Isoenzymes of N-acetyl-beta-hexosaminidase in complicated pregnancy

The activity of N-acetyl-beta-hexosaminidase was found to be significantly higher in the placentas collected after delivery from women in puerperium with symptoms of prolonged pregnancy or complicated by EPH gestosis, than in placentas from normal pregnancy. Isoelectrofocusing of placenta homogenates showed the presence of isoenzymes A, P and B of N-acetyl-beta-hexosaminidase. Different isoenzyme A patterns in homogenates were observed in placentas obtained from normal and prolonged pregnancies and in those complicated by EPH gestosis.     

Partial characterization of intracellular and secreted glycosidases from rabbit articular chondrocytes in culture

N-Acetyl-beta-hexosaminidase, beta-galactosidase and beta-glucuronidase activities were shown to be present in cultured rabbit articular chondrocytes. Secretion of enzyme activity seems to preferentially result in the accumulation of N-acetyl-beta-hexosaminidase. Three days after seeding, the amount of N-acetyl-beta-hexosaminidase activity found in the medium accounts for about 140% of the total N-acetyl-beta-hexosaminidase activity after complete disruption of the cell pellet. Optimal conditions of incubation time, cell numbers, substrate concentration, and pH for glycosidase activities were determined in 0.1% Triton X-100. Intracellular and secreted glycosidases have shown similar elution profiles by chromatofocusing. N-acetyl-beta-hexosaminidase exhibits two major forms which may play a role in the catabolism of glycosaminoglycans.     

Isoenzymes of N-acetyl-beta-hexosaminidase

Biological significance, structure and posttranslational processing of N-acetyl-beta-hexosaminidase isoenzymes are described. Clinical application of N-acetyl-beta-hexosaminidase is also reviewed.     

N-acetyl-beta-hexosaminidase B deficiency in cultured fibroblasts from a patient with progressive motor neuron disease

A patient with a 20-year history of progressive motor neuron disease was previously found to have profoundly low levels of N-acetyl-beta-hexosaminidase (Hex) in serum and leukocytes; Hex activity in cultured skin fibroblasts was in the low normal range. By thermal inactivation and cellulose acetate electrophoresis, the residual activity appeared to be Hex A. In the present study, the residual activity in cultured skin fibroblasts was further characterized as Hex A by thermal inactivation at reduced temperatures and ion exchange chromatography; no evidence was obtained for a diffusible inhibitor of Hex activity. After labeling with [3H]leucine, immunoprecipitation with polyclonal antibody to Hex B, and SDS-polyacrylamide gel electrophoresis, both alpha and beta polypeptide chains were visualized, confirming the presence of Hex A. The results suggest that, in the patient's fibroblasts, a defect in beta-chain synthesis or processing precludes the self-association of beta-chains to form Hex B, but does not prevent the association of alpha- and beta-chains to form Hex A.     

The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.     

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

n-acetyl-beta-hexosaminidase activity in human breast milk

1. The lysosomal enzyme, N-acetyl-beta-hexosaminidase (HEX) is present in human breast milk. It is composed predominantly of "A" (heat-labile) and "B" (heat-stable) isozymes which coelute with the corresponding major serum isozymes on DE-52 ion-exchange chromatography. 2. Total HEX activity in "early" milk obtained at 2.8 +/- 1.4 weeks post partum, is approx. 2.5-fold higher (87 +/- 29 nmol/60'/mg protein. n = 10) than that of pregnancy serum (35.7 nmol/60'/mg protein) prior to delivery. 3. These levels increase to greater than 3-fold (110 +/- 20 nmol/60'/mg protein, n = 13) as the milk matures (10.3 +/- 4.2 weeks). 4. The specific activity of HEX A in the milk changes little with time post partum, because absolute 5. In contrast, HEX B specific activity is increased, as absolute levels (per volume) remain constant in the face of decreasing milk protein content, 5. These changes result in a high degree of correlation (r = 0.81) between time of lactation and % HEX A observed.     

The interaction of a carbohydrate-binding module from a Clostridium perfringens N-acetyl-beta-hexosaminidase with its carbohydrate receptor

Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-beta-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc (beta-d-galactosyl-1,4-beta-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 A, respectively.     

Separation of N-acetyl-beta-hexosaminidase isoenzymes. Methological note

We examined the activity of total N-acetyl-beta-hexosaminidase and of its isoenzyme forms, that represent different stages of the maturation of the lysosomal hydrolase. In both methods the enzyme catalyzes the separation of 4-methylumbelliferone, a fluorescent substance, from 4-methylumelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. We used Leaback's method for the fluorimetric assay of total enzyme, and Ellis's DEAE-cellulose microcolum chromatography for the assay of its components. We obtained a clear separation of each fraction. We will apply these methods in our further studies of children with renal damage, because hexosaminidase seems to be one of the most sensitive markers of tubular damage.     

Cytochemical and Histochemical Aspects of the Digestive Gland Cells of the Mussel Mytilus galloprovincialis (L.) in Relation to Function

The epithelium of the digestive tubules of the mussel Mytilus galloprovincialis is comprised of two cell types, namely digestive and basophilic cells. In basophilic cells, the secretory granules are beta-glucuronidase immunoreactive, a fact that enhances the hypothesis that beta-glucuronidase is synthesized in basophilic cells. A novel observation at the ultrastructural level is the pinocytic activity associated with the formation of coated pits. This observation constitutes direct evidence for endocytic processes taking place in basophilic cells. The use of cryostat sections from the same digestive tubules reveals, in many instances, a very pronounced neutral lipid accumulation in the same structures giving a positive reaction for N-acetyl-beta-hexosaminidase, indicating the association of those lipids with lysosomes. In some mussels, a high content of lipofuscin was observed in the lysosomes of the digestive cells. In these cases, the lysosomal structures show a limited neutral lipid content, and a weaker N-acetyl-beta-hexosaminidase reaction. In the digestive cells, the carbohydrate content of the lysosomes, and very well-developed canal system in the apical part of cells are discussed in relation to their function.     

The effects of N-hexyl-O-glucosyl sphingosine on normal cultured human fibroblasts: a chemical model for Gaucher's disease.

Normal human skin fibroblasts were grown in the presence of N-hexyl-O-glucosyl sphingosine (HGS), an inhibitor of aryl glucosidase and glucocerebrosidase. Tests of the cells with aryl glycosides showed that beta-glucosidase activity in the cells was drastically reduced while other enzyme activities (alpha-glucosidase, beta-galactosidase, and N-acetyl-beta-hexosaminidase) were normal or elevated. Exposure of cells to HGS for 28 days resulted in increased values for cell weight per plate, glucocerebroside concentration, and galactosyl-galactosylglucosyl ceramide concentration. The concentrations of total lipid, cholesterol, and protein were unchanged, as was the fatty acid distribution within the glycolipids. Chemically, the inhibitor-treated cells exhibited a model form of Gaucher's disease. Although many membranous cytoplasmic inclusions were induced by HGS, they were unlike the characteristic inclusions seen in individuals with the genetic disorder. Skin fibroblasts from a Gaucher patient showed no abnormalities in composition or appearance.     

N-acetylneuraminic acid accumulation in a buoyant lysosomal fraction of cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured fibroblasts from control individuals and two patients affected with the infantile variant of generalized N-acetylneuraminic acid (NeuAc) storage disease were disrupted by nitrogen cavitation, and the post-nuclear supernatant fractions were subjected to subcellular fractionation on Percoll gradients. Accumulating NeuAc in affected fibroblasts (approx. 150 nmol/mg protein) co-localized with the lysosomal marker N-acetyl-beta-hexosaminidase (Hex), in a fraction with a mean density of 1.035 g/ml. In contrast, more than 70% of the Hex activity of control cells sedimented in comparable gradients with a density of more than 1.07 g/ml. The lysosomal localization of NeuAc accumulation in affected fibroblasts was confirmed by treatment of post-nuclear supernatant fractions with 0.5 mM Gly-Phe-beta-naphthylamide (20 min, 37 degrees C) prior to centrifugation, which resulted in the simultaneous loss of latency of Hex and free NeuAc, and their association with the soluble fraction on Percoll gradients. The results provide direct evidence for the accumulation of free NeuAc in a unique buoyant lysosomal fraction of affected fibroblasts.     

3Development-Dependent Regulation of N-Acetyl-β-d-Hexosaminidase of Cerebellum and Cerebrum of Normal and Staggerer Mutant Mice

Distinctive activities of various glycosidases were expressed in the cerebellum and cerebral cortex of mice during their development. In particular, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) appeared to be developmentally regulated. A transient peak of enzyme activity at postnatal day 7 was characteristic for the cerebellum, whereas the activity in the cerebral cortex gradually increased through the 1st postnatal month and was maintained at a high level of activity throughout adulthood. The regulation of N-acetylhexosaminidase activity in the developing cerebellum of the staggerer mouse deviated clearly from enzyme activities in the wild-type, whereas the activity pattern in the staggerer cerebral cortex remained unaffected. In experiments mixing wild-type and staggerer cerebellum homogenates, the specific activity was additive. Thus, involvement of inhibitors or activating molecules can be excluded. This developmentally controlled regulation or disregulation in staggerer appears to be enzyme specific, sine beta-glucosidase, alpha-glucosidase, and beta-galactosidase did not exhibit such a pattern in either normal or staggerer mice. In the mutation weaver that, like staggerer, loses the majority of its cerebellar granule cells, N-acetyl-beta-hexosaminidase activity of the cerebellum was not elevated, indicating a specific defect in staggerer rather than a general effect on lysosomal enzymes due to cell death.     

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

Purification of ricin A1, A2, and B chains and characterization of their toxicity

This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains.     

Diplococcal beta-galactosidase with a specificity reacting to beta 1-4 linkage but not to beta 1-3 linkage as a useful exoglycosidase for the structural elucidation of glycolipids

Diplococcal beta-galactosidase, which is known to be useful for the structural studies of glycoprotein-linked oligosaccharides, was found to show the same substrate specificity in cleaving Gal beta 1-4 linkages of glycolipids as that of the oligosaccharides. The optimum conditions of beta-galactosidase in the 80% ammonium sulfate precipitates of the culture medium of Streptococcus (Diplococcus) pneumoniae were determined with nLcOse4Cer radiolabeled by the galactose oxidase-NaB3H4 procedure. Detergent was required for the highest activity, and different combinations of several buffers and detergents showed different properties in stimulating beta-galactosidase, and in enhancing or suppressing N-acetyl-beta-hexosaminidase which was contaminated in the enzyme preparation. The optimum pH was found to be at 6.5, and specific activity and Km were 8.1 nmol/mg protein/h and 1 nmol, respectively. While more than 70% of beta-galactose was liberated from LacCer and nLcOse4Cer within 1 h under the optimum conditions to form GlcCer and nLcOse3Cer, respectively, none was liberated from LcOse4Cer, GalCer, GgOse4Cer, GbOse3Cer, IV3 alpha GalnLcOse4Cer, and Il3NeuAcGgOse4Cer, showing the substrate specificity solely to Gal beta 1-4 linkage.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.     

The existence of atypical blood group galactosyltransferase which causes an expression of A2 character in A1B red blood cells

It is generally accepted that the blood group subtypes A1 and A2 expressions are controlled by two different blood group N-acetylgalactosaminyl-transferases, that is, A1-enzyme and A2-enzyme, respectively, and that the two types of enzymes are governed by the allelic A1 and A2 genes. The observed frequencies of blood types in Caucasians are compatible to this model. However, the subtype A2 character is far more frequently observed in AB red cells than in A red cells in some black and Oriental populations. Two black blood samples with phenotype A2B contained A1-enzyme, but not A2-enzyme, and exhibited several times higher B-enzyme activity than control AB and B blood. The kinetic properties, that is, pH-activity profile and Km for UDP-Gal, of the B-enzyme from these two A2B subjects differed from that of control B-enzyme. In these two cases, therefore, the A2 character was not caused by the subactive A2-enzyme, but because of an insufficient formation of the A-substances in red cell membranes presumably caused by the competition between the A1-enzyme and the super active atypical B-enzyme at the common H-sites. The results suggest that the B gene can be subdivided into usual B1 and atypical B2, and that not only A2B subjects but also A1B2 subjects could express A2 character in their red cells. The B2 gene may be common in certain black and Oriental populations.     

Blood group A and B glycosyltransferases synthesize A and B determinants on different acceptor polyglycosyl peptidesin vitro

The glycosylation of polyglycosyl chains from human erythrocytes by human plasma blood group A and B glycosyltransferases was studied in order to clarify why human blood group AB erythrocyte polyglycosyl peptides carry only either A or B determinants [Eur J Biochem (1981) 113:259–65].The blood group A transferase was able to add radioactiveN-acetylgalactosamine from labeled UDP-N-acetylgalactosamine to B-type erythrocytes which had been treated with -galactosidase in order to cleave the B determinant sugar from the erythrocytes. This suggests that the enzymes specified by theA andB genes utilize the same acceptor molecules on erythrocyte membranes. Polyglycosyl peptides isolated from blood group B erythrocytes acted as acceptors for blood group A glycosyltransferase and the generation of hybrid structures containing both A and B determinants was demon-strated. When blood group O polyglycosyl peptides were used as acceptors in the simultaneous presence of both blood group A and B glycosyltransferases, however, the A and B determinant sugars were found in different polyglycosyl peptides. It is suggested that the enzyme-acceptor complex does not dissociate until the final number of determinants has been added.