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1.
Circadian variations of mouse liver, brain and heart lysosomal susceptibility to hypoxia were investigated. Lysosomal disruption during hypoxia was estimated on the basis of the following measurements: changes in percentage free activity of β-galactosidase and acid phosphatase, tissue loss of both lysosomal enzymes and accumulation of serum β-galactosidase. When exposure to hypoxia took place at the end of the rest phase or at the beginning of the active phase, it was accompanied by maximum increase of percent free activity. This, presumably represents a diffusion of enzymes from lysosomes due to altered membrane permeability. However, hypoxia when occurring during the second part of the active phase and first part of the rest phase resulted in tissues loss of lysosomal enzymes and accumulation of serum lysosomal enzymes. This is believed to represent the release of lysosomal enzymes in bulk from damaged or ruptured lysosomal membranes. 相似文献
2.
In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β- N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values. 相似文献
3.
Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active
β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by
cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a
3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having
88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose,
ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and
thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when
compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic
and thermophilic enzymes. 相似文献
4.
A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β- N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders. 相似文献
5.
Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β- N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes. 相似文献
6.
Cultured human diploid fibroblasts were grown for 4 days in media at five different pH values between pH 6.8 and 8.0. The specific activities of α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase and hexosaminidase were determined. Increasing environmental pH levels produced increased levels of β-glucosidase activity and decreased levels of activity for the other five enzymes. 相似文献
7.
Staining for β-galactosidase activity for whole tissues, sections, and cells is a common method to detect expression of β- galactosidase reporter transgene as well as senescence-dependent β-galactosidase activity. Choice of fixatives is a critical step for detection of β-galactosidase activity, subsequent immunostaining, and enzymatic digestion of tissue to dissociate cells. In this report, the authors examined several aldehyde and alcohol fixatives in mouse skeletal muscle tissues for their efficiency at improving detection of β-galactosidase activity as well as detection by immunostaining. In addition, fixatives were also analyzed for their efficiency for collagenase digestion to isolate single muscle fibers on postfixed β-galactosidase-stained whole skeletal muscle tissues. The results show that fixing cells with isopropanol yields the greatest reliability and intensity in both β-galactosidase staining as well as double staining for β-galactosidase activity and antibodies. In addition, isopropanol and ethanol, but not glutaraldehyde or paraformaldehyde, allow for the isolation of single muscle fibers from the diaphragm and tibialis anterior muscles following postfixed β-galactosidase staining. Using this method, it is possible to identify the amount of cells that occupy the satellite cell compartment in single muscle fibers prepared from any muscle tissues, including tibialis anterior muscle and diaphragm. 相似文献
8.
Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. 相似文献
9.
Trypsin released from the surface of intact human skin fibroblasts β- N-acetylglucosaminidase. The amount of trypsin removable β- N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β- N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β- N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β- N-acetylglucosaminidase, α- N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β- N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β- N-acetylglycosaminidase, α- N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface. 相似文献
10.
Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5m m-mannose 6-phosphate, which decreases β- N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50m m-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular β- N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun. 49, 992–999]. We propose that ( a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, ( b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and ( c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle. 相似文献
11.
The present study examined the acute effects of hypoxia on the regulation of skeletal muscle metabolism at rest and during 15 min of submaximal exercise. Subjects exercised on two occasions for 15 min at 55% of their normoxic maximal oxygen uptake while breathing 11% O(2) (hypoxia) or room air (normoxia). Muscle biopsies were taken at rest and after 1 and 15 min of exercise. At rest, no effects on muscle metabolism were observed in response to hypoxia. In the 1st min of exercise, glycogenolysis was significantly greater in hypoxia compared with normoxia. This small difference in glycogenolysis was associated with a tendency toward a greater concentration of substrate, free P(i), in hypoxia compared with normoxia. Pyruvate dehydrogenase activity (PDH(a)) was lower in hypoxia at 1 min compared with normoxia, resulting in a reduced rate of pyruvate oxidation and a greater lactate accumulation. During the last 14 min of exercise, glycogenolysis was greater in hypoxia despite a lower mole fraction of phosphorylase a. The greater glycogenolytic rate was maintained posttransformationally through significantly higher free [AMP] and [P(i)]. At the end of exercise, PDH(a) was greater in hypoxia compared with normoxia, contributing to a greater rate of pyruvate oxidation. Because of the higher glycogenolytic rate in hypoxia, the rate of pyruvate production continued to exceed the rate of pyruvate oxidation, resulting in significant lactate accumulation in hypoxia compared with no further lactate accumulation in normoxia. Hence, the elevated lactate production associated with hypoxia at the same absolute workload could in part be explained by the effects of hypoxia on the activities of the rate-limiting enzymes, phosphorylase and PDH, which regulate the rates of pyruvate production and pyruvate oxidation, respectively. 相似文献
12.
Rat mammary tumours induced by 7,12-dimethylbenz[ a]anthracene can undergo repeated growth and regression during successive pregnancies. In a 10-day period after birth about half of the tumours regressed 50% or more. The concentrations of the lysosomal enzymes increased in regressing mammary tumours to the following multiples of the initial values: β-glucuronidase, 7·7; β-galactosidase, 3·9; cathepsin, 2·9; acid ribonuclease, 2·1; arylsulphatase A, 1·5; acid phosphatase, 1·4. In contrast, several non-lysosomal enzymes failed to increase. Activities in the post-partum uterus increased to the following multiples of the initial values: β-glucuronidase, 5·8; cathepsin, 5·5; acid ribonuclease, 4·3; β-galactosidase, 2·2; acid phosphatase, 1·8. Arylsulphatase A in the post-partum uterus decreased significantly, suggesting a non-lysosomal distribution or a special function related to pregnancy. No other significant changes were observed in the lysosomal or non-lysosomal enzymes in the hormone-independent liver or hormone-dependent normal mammary gland. The ratio of free to bound arylsulphatase A and acid ribonuclease decreased slightly 1–3 days after birth because of problems in homogenizing the tumours. At days 4–8, however, there was a dramatic increase in the ratio of the free to bound activities. The results can be explained in terms of the lysosomal theory of intracellular digestion. 相似文献
14.
Colloidal gold is accumulated in much smaller concentrations in lysosomes of hepatocytes of the ‘dense body’ type than in the larger lysosomes of sinus endothelial cells. These differences were used for labeling of lysosomal subpopulations as well as for the isolation of a hepatocytederived lysosomal subpopulation by combined differential and density gradient centrifugation. The origin of isolated lysosomes was determined by measurements of the gold-grain content and by cytomorphometric analyses giving size distribution curves of lysosomal profiles before and after isolation. The specific activities of β-galactosidase, acid phosphatase and especially of β- N-acetyl-glucosaminidase and β-glucuronidase were lower in lysosomal subfractions derived mainly from hepatocytes than in a mixed lysosomal fraction containing both lysosomes from hepatocytes and sinus endothelial cells. 相似文献
15.
The enzymes β-glucosidase, α-glucosidase, β-galactosidase, α-galactosidase, and β-xylosidase were detected in Phaseolus vulgaris L. var. Red Kidney bean hypocotyl tissue throughout the first 13 days of development with p-nitrophenyl glycosides as substrates. Activities of all enzymes except β-glucosidase declined as a function of increasing tissue age. In contrast, β-glucosidase activity increased rapidly 3 days after imbibition to a maximal activity at 5 days and then subsided to one-third the maximum by day 7. This activity peak immediately preceded the logarithmic phase of hypocotyl growth. This enzyme is strongly associated with cell walls during extraction, suggesting that it is wall-bound in situ. Various polysaccharide substrates were used to evaluate the specificity of this enzyme. 相似文献
16.
Oxygen-sensing mechanisms are often dysfunctional in tumours. Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor α (HIF-α) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes ( SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor α-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. α-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFα and/or resistance to EglN3-mediated apoptosis. 相似文献
17.
A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes. 相似文献
18.
With the aim to improve the performance of enzyme bound to hydrophilic solid phases, their immobilization with polyethylene glycol (PEG) tether have been studied. Sweet potato β-amylase, which hydrolyses the high molecular weight substrate starch and β-galactosidase, which acts on low molecular weight substrates, were used as model enzymes and beaded thiol–agarose as solid phase. Several two step methods for the introduction of the tether using a bis-oxirane homobifunctional PEG as well as a heterobifunctional derivative with a hydroxysuccinimide ester and a maleimide group have been evaluated. Amino groups, native and de novo thiol groups in the enzymes were utilized for immobilization. The best approach was found to be to first introduce the PEG derivative via one of its reactive groups to the enzyme. Subsequently the formed conjugate was bound to the solid phase by the remaining reactive group. Attempts to first introduce the PEG tether into the solid phase were not successful. A high degree of substitution with PEG chains on the enzyme leads to high immobilization yields for both β-amylase and β-galactosidase, but relatively lower gel-bound activity for the former enzyme which is acting on a high molecular weight substrate and thus more sensitive for steric shielding effects. With optimal degree of PEG substitution (which occurred at five times molar excess of the heterobifunctional reagent) the gel-bound activity of β-amylase was increased from 12% (for the derivative without tether) to 31%. 相似文献
19.
Mammalian lysosomal sialidase exists as an enzyme complex with β-galactosidase and carboxypeptidase, so-called “protective protein.” In this article, we report that chicken sialidase also occurs as a complex with β-galactosidase and protective protein. The purified sialidase complex had a molecular weight > 700 kDa on gel filtration and showed four protein components of 76, 65, 54 and 48 kDa on SDS-PAGE under nonreducing conditions. N-Terminal sequences of the 65- and 48-kDa proteins were homologous to human lysosomal β-galactosidase and protective protein precursor, respectively. The purified sialidase complex also had carboxypeptidase activity. Both sialidase and carboxypeptidase activities were precipitated together by an antibody against chicken β-galactosidase. The complex reversibly dissociated into 120-kDa β-galactosidase dimer and 100-kDa carboxypeptidase dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. These findings indicate that chicken sialidase exists as a multienzyme complex, by which the sialidase activity appears to be stabilized. 相似文献
20.
BackgroundGM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid β-galactosidase (βgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis. Methodology/Principal FindingsIn this study we examined the effect of thalamic infusion of AAV2/1-βgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice. Conclusions/SignificanceOur studies show that the AAV-modified thalamus can be used as a ‘built-in’ central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice. 相似文献
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