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PubMed1989

Processing enzymes acting on carbohydrate moiety of lysosomal hydrolases in leukemic cells: elevated activity of N-acetylglucosamine-1-phosphotransferase.

We previously demonstrated that an acidic variant form of lysosomal arylsulfatase B accumulated in chronic myelogenous leukemia (CML) cells was highly phosphorylated at its carbohydrate moiety (Uehara Y, et al, Cancer Res 43:5618, 1983). Since lysosomal hydrolases including the sulfatase underwent the posttranslational phosphorylation processing at the carbohydrate moiety, we investigated two enzymes acting on the processing in peripheral leukocytes from leukemia patients. The activity level of the first enzyme in the processing, an N-acetylglucosamine-1-phosphotransferase to form phosphodiester at the carbohydrates, was significantly higher in CML cells than in normal control. The transferase level in CML cells was also higher compared with that in normal bone marrow cells, which include myeloid progenitor cells. However, the activity of the second processing enzyme, a phosphodiester glycosidase that converts a phosphodiester to a phosphomonoester, showed no consistent change in CML cells. Thus, increment of the sulfatase variant containing phosphomonoesters and diesters in CML cells is most probably associated with elevated activities of the phosphotransferase. In two cases of CML in blastic crisis and a case of acute myelogenous leukemia (AML), activity of the processing enzyme was considerably decreased concomitant with reduction of peripheral blastic cells by chemotherapy.

Adult↗
PubMedDate not supplied

Phase II biotransformation of carcinogens/atherogens in cultured aortic tissues and cells. I. Sulfation of 3-hydroxy-benzo(a)pyrene.

The capacity of polynuclear aromatic hydrocarbons to elicit profound effects on the development of avian aortic atheromata has raised questions regarding the biotransformation of polynuclear aromatic hydrocarbons in the target (aortic) tissue. Results of this investigation demonstrate the capacity of aortic enzymes to affect the sulfoconjugation of 3-hydroxy-benzo(a)pyrene and describe several characteristics of the aortic sulfotransferase activity. Conjugating activities measured in avian aortic tissues were approximately 10-20% of those assayed in corresponding preparations of avian hepatic tissues under the same reaction conditions. Activities were measured in homogenates, in a series of homogenate subfractions, in whole organ cultures, in cultured aortic endothelial cells, and in cultured aortic smooth muscle cell preparations. Sulfoconjugation was localized in the cytosolic fraction and kinetics in this fraction yielded a range of apparent Km values from 9 to 16 microM (mean = 11.8 +/- 3.1, n = 4) and a range of apparent Vmax values from 281 to 457 pmol/mg of protein/30 min (mean = 360 +/- 49, n = 4). Abdominal and thoracic segments of the aorta exhibited virtually identical specific activities. Also, activities assayed in cultured aortic smooth muscle cells were similar to those measured in cultured aortic endothelial cells. Capacity to generate adenosine 3'-phosphate 5'-phosphosulfate (PAPS) appeared to limit the reaction rate as judged by comparative investigations with PAPS and a PAPS-generating system. Aortic sulfatases actively hydrolyzed benzo(a)pyrene-3-O-sulfate. The sulfatase activity appeared to partially mask sulfotransferase activities measured in organ and cell culture preparations and in particulate subfractions of cellular homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Hepatic drug-metabolizing enzymes in primary and secondary tumors of human liver.

Significant increases in activities of epoxide hydrolase, UDP-glucuronosyltransferase, and glutathione S-transferase, and marked reductions in cytochrome P-450 mixed-function oxidase systems occur in hyperplastic nodules induced in rat liver by chemical mutagens. In contrast, activities of both oxidative (Phase I) and conjugative (Phase II) enzymes are decreased in hepatocellular carcinomas induced by peroxisome proliferators. The present work compares alterations induced by chemical mutagens or peroxisome proliferators with changes in enzyme activities that occur in primary and secondary hepatic tumors in man. The above activities, along with beta-glucuronidase and arylsulfatase, were measured in liver samples from 6 normal livers obtained at immediate autopsy, and liver specimens obtained by surgical biopsy from the following patients: 8 with hepatomas, 5 with nonmetastatic colorectal carcinomas, and 14 with metastatic colorectal carcinomas. Cytochromes P-450MP and P-450NF in addition to epoxide hydrolase were measured by immunoquantitation. Enzymes involved in conjugation reactions were either assayed fluorometrically (UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase) or spectrophotometrically (glutathione S-transferase) using umbelliferyl substrates or 1-chloro-2,4-dinitrobenzene. Secondary hepatic tumors showed no significant change in drug-metabolizing enzymes, in contrast to primary hepatomas, which displayed decreases in all of the measured drug metabolizing enzymes. Arylsulfatase was markedly depressed in primary hepatomas (14% of normal values). Thus, activities of drug-metabolizing enzymes in human primary tumors resemble those associated with altered hepatic foci induced by peroxisome proliferators such as ciprofibrate. The marked decreases in sulfatase that occurred in primary but not in secondary human tumors suggest that sulfation of endogenous compounds and xenobiotics may differ in patients with primary and secondary hepatic tumors.

Adenocarcinoma↗
PubMed1987

[Changes in steroid enzyme activity in the human endometrium during the menstrual cycle].

Sulfokinase, sulfatase, 17 beta-HSD, 20 alpha-HSD, 3 beta-HSD and 5 alpha-reductase activity and steroid concentrations including estradiol, estrone, estrone-sulfate, progesterone, 20 alpha-dihydroprogesterone, DHA and DHA-sulfate in endometrial tissue were examined in order to study the changes in steroid metabolism in relation to the menstrual cycle in the human endometrium. Thirty-one (14) proliferative and 17 secretory) endometrial tissue samples were obtained from women who underwent hysterectomy. Low enzymatic activity of sulfokinase, sulfatase and 17 beta-HSD activity were observed in the proliferative phase (0.25, 8.5, 3.1 nmole/mg protein/h). A pronounced increase in enzymatic activity was observed in the early secretory phase and activity gradually decreased toward the mid and late secretory phase. On the other hand, 20 alpha-HSD and 3 beta-HSD activity did not change during the cycle. 5 alpha-reductase activity was not detectable under the conditions used. The concentration of progesterone in the secretory phase was significantly higher than that in the proliferative phase. The concentration of estradiol in the proliferative phase was significantly higher than that in the secretory phase. There was no significant change in the concentration of estrone, estronesulfate, 20 alpha-dihydroprogesterone, DHA or DHA-sulfate during the cycle. The relationship between the steroid concentration and the enzymatic activity was discussed. The results suggested an active role of the endometrium in controlling the biological effect of steroids.

Adult↗
PubMed1986

Aromatase inhibitors for treatment of breast cancer: current concepts and new perspectives.

Estrogens provide the major hormonal support for endocrine-dependent human mammary neoplasms. In postmenopausal women, the extraglandular aromatization of the adrenal prehormone, androstenedione to estrone is the major pathway for estrogen biosynthesis. Estrone can then be converted into estradiol or into an inactive conjugate, estrone sulfate. Recent data suggest that the estrogens may also be synthesized in situ by human breast tumors, either from androstenedione via aromatase, or from estrone sulfate via the enzyme, sulfatase. Our enzyme kinetic studies support the predominance of the sulfatase pathway for in situ estrogen biosynthesis. The ability of estrone sulfate to stimulate colony formation of the nitrosomethylurea-induced rat mammary tumor in the clonogenic assay, suggests that this in situ pathway has biologic relevance. Aromatase inhibitors can be used to suppress the levels of circulating estrone, estrone sulfate, and estradiol in postmenopausal women. Aminoglutethimide, the major inhibitor currently used clinically, acts in a competitive fashion and blocks cholesterol side chain cleavage and 11 beta-hydroxylase as well as aromatase. Clinical studies indicate that the combination of aminoglutethimide plus replacement glucocorticoid causes breast tumor regression with the same frequency and for the same duration as surgical ablative therapies such as adrenalectomy or hypophysectomy. Aminoglutethimide also induces a similar rate of tumor regression as achieved with the antiestrogen, tamoxifen. However, because tamoxifen is associated with fewer side effects, this antiestrogen is to be preferred over use of aminoglutethimide as first-line hormonal treatment for women with breast cancer. Several specific suicide inhibitors of aminoglutethimide such as 4-hydroxy-androstenedione are being developed and have proven effective in early clinical trials with breast cancer patients. Further development of active aromatase inhibitors should allow precise control of estradiol levels in women with breast cancer. This ability to perform an 'estrogen clamp' may allow new strategies to be developed in which hormone depletion followed by repletion can produce a synchronization of tumor cell DNA synthesis. If achievable, such manipulations may allow potentiation of the effects of cytotoxic chemotherapy. This latter concept is currently being rigorously tested in basic and in clinical investigative studies.

Adrenalectomy↗
PubMed1986

Hydrolysis of organic sulfates in periportal and pericentral regions of the liver lobule: studies with 4-methylumbelliferyl sulfate in the perfused rat liver.

The hydrolysis of 4-methylumbelliferyl sulfate by liver sulfatases to free fluorescent 4-methylumbelliferone and the subsequent formation of the glucuronide conjugate were studied in the isolated perfused rat liver. In livers from fed, phenobarbital-treated rats, 4-methylumbelliferyl sulfate (0.25-1.5 mM) was hydrolyzed rapidly to free 4-methylumbelliferone at maximal rates of about 5 mumol/g/hr. A major fraction of the free 4-methylumbelliferone formed was converted to the glucuronide at maximal rates around 20 mumol/g/hr. Similar rates of hydrolysis were observed in livers from fasted, phenobarbital-treated or normal rats, although the ratio of glucuronide to free product was decreased markedly by fasting. In liver homogenates, however, rates of organic sulfate hydrolysis exceeded those observed in the perfused liver by at least 2-fold, suggesting that 4-methylumbelliferyl sulfate content is an important determinant of rates of hydrolysis in the perfused liver. There was a good correlation (r = 0.91) between rates of product formation and fluorescence of 4-methylumbelliferone detected from the liver surface with fiber optic light guides. Fluorescence of 4-methylumbelliferone produced from hydrolysis of 4-methylumbelliferyl sulfate was also monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule for the estimation of local rates of product formation. When perfusions were in the anterograde direction, desulfation of 4-methylumbelliferyl sulfate was about 50% higher in pericentral (28.8 +/- 9.3 mumol/g/hr) than in periportal (18.2 +/- 2.7 mumol/g/hr) areas. Furthermore, 4-methylumbelliferyl sulfate content determined in microdissected samples was 1.5- to 2-fold higher in pericentral than in periportal regions of the liver lobule but the activity of 4-methylumbelliferyl sulfate sulfatase was identical in both zones of the liver lobule. We conclude, therefore, that the local substrate content is an important determinant of rates of 4-methylumbelliferyl sulfate hydrolysis in sublobular zones of the liver.

Animals↗
PubMed1979

Chondroitin SO4 catabolism in chick embryo chondrocytes.

An enzyme preparation from cultured chick embryo vertebral chondrocytes attacks chondroitin SO4 oligosaccharides from the nonreducing terminal in a recycling pathway involving the sequential action of a beta-glucuronidase, a 4- or a 6-sulfatase, and a beta-N-acetylgalactosaminidase. The sequence is blocked by saccharo-1,4-lactone, an inhibitor of the beta-glucuronidase, or by 2-acetamido-2-deoxy-D-galactonolactone, an inhibitor of the beta-N-acetylgalactosaminidase. The level of 4-sulfatase activity is low relative to the other activities and limits the rate of catabolism of hybrid oligosaccharide structures containing both 6-sulfated galactosamine residues and 4-sulfated galactosamine residues. This results in the accumulation of shortened oligosaccharides, most of which have galactosamine-4-SO4 residues at their nonreducing terminals. In the presence of the lactone inhibitors, polymeric chondroitin SO4 is broken down by the enzyme preparation to oligosaccharides which are 10 to 15 monosaccharides long, indicating that degradation of chondroitin SO4 chains is initiated by an endoglycosidase which generates oligosaccharide substrates for the recycling exoglycosidase system.

Acetylgalactosamine↗
PubMed1983

N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are desulfated by the same enzyme.

We have prepared a series of oligosaccharides to assess the substrate specificity of exo sulfatase activity in cultured human skin fibroblasts toward N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from HS) were desulfated by a specific sulfatase that when deficient leads to the accumulation of HS and the expression of mucopolysaccharidosis type IIID (Sanfilippo D). Under the in vitro conditions studied there are two pathways for the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4 residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4 residue is desulfated and then cleaved by the action of an beta-N-acetylglucosaminidase activity. There was no detectable beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides. There was approximately 10% of this normal beta-N-acetylglucosaminidase activity in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a Sanfilippo D patient we propose that KS degradation in these patients proceeds by the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4 which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1984

A new method for the simultaneous analysis of free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine in human urine by HPLC with electrochemical detection.

A sensitive and specific assay for free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine was achieved by tetraphenylboron complexing and by high-performance liquid chromatography with electrochemical detection. Sulfoconjugated metabolites were hydrolyzed by sulfatase. Complexing the 3-O-methylated catecholamines with tetraphenylboron followed by their extraction in either and reextraction in dilute hydrochloric acid succeeded in partially purifying the sample without the use of a prepacked column. Detection by electrochemical technique additionally eliminates unoxidable components in the urine. The relatively high sensitivity of this method permits measurement of metabolite concentrations as low as 10 ng/ml, allowing for the first time the determination of free 3-O-methylated catecholamines in human urine. Hydrolysis by sulfatase specifically identifies the form of the conjugated metabolite. The mean total (free plus sulfoconjugated) values of normetanephrine, metanephrine, and 3-methoxytyramine obtained from 25 normal volunteers were 214, 102, and 227 micrograms/day, respectively. Values were greatly increased in patients with pheochromocytoma. The degree of sulfoconjugation varied, and each individual metabolite was highest with normetanephrine (86%). This method is sensitive, rapid, simple, and can be easily standardized for clinical investigation of pheochromocytoma.

Adrenal Gland Neoplasms↗
PubMedDate not supplied

[Properties of a bacterial enzyme preparation of alkylsulfatase].

A preparation of primary alkyl sulfatase was obtained from the culture of Pseudomonas species 2T/1. It can hydrolyze alkyl sulfates, which belong to anion surface-active compounds, to sulfate ion and fatty alcohol, and as a result the harmful for biosphere property of the surface activity is gone. pH and temperature of the incubation mixture, the presence of ions of some bivalent metals and components of synthetic detergents (SD), composition of the buffer mixture and substrate concentration affect the rate of sodium dodecylsulfate (SDS) hydrolysis. The alkyl sulfatase preparation is relatively stable. The maximum rate of SDS hydrolysis was found to be at 70 degrees C. The preparation catalyzes the hydrolysis of some alkyl sulfate homologues and industrial alkyl sulfates. The temperature optimum of the preparation is 40 degrees C, the pH-optimum is 8.0-9.0.

Hydrogen-Ion Concentration↗
PubMed1993

Prenatal diagnosis of Sanfilippo syndrome type A by early amniocentesis.

Early amniocentesis performed at 13 weeks gestation was utilized to obtain amniocytes for culture. Sonicates of cultured amniocytes were used to measure heparin sulfamidase activity for assessment of the status of an at risk pregnancy for Sanfilippo syndrome, type A. The heparin sulfamidase activity was not detectable in cultured amniocytes of the fetus at risk while another enzyme, N-acetylglucosamine 6-sulfatase, was comparable to that of the control. Following termination of the pregnancy, various tissue from the fetus were used for assay of both enzymes. The sulfamidase activity was not detectable in any of the fetal tissue while the 6-sulfatase activity was present in all fetal tissue but varied in activity depending on the type of tissue. Cultured fetal skin and brain contained the highest enzyme activity while skin and liver contained the lowest.

Amniocentesis↗
PubMedDate not supplied

[Mutation analysis in Hunter patients].

Mutations of the iduronate-2-sulfatase gene have been identified as responsible of Hunter syndrome or mucopolysaccharidosis type II. About 20% of the patients have deletions of the whole gene or other major structural alterations. The mutations found so far include: 34 missense, 8 nonsense, 11 small deletions from 1 to 3 bp, 2 deletions of 8 pb, 2 insertions of 1 bp and 2 insertions of 14 bp, with most leading to a frameshift and premature chain termination. Also 8 different splice-site mutations leading to insertions or deletions in the mRNA have been tabulated. Knowledge of the primary genetic defect allows insight into genotype-phenotype correlation and allows a better understanding of the structure and function of iduronate-2-sulfatase.

Base Sequence↗
PubMed1998

Mucopolysaccharidosis type II (Hunter's syndrome) in Taiwan.

The mucopolysaccharidoses are a group of inherited disorders of lysosomal storage of glycosaminoglycans. Among them, mucopolysaccharidosis (MPS) type II (Hunter's syndrome), caused by a deficiency in iduronate sulfatase, is the only one inherited in an X-linked recessive manner. We describe 12 Hunter's syndrome patients and seven carriers, with precise analysis of glycosaminoglycan content in urine and iduronate sulfatase activity in cultured fibroblasts and plasma. Their ages at the time of diagnosis ranged from 1 year 10 months to 11 years (mean 4.3 yr). The delay in diagnosis was from 1 month to 5 years (mean 2.1 yr) after the initial presentation. The most frequent initial complaints of the patients were delayed developmental milestones (75%) and speech (67%), although all patients were found to have coarsening of facial features at diagnosis. The difficulties in disease recognition allowed disease recurrence in four of the 11 families. Prompt clinical suspicion and referral will be important in genetic counseling for MPS type II and its management, if definitive therapy becomes available.

Adolescent↗
PubMed2005

Tumor necrosis factor inhibits conversion of dehydroepiandrosterone sulfate (DHEAS) to DHEA in rheumatoid arthritis synovial cells: a prerequisite for local androgen deficiency.

OBJECTIVE: Use of anti-tumor necrosis factor (anti-TNF) antibody therapy in rheumatoid arthritis (RA) has expanded our understanding of possible mechanisms by which this treatment reduces inflammation. Beyond its effects on local immune responses, anti-TNF treatment may also modulate the local hormone supply. Because androgens are thought to inhibit immune responses, their presence in inflamed tissue is an additional important antiinflammatory factor. METHODS: We investigated conversion of the ubiquitous dehydroepiandrosterone sulfate (DHEAS), the biologically inactive precursor of DHEA, to the androgen DHEA in mixed synovial cells from patients with RA and patients with osteoarthritis (OA), making use of thin-layer chromatography and phosphorimaging. Using immunohistochemical analysis, we detected the key enzyme, steroid sulfatase. RESULTS: DHEAS-to-DHEA conversion in synovial cells from patients with RA was significantly lower than that in synovial cells from patients with OA (mean +/- SEM 3.3 +/- 0.5% versus 6.0 +/- 0.9% of applied (3)H-DHEAS per 10(6) synovial cells; P = 0.042). In RA, but not in OA, the level of converted (3)H-DHEA was inversely correlated with the density of synovial macrophages (for RA, R(rank) = -0.725, P = 0.005; for OA, R(rank) = 0.069, P not significant [NS]) and T cells (for RA, R(rank) = -0.621, P = 0.024; for OA, R(rank) = 0.247, P NS). Double immunohistochemistry analysis revealed that steroid sulfatase was located mainly in synovial macrophages but was also observed in fibroblasts. Neutralization of TNF largely up-regulated the conversion of DHEAS to DHEA in RA, but not in OA. A similar neutralizing effect was observed with polyclonal human immunoglobulins; this effect is most probably mediated via TNF neutralization at low TNF concentrations. CONCLUSION: These data indicate that TNF inhibits the conversion of DHEAS to DHEA in RA synovial cells. Because androgens are antiinflammatory mediators, TNF-induced inhibition of the local androgen supply is a supplementary proinflammatory factor. Consequently, anti-TNF strategies may also exert their positive effects by increasing tissue androgens.

Aged↗
PubMed2005

Mutation and polymorphism spectrum of the GALNS gene in mucopolysaccharidosis IVA (Morquio A).

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal-recessive disorder caused by a deficiency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS; E.C.3.1.6.4). GALNS is required to degrade glycosaminoglycans, keratan sulfate (KS), and chondroitin-6-sulfate. Accumulation of undegraded substrates in lysosomes of the affected tissues leads to a systemic bone dysplasia. We summarize information on 148 unique mutations determined to date in the GALNS gene, including 26 novel mutations (19 missense, four small deletions, one splice-site, and two insertions). This heterogeneity in GALNS gene mutations accounts for an extensive clinical variability within MPS IVA. Seven polymorphisms that cause an amino acid change, and nine silent variants in the coding region are also described. Of the analyzed mutant alleles, missense mutations accounted for 78.4%; small deletions, 9.2%; nonsense mutation, 5.0%; large deletion, 2.4%; and insertions, 1.6%. Transitional mutations at CpG dinucleotides accounted for 26.4% of all the described mutations. The importance of the relationship between methylation status and distribution of transitional mutations at CpG sites at the GALNS gene locus was elucidated. The three most frequent mutations (over 5% of all mutations) were represented by missense mutations (p.R386C, p.G301C, and p.I113F). A genotype/phenotype correlation was defined in some mutations. Missense mutations associated with a certain phenotype were studied for their effects on enzyme activity and stability, the levels of blood and urine KS, the location of mutations with regard to the tertiary structure, and the loci of the altered amino acid residues among sulfatase proteins.

Animals↗
PubMed2004

Differentiation of sulfate and phosphate by H/D exchange mass spectrometry: application to isoflavone.

Often phosphorylation or sulfation is an important step which occurs in the signal transduction and cascade of metabolic pathways. Some natural products and metabolites contain one or more sulfate or phosphate groups. Isoflavone sulfate has been identified from high-resolution mass spectrometry (HRMS) and enzymatic digestion by sulfatase. We previously reported the new water-soluble isoflavone analogs, daidzein 7-O-phosphate and genistein 7-O-phosphate, which were surprisingly hydrolyzed by sulfatase. In this previous study, we could not determine the phosphate from the results of HRMS and enzymatic digestion, that is, HRMS and enzymatic digestion did not provide clear evidence. In this case, we drew conclusions from NMR analysis. HRMS has been ineffective with a regular fast atom bombardment (FAB) mass spectrometer to distinguish between phosphate and sulfate since the mass difference is only 0.009 mass units. There was, however, no conventional method of microanalysis to distinguish phosphate from sulfate owing to the same nominal mass. It is still very difficult to determine by negative FABMS [--O--P(==O)(OH)(2)] = 80 and [--O--S(==O)(2)OH] = 80. In this paper, we report a method to distinguish between these groups by using a popular low-resolution mass instrument; thus, phosphate and sulfate were measured by H/D exchange mass spectrometry at the picomole level to differentiate [--O--P(==O)(OD)(2)] = 82 and [--O--S(==O)(2)OD] = 81, respectively. This method is applicable not only to the isoflavone, but also to other phospho and sulfo compounds.

Genistein↗
PubMed1997

Lysosomal biogenesis in lysosomal storage disorders.

Lysosomal biogenesis is an orchestration of the structural and functional elements of the lysosome to form an integrated organelle and involves the synthesis, targeting, functional residence, and turnover of the proteins that comprise the lysosome. We have investigated lysosomal biogenesis during the formation and dissipation of storage vacuoles in two model systems. One involves the formation of sucrosomes in normal skin fibroblasts and the other utilizes storage disorder-affected skin fibroblasts; both of these systems result in an increase in the size and the number of lysosomal vacuoles. Lysosomal proteins, beta-hexosaminidase, alpha-mannosidase, N-acetylgalactosamine-4-sulfatase, acid phosphatase, and the lysosome-associated membrane protein, LAMP-1, were shown to be elevated between 2- and 28-fold above normal during lysosomal storage. Levels of mRNA for the lysosome-associated membrane proteins LAMP-1 and LAMP-2, N-acetylgalactosamine-4-sulfatase, and the 46- and 300-kDa mannose-6-phosphate receptors were also elevated 2- to 8-fold. The up-regulation of protein and mRNA lagged 2-4 days behind the formation of lysosomal storage vacuoles. Correction of storage, in both systems, resulted in the rapid decline of the mRNA to basal levels, with a slower decrease in the levels of lysosomal proteins. Lysosomal biogenesis in storage disorders is shown to be a regulated process which is partially controlled at, or prior to, the level of mRNA. Although lysosomal proteins were differentially regulated, the coordination of these events in lysosomal biogenesis would suggest that a common mechanism(s) may be in operation.

Antibodies, Monoclonal↗
PubMed1987

Hydrolysis of 4-methylumbelliferyl sulfate in periportal and pericentral areas of the liver lobule.

4-Methylumbelliferyl sulfate was used to characterize sulfatase activity in periportal and pericentral regions of the liver lobule in the perfused rat liver. Following infusion of 1.5 mM of this organic sulfatester, free 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were formed at rates of 13 and 9 mumoles/g/h, respectively, in livers from fasted, phenobarbital-treated rats. 5-Pregnen-3 beta-ol, 20-one sulfate inhibited hydrolysis and metabolite production completely, whereas perfusion with nitrogen-saturated perfusate or FCCP decreased total metabolite formation by only 30%. 4-Methylumbelliferone formed from the hydrolysis of 4-methylumbelliferyl sulfate was monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule. Detection of the desulfated product was always greater in the downstream region, i.e., infusion of 4-methylumbelliferyl sulfate produced a higher fluorescence signal in pericentral areas when perfusion was in the anterograde direction, while periportal areas demonstrated higher activity during perfusion in the retrograde direction. Perfusion with nitrogen-saturated perfusate abolished these differences. Taken together, these data suggest that uptake of organic sulfateesters is partially energy dependent, follows the hepatic oxygen gradient inversely, and is a major rate determinant for sulfatase activity in the liver.

Animals↗