Ultrastructural localization of acid phosphatase in rosetted T and B lymphocytes of normal human blood. (49/74)

Using electron microscopy and cytochemical techniques, the authors determined the distribution of acid phosphatase (AcPase) within the organelles of lymphocytes from blood rosetted with either neuraminidase-treated sheep erythrocytes (En) or sheep erythrocytes coated with antibody and complement (EACs). Subsequently, the various reactive organelles of the rosetted lymphocytes were counted, affording a comparison of T and B cells. It was found that AcPase was present in approximately 80% of T cells and 45% of B cells and was most frequently observed in secondary lysosomes of varying size and content. Although more T cells than B cells were reactive for AcPase, the extent of reaction in some B cells clearly precludes the use of AcPase for differentiating the two cell lines. It should be recognized that while the En rosetting procedure detects T cells in a nonselective manner, the EAC rosette is a marker of a major subpopulation of B lymphocytes, ie, those bearing complement receptors. We believe that the distribution of lysosomal enzymes in B and T lymphocytes probably reflects the functional state of individual cells rather than being a reliable indicator of cell lineage. A surprising finding (which could be established only by a fine-structural study) was the fact that 20% of circulating "resting" T cells contained reaction product for AcPase within endoplasmic reticulum and the perinuclear cisterna indicating that these cells are actively synthesizing AcPase, probably due to a foregoing inductive event. Such stimulus could be the result of recent endocytosis of surface receptors in combination with antigen, antibody, or immune complexes and/or recent mitosis, or possibly some unrelated autophagic incident.  (+info)

Purification and properties of feline and human arylsulfatase B isozymes. Evidence for feline homodimeric and human monomeric structures. (50/74)

Normal feline and human arylsulfatase B isozymes were purified to homogeniety from liver. The specific activities of the feline and human enzymes toward p-nitrocatechol sulfate were 1,100,000 and 800,000 units/mg of protein, and toward UDP-N-acetylgalactosamine-4-sulfate were 5,500 and 4,000 units/mg of protein, respectively. Although both enzymes had the same pH optimum (5.7), the feline enzyme was more electronegative than the human enzyme when electrophoresed on polyacrylamide gels. Compared to the human isozyme, feline arylsulfatase B had a lower Km toward p-nitrocatechol sulfate (1.2 versus 3.6 mM), was more thermostable at 60 degrees C (68 versus 30 min), and had a slightly lower pI (7.8 versus 8.0). The native molecular weight of the feline enzyme was estimated to be about twice that the human isozyme by gel filtration, analytical polyacrylamide gel electrophoresis, and sucrose density-gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single protein bands of Mr = 41,000 and 38,000 for the feline and human isozymes, respectively. Alkylation and cross-linking experiments were consistent with the feline enzyme being a homodimer and the human enzyme a monomer. Amino acid compositional analyses revealed few significant differences between the two isozymes.  (+info)

Lysosomal enzyme content of Kupffer and endothelial liver cells isolated from germfree and clean conventional rats. (51/74)

Rats kept under germfree conditions showed lower specific lysosomal enzyme activities in liver endothelial cells, but not in Kupffer cells.  (+info)

Preliminary characterization of the oligosaccharide component of arylsulfatase B from human placenta. (52/74)

Isoelectric focusing of homogenous arylsulfatase B from human placenta pointed to the presence of enzymatically active and inactive forms of high pI (pH 9-8) and of lower pI (pH 6.5-5.5). Glycan chain analysis performed with the use of a Glycan Differentiation Kit showed that basic forms of arylsulfatase B from human placenta contained mostly high mannose/hybrid type glycans, with 6-O-L-fucose bound to the innermost N-acetylglucosamine residue, whereas acidic forms of the enzyme contained complex type glycans containing fucose and sialic acid. However, the latter forms constitute a small percentage of the total carbohydrate component. Lectin affinity chromatography of the native enzyme confirmed the presence of a core fucose and a sialic acid.  (+info)

Autophagy-related changes of arylsulphatases A and B in rat liver lysosomes. (53/74)

The total arylsulphatase activity and the relative activities of lysosomal arylsulphatases A and B were measured in the liver of control rats and rats subjected to treatments that provoke hepatic autophagocytosis. The total liver arylsulphatase activities were increased in starved and starved glucagon-treated rats, but not in sham-operated and hepatectomized rats. Arylsulphatases A and B in the mitochondrial-lysosomal (M-L) fraction were separated by polyacrylamide-gel electrophoresis at pH 8.8; they were made visible by incubating the gels with p-nitrocatechol sulphate as substrate, and measured by quantitative densitometry. In untreated controls, arylsulphatases A and B comprised 41.4 +/- 0.5% and 58.6 +/- 0.5% of the total arylsulphatase activity respectively; the arylsulphatase A/arylsulphatase B activity ratio was 0.71. All experimental treatments produced a significant decrease in the percentage of lysosomal arylsulphatase present as the A form and an increase in that present as the B form, and the activity ratio of arylsulphatase A/arylsulphatase B declined. The magnitude of these changes increased in the following direction: starvation for 24h=sham hepatectomy less than glucagon + starvation less than subtotal hepatectomy. These results indicate that the arylsulphatase A/arylsulphatase B activity ratio in liver lysosomes of normal rats is maintained within rather narrow limits, and this ratio declines during enhanced autophagocytosis. These findings, together with observations that suggest that arylsulphatase B may be a partially degraded form of arylsulphatase A, are consistent with the view that the A form is more rapidly converted into the B form during autophagy, owing to the digestive activity of the other lysosomal hydrolases present in autophagic vacuoles.  (+info)

Boar seminal vesicles secrete arylsulfatases into seminal plasma: evidence that desulfation of seminolipid occurs only after ejaculation. (54/74)

The presence and composition of arylsulfatases in secretions of various glands of the boar genital tract were studied. Arylsulfatase A was present in seminal plasma but not in extracellular fluids of the testis and epididymis nor in blood serum of boars. On the other hand, arylsulfatase B was present in both seminal plasma and extracellular fluids of the testis but was completely resorbed in the epididymis. The acrosomal arylsulfatase A did not leak out of spermatozoa before ejaculation. We conclude that arylsulfatases A and B present in seminal plasma are secreted by the seminal vesicles, for three reasons: 1) secretions from seminal vesicles contained 2.3-fold higher arylsulfatase activities than did those from seminal plasma, but had an identical composition; 2) cauda epididymal fluids did not contain arylsulfatase; and 3) other accessory glands of the boar genital tract did not secrete arylsulfatase. When intact boar spermatozoa were incubated with arylsulfatase A, complete desulfation of seminolipid was observed. The most important arguments favoring our hypothesis that desulfation of seminolipid does not start before ejaculation are the following: 1) desulfoseminolipid is not detectable in epididymal or freshly ejaculated sperm samples; 2) the acrosomal arylsulfatase A cannot desulfate seminolipid present at the surface of the plasma membrane of intact spermatozoa because of its intracellular localization; 3) extracellular arylsulfatase A is stored in seminal vesicles and thus can interact with spermatozoa during and after ejaculation.  (+info)

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase B gene alleles causing variable disease phenotypes. (55/74)

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase B (ASB), also known as N-acetylgalactosamine-4-sulfatase. Multiple clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of the human ASB gene facilitated the analysis of molecular defects underlying the different phenotypes. Conditions for PCR amplification of the entire open reading frame from genomic DNA and for subsequent direct automated DNA sequencing of the resulting DNA fragments were established. Besides two polymorphisms described elsewhere that cause methionine-for-valine substitutions in the arylsulfatase B gene, six new mutations in six patients were detected: four point mutations resulting in amino acid substitutions, a 1-bp deletion, and a 1-bp insertion. The point mutations were two G-to-A and two T-to-C transitions. The G-to-A transitions cause an arginine-for-glycine substitution at residue 144 in a homoallelic patient with a severe disease phenotype and a tyrosine-for-cysteine substitution at residue 521 in a potentially heteroallelic patient with the severe form of the disease. The T-to-C transitions cause an arginine-for-cysteine substitution at amino acid residue 192 in a homoallelic patient with mild symptoms and a proline-for-leucine substitution at amino acid 321 in a homoallelic patient with the intermediate form. The insertion between nucleotides T1284 and G1285 resulted in a loss of the 100 C-terminal amino acids of the wild-type protein and in the deletion of nucleotide C1577 in a 39-amino-acid C-terminal extension of the ASB polypeptide. Both mutations were detected in homoallelic patients with the severe form of the disease. Expression of mutant cDNAs encoding the four amino acid substitutions and the deletion resulted in severe reduction of both ASB protein levels and arylsulfatase enzyme activity in comparison with a wild-type control. The six mutations described in the present study were unique among 25 unrelated mucopolysaccharidosis VI patients, suggesting a broad molecular heterogeneity of the Maroteaux-Lamy syndrome.  (+info)

Juvenile form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). A C-terminal extension causes instability but increases catalytic efficiency of arylsulfatase B. (56/74)

A deficiency of the enzyme arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type arylsulfatase B.  (+info)