level significantly differed from that of the month immediately preceding.

Gangliosides, neuraminidase and sialyltransferase at the nerve endings.

Gangliosides are characteristic glycolipid components of the plasma membranes of mammalian cells. They are particularly abundant in the nervous tissue, specially the grey matter, where their concentration is about one tenth that of total phospholipids. The high content of gangliosides in the neuronal membranes, the great variety in the composition of their oligosaccharide chains, and their peculiar location in the outer membrane surface are enough evidence to stimulate research and speculation on the possible involvement of gangliosides in brain specific functions. As a matter of fact, gangliosides are just located- the synaptic junctions-where a specialized physiological event takes place, and definitely synaptic membranes would be and would behave differently without gangliosides. However, in order to provide a plausible working hypothesis for any specific roles of gangliosides in brain function, a more precise knowledge on the contribution given by gangliosides to the local environment of the membrane, in terms of capability and quality of interactions with both the lipid and protein components of the membrane itself, is required.