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Biomedical subjects

J A Lowden

Publications and source records attributed to J A Lowden.

At least 37 records · Page 2Linked to original sources
PubMed1985

Characterization by nuclear magnetic resonance of the concanavalin A binding oligosaccharide on the beta b chain of placental beta-hexosaminidase B: lectin binding to the separated polypeptide chains of hexosaminidases A and B.

The type and distribution of the oligosaccharides on each polypeptide of human placental beta-hexosaminidases A (alpha (beta a beta b)) and B (2(beta a beta b)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the beta a chain contains negligible carbohydrate, the beta b chain contains both the high-mannose and a complex type oligosaccharide, and the alpha chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the beta b polypeptide of beta-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.

Autoradiography↗
PubMed1985

The lysosomal hexosaminidase isozymes.

In the 15 years since the demonstration that HEX A is the defective enzyme in patients with TSD, intensive efforts in many laboratories have revealed much about the HEX group of enzymes. In contradistinction to the two isozymes described by Robinson and Stirling [1968], we now know that there are several different species. They include the products of at least three genes which code for the alpha and beta polypeptides as well as for an enzyme that we have called HEX D. The different species of HEX found in human tissues and fluids include significant amounts of larger, unprocessed polypeptides as well as mature enzyme. Thus the HEX A of serum (HEX AS) is a more primitive form of the enzyme than that found in lysosomes. The role of biosynthesis in the formation of multiple species of HEX is not confined to the polypeptide chains of the enzyme. All lysosomal enzymes are glycosylated and HEX is not an exception. The carbohydrate side-chains are essential to the packaging process that places HEX in the lysosome. Carbohydrates on lysosomal HEX species clearly differ from those on HEX in serum. Characterization of HEX oligosaccharides is still in the preliminary stages. Many minor species of HEX have been described. The more important ones are the intermediate isozymes (HEX Is). In tissues the HEX Is appear to contain mixtures of processed and unprocessed alpha and beta polypeptides. In serum, on the other hand, they contain unprocessed beta chains and differ in the carbohydrate side-chains. Most species of HEX show microheterogeneity. Native, mature HEX B separates into several bands on isoelectric focusing. The nature of this microheterogeneity has not been defined. Clear differences have been described, however, between the two chains in the beta subunit. The chains are always united in non-random fashion and are probably derived by cleavage of a single gene product. Studies of hydrolytic activity have been interesting. Like other lysosomal enzymes, HEX A requires a specific protein activator for optimum activity. This small polypeptide has been partially characterized but its mode of action is as yet unclear. Defects in activator synthesis lead to a form of GM2 ganglioside storage disease. Clinically many different phenotypes have been identified which appear to result from defects in each of the HEX genes. The differences between the defects have not been characterized in molecular terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

The beta 2 subunit of human placenta hexosaminidase (Hex) A and B is a non-random association of the two polypeptides beta a and beta b.

The subunit structures of placental Hex A and B have previously been assigned as alpha beta 2 and 2 beta 2, respectively. The beta 2 subunit is composed of two non-identical polypeptide chains, beta a and beta b. Purified Hex A and B were fractionated on a chromatofocusing column, and the fractions were reduced and then alkylated with iodo-1-14C-acetamide. The polypeptide chains were separated by polyacrylamide-gel isoelectric focusing. From the radioactivity measurements of the polypeptides a constant value for beta a/beta b was obtained in all the chromatofocusing fractions, demonstrating a non-random structure of (beta a beta b) in each beta 2 subunit.

Female↗
PubMed1984

Congenital ascites as a presenting sign of lysosomal storage disease.

Neonatal ascites is usually attributed to hematologic, genitourinary, gastrointestinal tract, or congenital heart disease. When these lesions have been excluded, metabolic storage disorders should be considered in the differential diagnosis. We report eight cases of neonatal ascites associated with different types of lysosomal storage disease: infantile sialidosis, Salla disease, GM1 gangliosidosis, and Gaucher disease. In each case there was a history of sibling of perinatal death resulting from the disease. In three cases the diagnosis of ascites was made in utero by ultrasound examination. These diseases are characterized by excretion in the fetal urine of abnormal catabolic products or by measurement of decreased activity of specific lysosomal hydrolases in cultured amniocytes. Thin-layer chromatography of the oligosaccharides in amniotic fluid may be indicated when a diagnosis of persistent fetal ascites has been established.

Adult↗
PubMed1984

Human placental beta-galactosidase: structural and immunological observations.

beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

Amino Acids↗
PubMed1983

HPLC analysis of neutral glycolipids: an aid in the diagnosis of lysosomal storage disease.

Concentrations of GL-la (glucocerebroside) (8.36 nmol/ml), GL-2a (lactosylceramide) (4.03 nmol/ml), GL-3a (globotriosylceramide) (2.25 nmol/ml) and GL-4a (globotetraosylceramide) (2.87 nmol/ml) have been determined in normal plasma and compared to concentrations in the plasma from patients with Gaucher, Krabbe, Fabry, Sandhoff and Tay-Sachs diseases as well as with hypercholesterolemia. HPLC analysis of perbenzoylated glycolipid derivatives (isolated and purified by modification of an existing procedure) was performed on samples equivalent to 50 to 100 microliter of plasma. The sensitivity could be readily increased ten-fold. We have employed a novel internal standard-monogalactosyl diglyceride, a plant glycolipid, commercially available in pure form. Analysis was performed on a 5 micron ultrasphere silica column, using a gradient of isopropanol in hexane rather than the more usual dioxane in hexane. Our gradient exhibited an essentially flat baseline precluding the necessity of a reference cell. Recoveries of glycolipids added to plasma (95%), experimental yields (60%) and standard curves are presented and discussed. A method is also presented for the separation of GL-la and monogalactosyl diglyceride derivatives for rapid (8 minute) isocratic analysis of multiple samples from Gaucher patients. The benefits of such a simple, reproducible HPLC technique are discussed.

Chromatography, High Pressure Liquid↗
PubMed1983

Biliary disease in metachromatic leukodystrophy.

This paper illustrates the previously unreported sonographic changes in the biliary tract in metachromatic leukodystrophy (MLD). Gallbladder wall thickening due to sulfatide deposition, intraluminal globules of sulfatide or papillomatosis may cause symptoms referrable to the biliary tract in these patients. A series of patients whom we have studied is briefly alluded to in order to bring to the attention of the radiologist MLD as a rare cause of biliary disease in children.

Adolescent↗
PubMed1983

Characterization of polypeptides serologically and structurally related to hexosaminidase in cultured fibroblasts.

Human fibroblasts synthesize several polypeptides that assort into the various forms of hexosaminidase (hex). We report here the occurrence of three newly identified, hexosaminidase-related polypeptides resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of immunoprecipitates from [35S]methionine-labeled cell extracts. These polypeptides, called band 2 (75,000), band 3 (70,000), and band 4 (63,000), were immunoprecipitated by an antiserum specific to placental hex I2. They are distinct from pre-alpha- (60,000) and pre-beta- (58,000) precursor polypeptides and the alpha- (56,000), beta a- (27,000), and beta b- (27,000) polypeptides of the mature hex A (alpha beta a beta b) and hex B (2[beta a beta b]). When fibroblast extracts were chromatographed on DEAE-Sepharose, bands 2, 3, and 4 were eluted together in fractions before hex A, in a position characteristic of serum and placental hex I2 and serum hex P. This suggests that bands 2, 3, and 4 might represent the polypeptides of a fibroblast hex I. The analysis of partial proteolytic digests of the radioactively labeled polypeptides revealed that bands 2 and 3, pre-beta, and beta a had several peptides in common, suggesting that they are structurally related to each other. However, bands 2, 3, and 4 were present in extracts of Tay-Sachs (pre-alpha and alpha deficiency) and Sandhoff cells (pre-beta, beta a, and beta b deficiency) and appeared later than pre-beta in pulse-chase experiments. These results suggest that bands 2 and 3 occur independently of pre-beta and beta a and are probably specified by different mRNA, whether from the same gene or distinct but homologous genes.

Animals↗
PubMed1983

Stability of the paraoxonase phenotyping ratio in collections of human sera with differing storage times.

Results from automated testing of 673 human sera, divided into four batches by duration of storage at -20 degrees C, indicated that paraoxon-hydrolysing activity, a, decreased with increased storage time. By contrast, the paraoxonase phenotyping ratio, c/b, a ratio designating enzyme activation by Na+ in the presence of Ca2+, was stable over a four year storage period.

Aryldialkylphosphatase↗
PubMed1983

The Tay-Sachs disease gene in North American Jewish populations: geographic variations and origin.

From data collected in a North American Tay-Sachs disease (TSD) heterozygote screening program, the TSD carrier frequency among 46,304 Jewish individuals was found to be .0324 (1 in 31 individuals). This frequency is consistent with earlier estimates based on TSD incidence data. TSD carrier frequencies were then examined by single country and single region of origin in 28,029 Jews within this sample for whom such data were available for analysis. Jews with Polish and/or Russian ancestry constituted 88% of this sample and had a TSD carrier frequency of .0327. No TSD carriers were observed among the 166 Jews of Near Eastern origins. Relative to Jews of Polish and Russian origins, there was at least a twofold increase in the TSD carrier frequency in Jews of Austrian, Hungarian, and Czechoslovakian origins (P less than .005). These findings suggest that the TSD gene proliferated among the antecedents of modern Ashkenazi Jewry after the Second Diaspora (70 A.D.) and before their major migrations to regions of Poland and Russia (before 1100 A.D.).

Female↗
PubMed1982

The assay of glucocerebrosidase activity using the natural substrata.

We examined eight published methods for the assay of glucocerebrosidase using the natural substrate glucocerebroside and tabulated the variable components and conditions while comparing these methods to ours. In each case assays were performed using our 8000-fold, partially purified human placental enzyme, having a pH optimum of 5.4-5.6 and a Km of approximately 0.60 mmol per liter. Results were obtained by following the release of radioactive ceramide or of unlabelled glucose. In many cases published results had been based on a one- or two-hour incubation time. Apparent specific activities varied over a 70-fold difference between the various procedures. When we measured activities from the linear (15-30 min) portion of rate curves the values increased by 1.4 to 3 times but still ranged from 6 X 10(3) - 180 X 10(3) nmol . mg-1 protein . h-1. We obtained maximum velocity using 1.2 mmol glucocerebroside, 0.5% (w/v)crude taurocholate and 2 microgram enzyme protein/ml. Specific activities reported from different laboratories are not directly comparable. Conditions for assay should be optimized for the enzyme preparation to be studied.

Cerebrosides↗
PubMed1982

Evidence for two dissimilar polypeptide chains in the beta 2 subunit of hexosaminidase.

The major isoenzymes of human hexosaminidase have the structures alpha beta 2 (hex A) and 2 beta 2 (hex B). In this study, we present evidence that the beta 2 subunit of hex B and hex BA (the form of hex B derived from hex A) is composed of two nonidentical polypeptide chains. We have called these chains beta a and beta b. They have similar molecular weights (25,000) but have pI values that differ by 1 unit. We have used a two-dimensional analytical gel electrophoresis method in combination with peptide mapping to compare the primary sequence structure of the two beta chains. In this method, the polypeptide chains of hex B or hex BA were first separated by isoelectric focusing in 8.5 M urea. The separated chains were subjected to partial proleolytic digestion in the stacking gel of a second NaDodSO4/polyacrylamide gel with subsequent separation of peptides by electrophoresis into the second gel. Partial digestion by protease V8 or papain showed that the beta a and beta b species have distinct primary structures, neither of which was similar to that of the alpha chain. On the basis of these results, we suggest that the beta 2 subunit of hexosaminidase has the structure of beta a beta b. The possibility that the distinct beta chains are encoded by a single gene is discussed in the light of genetic and other data.

Hexosaminidases↗
PubMed1982

Purification of glucosylceramidase by affinity chromatography.

Glucosylceramide: beta-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate--butanol or citrate--ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate--taurocholate buffer at low pH or with citrate--taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate--taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate--polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide--0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.

Chromatography, Affinity↗
PubMed1981

Radioimmunoassay and heat denaturation enzyme assay for the detection of Tay-sachs heterozygotes during pregnancy.

Tay-Sachs disease results from a loss of activity of hexosaminidase A (HEXA) in body tissues and fluids. Heterozygotes for the disease are usually identified by their relatively low ratio of heat-labile HEX A to total hexosaminidase. During pregnancy an intermediate isoenzyme (HEX I) increases in activity in serum and obscures the heterozygote status. HEX I dose not increase in leucocytes, tears and other body tissues but because of technical difficulties in these assays we examined the feasibility of using a radioimmunoassay for HEX A. By univariate analysis, the heat denaturation assay gave a lower cost of misclassification for non-pregnant normals while RIA did so for pregnant normals. A combination of both tests led to reduced cost of misclassification compared to either alone. Bayesian analysis of bivariate gaussian density functions for heat denaturation and for radioimmunoassay of HEX isoenzymes was employed to calculate misclassification frequencies. Among the parameters examined, HEX A measured by RIA and % HEX A by heat-denaturation assay were the two having the best discriminatory power.

Acetylglucosaminidase↗
PubMed1981

A structural difference between the beta-chains in hexosaminidase B and hexosaminidase A.

Hexosaminidase (Hex) BA was prepared from purified placental Hex A by treatment with merthiolate and rechromatography on DEAE Sepharose 6B-CL. The heat stability, isoelectric focussing pattern, and peptide map of this isoenzyme were compared with those of naturally occurring purified placental Hex B. Hex BA proved to be less heat stable at 60 degrees C, have a slightly lower pI, and have one more peptide spot than did Hex B. The lower pI of Hex BA is not caused by sialic acid residues since it was unaffected by neuraminidase treatment. The extra peptide spot in the Hex BA map cannot be explained by carbohydrate differences since no corresponding unpaired peptide spot was seen in the Hex B map. Though Hex B and BA are products of the same gene, there is evidence that both are derived from a larger precursor polypeptide chain. Our results indicate that Hex B and BA are processed differently resulting in a small peptide present in Hex BA that is not found in Hex B.

Drug Stability↗
PubMed1981

Type 2 GM1 gangliosidosis with long survival and neuronal ceroid lipofuscinosis.

Neurologic deterioration began in a girl before age 2 years. By 4 she was spastic and decerebrate. GM1 gangliosidosis was diagnosed by absence of beta-galactosidase activity in leukocytes and fibroblasts. She died at 17 years. Her small brain contained only 2.61 mumole glycolipid N-acetylneuraminic acid per gram, and was filled with autofluorescent material. GM1 gangliosidosis was confirmed by the presence of membranous cytoplasmic bodies, by the absence of beta-galactosidase, and by failure of complementation when the patient's fibroblasts were fused with cells from other forms of GM1 gangliosidosis. The autofluorescent material probably accumulated because of the long survival rather than the primary enzyme defect.

Brain↗