Thomas G. Warner

Partial characterization of the bile salt-dependent triacylglycerol lipase from the leopard shark pancreas

Leopard shark triacylglycerol lipase has been characterized as a crude pancreatic preparation. The enzyme demonstrated an absolute requirement for trihydroxy bile salts for activity with natural bile salts of the shark giving a 4-fold greater stimulation of activity than pure sodium taurocholate. Bile salts also protected the enzyme from apparent inactivation by p-chloromercuribenzoate and trypsin treatment. The shark lipase demonstrated a temperature optimum of 36 degrees C and was rapidly inactivated at 50 degrees C even in the presence of bile salts. Divalent metal ions were required for activity with Ca2+ providing the greatest stimulation. At 22 degrees C, pH 8.5 and in the presence of natural bile salts, the apparent V was about 0.6 mumol fatty acid released/min per mg protein. The shark enzyme hydrolyzed over 90% of the fatty acids from trioleovylglycerol and methyl esters of pancreatic lipase-resistant fatty acids were hydrolyzed at the same rate as typical fatty acid methyl esters. Hydrolysis of triacylglycerol proceeded about ten-times faster than wax ester hydrolysis. The kinetic properties of the leopard shark enzyme were compared to other bile salt-dependent lipolytic enzymes. Pancreatic lipase activity was not detected.

Prenatal diagnosis of sialidosis with combined neuraminidase and ø-galactosidase deficiency

Recently a combined deficiency of neuraminidase and P-galactosidase was reported in two patients (Wenger et al. 1978, O’Brien 1978), who had earlier been described as having variant types of Gq-gangliosidosis (Justice et al. 1977, Koster et al. 1976). We have found similar deficiencies in the second of two children who both died at birth with severe hydrops fetalis (Niermeijer et al. unpublished). The parents are consanguineous, of Turkish origin, and living in The Netherlands. Cultured skin fibroblasts and liver, obtained at autopsy, showed a deficiency of p-galactosidase with a residual activity of 5-10 % of normal. In addition, we found an almost complete deficiency of neuraminidase activity in the fibroblasts and an increased level of sialic acid in both fibroblasts and liver (3 and 23 times the normal levels, respectively). We now wish to report the results of the prenatal diagnosis which was offered to the parents during the third pregnancy. Amniocentesis was done in the 17th week (Dr. R. Vosters, University Hospital, Rotterdam). After 18 days of growth, the amniotic fluid cells were harvested in their first passage for enzyme assays. P-galactosidase and neuraminidase activities were determined using the fluorogenic 4-methyl-umbelliferyl substrates and micromethods, as described earlier for p-galactosidase in the prenatal analysis for GM1-gangliosidosis (Kleijer et al. 1976). The results are shown in Table 1. Both B-galactosidase and neuraminidase activity are strongly reduced in the amniotic fluid cells from the pregnancy at risk, as was the case in fibroblasts of the index patient. These results indicated that the fetus was affected with the variant type of sialidosis. Further evidence was obtained 1 week later by the demonstration of a 2-fold elevated sialic acid content of the amniotic fluid cells (44.3 nmol/mg protein, control: 22.1 nmoYmg). The parents decided on termination of the pregnancy, which was done by prostaglandin induction in the 21st week of pregnancy. The prenatal diagnosis was confirmed by the demonstration of reduced activities in the freshly obtained fetal liver and brain for P-galactosidase (5-10 % of normal activity) and neuraminidase (undetectable). The sialic acid content was 20-fold

Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains

Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [3H]glucosamine, [3H]mannose or [3H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.

Stable Isotope Dilution Analysis of Galactitol in Amniotic Fluid: an Accurate Approach to the Prenatal Diagnosis of Galactosemia

Summary: A stable isotope dilution assay for galactitol, in amniotic fluid has been developed using selected ion monitoring chemical ionization gas chromatography-mass spectrometry of the hexaacetate derivative. [1,1-2H2]Galactitol was synthesized for use as the internal standard. Galactitol is a component of normal amniotic fluid with a mean concentration of 0.70 ± 0.18 μmol/liter (n = 5). The amniotic fluid of a fetus with galactosemia had a concentration of 7.96 μmol/liter. Mannitol, sorbitol, and inositol were also found to be normal constituents of amniotic fluid. This stable isotope dilution assay is a rapid accurate method for measurement of galactitol in amniotic fluid for prenatal diagnosis of galactosemia.

Sialidosis: delineation of subtypes by neuraminidase assay.

A sensitive assay for acid neuraminidase using 4–methylumbelliferyl‐α‐D‐N‐acetylneura‐mink acid is described. In skin fibroblasts, patients with sialidosis Types 1 and 2 have severe deficiencies of neuraminidase activity compared with controls. Patients with Type 1 sialidosis have activities which are 10 times higher than those with Type 2 sialidosis, in keeping with their milder clinical involvement. Two Italian patients with Type I sialidosis had a Km which was one‐sixth normal; the other patients had a Km in the normal range.

Diagnosis of GM1 gangliosidosis based on detection of urinary oligosaccharides with high performance liquid chromatography

An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.

Alpha‐mannosidosis: analysis of urinary oligosaccharides with high performance liquid chromatography and diagnosis of a case with unusually mild presentation

Mannose containing oligosaccharides (OS) excreted in the urine of patients with alpha‐mannosidosis have been analyzed with high performance liquid chromatography (HPLC). The HPLC method provides a highly sensitive assay for detection of the urinary oligosaccharides and was employed for diagnosis of a fifteen‐year‐old female with an unusually mild presentation of the disease. Dysostosis multiplex and coarse facies were absent; mental impairment was particularly mild. The elution profile of the urinary OS from this patient and two, more severely affected, patients with mannosidosis were nearly identical, containing nine major OS fractions. The concentrations of the OS were eight fold lower in our patient hut, when calculated relative to creatinine, the levels of the urinary OS of all patients were similar.

Phosphatidylserine decarboxylase: Analysis of its action towards unsaturated and saturated phosphatidylserine and the effect of triton X-100 on activity

Abstract The membrane-bound enzyme phosphatidylserine decarboxylase ( Tetrahymena pyriformis ) was found to have activity both in a crude, particulate form and when it is in a soluble form in the presence of the nonionic surfactant Triton X-100. This surfactant has routinely been included in the assay of phosphatidylserine decarboxylases from all sources; its effect on the activity of the Tetrahymena enzyme has now been characterized and a detailed consideration of the functioning of this surfactant in the assay of this membrane-bound enzyme is presented. The activity of the enzyme towards natural phosphatidylserine is found to be greater than towards saturated phosphatidylserine, both with and without Triton present; this finding is considered in terms of the effect of the thermotropic phase transition of the saturated material on the physical state of the phospholipid, rather than simply in terms of the specificity of the enzyme for phosphatidylserine containing unsaturated fatty acid groups. At high molar ratios of Triton to phospholipid, the activity of the enzyme is dramatically decreased. The decreased activity of the enzyme toward unsaturated Phosphatidylserine is considered in terms of a surface dilution model and the greatly diminished activity towards the saturated analogue is suggested to be the result of lipid phase separation.