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PubMedDate not supplied

[Activity and appearance of isoenzyme spectrums of some lysosomal hydrolases in biopsy material of human chorion].

Activities and isozyme spectra of alpha-L-fucosidase and beta-D-hexosaminidase were similar both in biopsy material from fetal zone of placenta (chorion) and in chorion tissue obtained after abortion. Evaluation of the isozyme spectrum of these glycosidases in biopsy reeterial of chorion might be carried out for prenatal detection of fucosidosis (deficiency of alpha-L-fucosidase), Tay-Sachs disease (deficiency of hexosaminidase A) and of Sandhoff disease (deficiency of hexosaminidases A and B).

Abortion, Spontaneous↗
PubMed1984

Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts.

Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.

Cell Line↗
PubMed1981

Liberation of N-acetylglucosamine-6-sulfate by human beta-N-acetylhexosaminidase A.

The first step of the degradation of p-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside and of keratan sulfate-derived oligosaccharides bearing N-acetylglucosamine-6-sulfate residues at the nonreducing end was considered to be accomplished by the action of a specific sulfatase (Kresse, H., Paschke, E., von Figura, K., Gilberg, W., and Fuchs W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6822-6826). In purification from human placenta, however, this activity co-chromatographed with isoenzyme A of beta-N-acetylhexosaminidase and had the same electrophoretic mobility as the latter enzyme. The activity was precipitated by a specific antiserum against beta-N-acetylhexosaminidase. A pronounced enzyme deficiency was found in Tay-Sachs and Sandhoff fibroblasts. The purified enzyme released p-nitrophenol from the chromogenic substrate as well as a second product which contained equimolar amounts of hexosamine and sulfate. This product had the same electrophoretic and chromatographic behavior as sulfated N-acetylglucosamine. It could be degraded by periodate to a smaller charged fragment. Incubation of keratan sulfate-derived oligosaccharides with beta-N-acetylhexosaminidase A analogously resulted in the liberation of N-acetylglucosamine-6-sulfate. The enzyme showed the highest affinity towards a trisulfated tetrasaccharide and exhibited a similar Km for the sulfated and the unsulfated p-nitrophenyl derivative.

Acetylglucosamine↗
PubMed1980

Correction of I-cell defect by hybridization with lysosomal enzyme deficient human fibroblasts.

I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G(M2) gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mobility of the isoenzymes, and in a decreased activity in the medium. Fusion of I-cells with fibroblasts from G(M1) gangliosidosis, type 1, led to enhancement of beta-galactosidase (beta-gal) activity. This complementation must be the result of the presence of normal polypeptide chains in I-cells, whereas the other cell types provide a factor that causes the intracellular retention of the enzymes. Restoration of beta-gal was also observed in heterokaryons after fusion of I-cells with beta-galactosidase/neuraminidase-deficient (beta-gal(-)/neur(-)) variants, indicating that the neuraminidase(s) and the posttranslational modification of beta-gal are affected in a different way in I-cell disease and in beta-gal(-)/neur(-) variants. Fusion of I-cells with mannosidosis fibroblasts resulted in a restoration of the acidic form of alpha-mannosidase and in a decrease of the extracellular activity of both this enzyme and the hexosaminidase enzyme, indicating that fusion of I-cells with different types of fibroblasts with a single lysosomal enzyme deficiency not only leads to complementation for one particular enzyme but also to a correction of the basic defect in I-cells.

Cell Fusion↗
PubMed1982

Ultrastructural study of biopsy specimens of rectal mucosa. Its use in neuronal storage diseases.

Rectal mucosa biopsy specimens from patients with neuronal storage diseases were examined by electron microscopy. The diseases were Tay-Sachs disease, Sandhoff's disease, Niemann-Pick disease types B and C, late infantile metachromatic leukodystrophy, GM1 gangliosidosis type 1, beta-galactosidase-neuraminidase deficiency, I-cell disease, and mucopolysaccharidoses (Hunter's syndrome and Sanfilippo's syndrome type A). Unmyelinated nerve fibers, endothelial cells, fibroblasts, plasma cells, and histiocytes were seen in the specimens. Except for plasma cells, the results thus obtained for various cells were similar to those obtained from skin and conjunctival biopsy specimens, which have been already reported. There has been no report so far on ultrastructure of the plasma cell in these diseases. Storage materials, eg, dense bodies and membrane-bound vacuoles, were observed in the plasma cells in various storage diseases, with the exception of late infantile metachromatic leukodystrophy. Thus, electron microscopy of rectal mucosa is useful in making diagnoses and examining plasma cells in some neuronal storage diseases.

Adolescent↗
PubMed1995

In situ assessment of beta-hexosaminidase activity.

We have adapted two methods to evaluate the beta-hexosaminidase (HEX) enzymatic activity in cultured cells, based on the use of (i) the fluorogenic substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2- deoxy-beta-D-glucopyranoside (MU-GlcNAc-6-SO4) and (ii) the naphthol AS-BI-N-acetyl-beta-D-glucosaminide and hexazotized pararosaniline. We demonstrate that both methods could be used for the HEX isoenzymes by comparing wild-type and mutant human fibroblast cell lines, deficient for either an alpha or beta subunit from Tay-Sachs and Sandhoff patients. This in situ cytochemical assessment of HEX activity offers a rapid evaluation to study the expression of this enzyme in a heterogeneous cell population such as in gene transfer experiments.

Acetylglucosamine↗
PubMed1994

HPLC analysis of oligosaccharides in urine from oligosaccharidosis patients.

Analysis of urinary oligosaccharides by thin-layer chromatography (TLC) is used as screening procedure for 10 different lysosomal diseases. We tested the usefulness of HPLC in screening, using a CarboPac PA1 column (Dionex), pulsed amperometric detection (PAD), and post-column derivatization (PCD). Patterns from six types of oligosaccharidoses were compared with normal urinary patterns and with the TLC patterns. PAD appeared to be nonspecific and therefore is applicable only to desalted urine samples. PCD was more specific and applicable to nondesalted urine samples, albeit with a lower resolving power. Peaks in urines from oligosaccharidoses patients were identified on the basis of retention times of commercially available oligosaccharides or TLC bands after isolation and HPLC of the corresponding oligosaccharides. Abnormal oligosaccharide peaks were seen in urines from patients with alpha-mannosidosis, GM1-gangliosidosis (juvenile), GM2-gangliosidosis (Sandhoff disease), Pompe disease, and beta-mannosidosis. HPLC detected no abnormal oligosaccharides in urine from patients with fucosidosis. Although TLC is a simple and reliable screening procedure for detecting classical lysosomal diseases with oligosaccharide excretion, HPLC, by its higher resolution and possibility of quantification, can more generally be used for recognition of abnormal oligosaccharides or detection of increased excretion or content for known oligosaccharides in urine, other body fluids, and cells.

Adolescent↗
PubMed1994

Preferential beta-hexosaminidase (Hex) A (alpha beta) formation in the absence of beta-Hex B (beta beta) due to heterozygous point mutations present in beta-Hex beta-chain alleles of a motor neuron disease patient.

Deficiency of the lysosomal enzyme beta-hexosaminidase B (beta-Hex B) (a homodimer, beta beta), caused by a defect in the HEX B gene encoding the beta-chain, is usually accompanied by an absence of beta-Hex A (a heterodimer, alpha beta), thereby causing Sandhoff disease. However, we have earlier demonstrated the presence of partial beta-Hex A (30-50% of normal) even in the absence of beta-Hex B in an adult with motor neuron disease. The patient is a compound heterozygote with normal beta-chain message and one HEX B point mutation originating from each asymptomatic parent. Since the non-expression of beta-Hex B was post-transcriptional, we transfected COS-7 cells to understand the effect of each mutation on beta-Hex B activity. Transfection of the A1367-->C mutant (maternal allele) construct produced no overexpressed beta-Hex B, indicating that the encoded Tyr456-->Ser beta-chain was non-functional. Chou-Fasman analysis predicted that the Tyr456-->Ser mutation would cause a dramatic change in beta-chain folding (which often inhibits formation of functional dimers). This explains the complete lack of beta-Hex B in the transfectants and a partial deficiency of beta-Hex A and B (50% of normal) in the patient's mother. Since immunoprecipitated beta-Hex A (alpha beta) protein from patient fibroblasts showed the presence of mature beta-chains even though there was no beta-Hex B (beta beta) protein, the mutant beta-chain inherited from the father (who has normal beta-Hex A and B) must undergo preferential association with the normal alpha-chains in the patient, thus producing only beta-Hex A. Transient expression of the A619-->G mutant (paternal allele) construct produced beta-Hex B activity comparable to the wild type (approximately 10-20-fold over mock-transfected) whereas stable expression produced normal message but no beta-Hex B activity (wild type beta-Hex B expression: only 2-fold over mock-transfected). The lack of increased beta-Hex B after stable expression of the Ile207-->Val beta-chains at a lower copy number indicates the absence of self-association at low concentrations of Ile207-->Val beta-chain. In the patient who also has a non-functional Tyr456-->Ser allele, the effective concentration of beta-chains is reduced to 50% of normal and the remaining Ile207-->Val beta-chains fail to self-associate but can still dimerize with the abundant normal alpha-chains thus producing partial beta-Hex A and no beta-Hex B.

Adult↗
PubMed1993

Enzyme immunoassay of beta-hexosaminidase A and B in serum: carrier detection of GM2-gangliosidoses, and equivalence of enzyme activity and enzyme protein reactivity.

beta-Hexosaminidase (Hex; EC 3.2.1.52) isoenzymes A and B were analyzed in sera from a control group of 22 apparently healthy subjects, 13 obligate carriers of Tay-Sachs disease (TSD), 10 obligate carriers of Sandhoff disease (SHD), and 4 affected TSD patients by enzyme immunoassay methods based on enzyme activity. No Hex A activity was detected in the sera of patients with TSD. The activities of Hex A in the obligate carriers of TSD and SHD tended to be lower (nonsignificantly) than in the control group. Hex B activities tended to be higher in TSD patients as well as in carriers of TSD, although the mean activities did not significantly differ from the corresponding mean for the control group. However, Hex B activities were decreased in the carriers of SHD in comparison with the other groups. Sera from 900 postmenopausal women, all of age 55 years, were also analyzed for Hex isoenzymes; the results indicated a carrier frequency of about 1 in 200 for both TSD and SHD. We also compared the enzyme immunoassay method based on enzyme activity with one based on the antigenic (enzyme protein) reactivity alone. Because both methods yielded similar information, we conclude that no significant amounts of inactive enzyme protein are present in the circulation.

Adolescent↗
PubMed1995

Lysosomal storage disorders in Thailand: the Siriraj experience.

Lysosomal storage disorders are a heterogeneous group of biochemical genetic disorders; currently 40-50 are known. The clinical phenotype is determined by the tissue distribution of the storage material and degree of enzyme deficiency. The genetic transmission is mostly autosomal recessive. Lysosomal storage disorders can be divided into three groups according to the major organ system pathology: (1) Primary involvement of the central nervous system without significant somatic or skeletal pathology. Disorders of grey matter, eg gangliosidosis and disorders of white matter eg the leucodystrophy are the most common; (2) Primary involvement of the reticuloendothelial system with or without associated neuropathology, eg Niemann-Pick disease and Gaucher disease; (3) Multisystem involvement in which skeletal manifestations are prominent features. The mucopolysaccharidosis and mucolipidoses are the two major forms with this clinical phenotype. Lysosomal storage disorders identified at Siriraj Hospital are neuronal ceroid lipofuscinosis, GMI gangliosidosis, mucolipidosis II, Maroteaux-Lamy, sialidosis, Sly syndrome, Hunter syndrome, Morquio syndrome, Gaucher disease, Niemann-Pick, Sandhoff disease, Pompe's disease and many more. Most patients came from the provinces where consanguinity is common. Confirmation usually is done by enzyme assays using skin fibroblast culture or leucocytes. Genetic counseling is extremely important and prenatal diagnosis is recommended to high-risk couple.

Child↗
PubMed1997

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Endosomes↗
PubMed1980

Genetic diseases in Lebanon.

Lebanon has a high incidence of common and rare genetic diseases, due probably to the mosaic different ethnic origins and the high rate of consanguineous marriages in certain communities. Two major investigations, exploring the genetic structure of the Lebanese population, indicated the population to be caucasiods (though Oriental traits were found). These investigations involved studies of dermatoglyphics, and type and distribution of genetic markers and protein variants. Recorded genetic diseases, some characteristic of ethnic group or particular to a geographic region include familial paroxysmal polyserositis, familial hypercholesterolemia, hypothroidism, the Dyggve-Melchoir-Clausen syndrome, Sandhoff disease, and various genetic hematologic diseases. Genetic counseling and care to patients with genetic diseases is available in Beirut from the Faculty of Medicine at American University and St. Joseph University. Also, the Lebanese National Council for Scientific Research initiates and finances medical genetics programs.

Consanguinity↗
PubMed1987

Demonstration of myenteric plexus abnormalities in genetic diseases by a microdissection technique: preliminary studies.

Eighty-eight specimens of esophagus, small intestine, or colon from 45 patients, predominantly infants and children, with 30 different genetic diseases were analyzed by a microdissection technique for the following abnormalities of the Auerbach (myenteric) plexus: (1) abnormality of the pattern of the nervous network of the plexus, (2) abnormal fraction of neural tissue in the plane of the plexus, (3) abnormal size or appearance of the cytoplasm of the neurons of the plexus, and (4) abnormal number of neurons in the ganglia of the plexus. Seven of 8 specimens of esophagus from patients with neuronal storage diseases (infantile Niemann-Pick disease, Jansky-Bielschowsky disease, etc.) showed an increased fraction of neural tissue in the plane of the plexus, whereas 2 of 3 patients with Cockayne syndrome showed a reduced fraction, with abnormally slender interganglionic fibers. The fraction of neural tissue in the plane of the plexus was also abnormal at one or more levels in patients with adrenoleukodystrophy, ataxia telangiectasia, Krabbe disease, and juvenile metachromatic leukodystrophy. Abnormality of neuron size and cytology was seen in several neuronal lipidoses, including Jansky-Bielschowsky and Sandhoff diseases and juvenile GM2 gangliosidosis, with the most striking neuronal enlargement noted in infantile Niemann-Pick disease. Abnormalities of plexus mass or pattern, as well as those of neuronal cytoplasm and neuron number, offer improved insight into possible mechanisms producing gastrointestinal tract dysfunction (swallowing difficulty, gastroesophageal reflux, constipation, etc) in patients with genetic disorders.

Child↗
PubMedDate not supplied

Activity and multiple forms of alpha-L-fucosidase and hexosaminidase in chorion biopsy specimens and some fetal organs.

It was shown that activities and isoenzyme patterns of alpha-L-fucosidase and hexosaminidase were similar in biopsy specimens of chorion obtained immediately before induced abortion in normal pregnancy and in chorion tissue itself. A comparative study of isoenzyme patterns of these glycosidases showed their similarity in chorion and human fetal kidney, liver and lungs. The data obtained may be applied to the investigation of the multiple forms of alpha-L-fucosidase and hexosaminidase in chorion biopsy specimens for the prenatal diagnosis of fucosidosis (alpha-L-fucosidase deficiency), Tay-Sachs diseases (hexosaminidase A deficiency) and Sandhoff disease (deficiency of hexosaminidase A and B).

Biopsy↗
PubMed1992

Prenatal diagnosis of metabolic diseases on chorionic villi obtained before the ninth week of pregnancy.

Nine pregnancies at risk for various metabolic disorders were monitored by prenatal diagnosis on chorionic villi obtained between the sixth and ninth weeks of pregnancy. A diagnosis of an affected fetus was made in five cases (Sandhoff, Tay-Sachs (2), Pompe's, GM1), while metachromatic leukodystrophy, GM1 (2), and Pompe's were excluded in four cases. It is concluded that chorionic villi are a reliable tissue for prenatal diagnosis of metabolic disorders also when obtained before the ninth week.

Cerebroside-Sulfatase↗
PubMed1997

Generation and characterization of mouse monoclonal antibodies specific for N-linked neutral oligosaccharides of glycoproteins.

We generated four monoclonal antibodies (MAbs) specific for asparagine-linked neutral oligosaccharides of glycoproteins by immunizing mice with neoglycolipids, which were derived from glycoproteins by conjugation to phosphatidylethanolamine dipalmitoyl. The binding specificity of these MAbs was determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. The four MAbs designated OMB3, OMB4, OMR5, and OMR6 reacted strongly with the neoglycolipids, Gal beta1-4GlcNAc beta1-2Man alpha1-6(Gal beta1-4GlcNAc beta1-2Man alpha1-3)Man beta1-4GlcNAc-PD, GlcNAc beta1-2Man alpha1-6(GlcNAc beta1-2Man alpha1-3)(GlcNAc beta1-4)Man beta1-4GlcNAc beta1-4GlcNAc-PD, Man alpha1-6Man beta1-4GlcNAc beta1-4(Fuc alpha1-6)GlcNAc-PD, and Man alpha1-3Man beta1-4GlcNAc-PD, respectively, that were used as immunogens. All of these MAbs exhibited a high binding specificity. The epitopes of the MAbs OMB3 and OMB4 were suggested to be nonreducing terminal trisaccharides, Gal beta1-4GlcNAc beta1-2Man-, and nonreducing beta-GlcNAc residues, respectively. MAbs OMR5 and OMR6 showed a highly restricted binding specificity, reacting only with the immunizing neoglycolipids. Subsequently, MAbs OMB3 and OMB4 were shown to react strongly with asialo-alpha1-acid-glycoprotein and asialo-agalacto-alpha1-acid-glycoprotein, respectively, by Western blotting. Furthermore, it was shown that these MAbs reacted specifically with the epitope on Chinese hamster ovary cells by an immunofluorescence technique. MAb OMB4 was also shown to detect the accumulated oligosaccharides with nonreducing terminal beta-GlcNAc residues as granular inclusions in the cultured fibroblasts from a classical Sandhoff disease patient.

Animals↗
PubMed1994

Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb.

Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1984

The effects of sucrose loading on lysosomal hydrolases.

The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM1-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH4Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of alpha-mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of alpha-mannosidase activity. However, some induction still exists in beta-galactosidase and alpha-fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process. In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism of the induction of lysosomal hydrolase activities by sucrose loading.

2,4-Dinitrophenol↗