GALACTOSIDASE
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Vypracovala sa jednoduchá, citlivá a reprodukovateľná metóda dôkazu extracelulárnej β-galaktozidázy. Kalusovou kultúrou, resp. koreňom klíčnych rastlín sekrétovaná β-galaktozidáza hydrolyzuje substrát (6-bróm-2-naftyl-β-D-galaktopyranozid) na β-D-galaktózu a 6-bróm-2-naftol, ktorý po simultánnej azokopulácii s hexazotovaným p-rozanilínom, resp. bázickým fuksínom vytvára červenohnedú vo vode takmer nerozpustnú zlúčeninu (dÍEizóniovú soI). Zafarbenie pod kalusovou kultúrou a okolo nej, ako aj koreňa iďíčnych rastlín je prejavom činnosti extracelulárnej β-galaktozidázy Študovanými objektmi. Intenzita zafarbenia je mierou ich enzýmovej aktivity. Intracelulárne lokalizovaný enzým má majoritné, kým extracelulárna β-galaktozidáza minoritné zastúpenie
A simple, sensitive and reproducible method of detection of extracellular β-galactosidase was worked out. β-Galactosidase secreted by the callus culture, or the roots of sprouting plants, hydrolyzes the substrate (6-bromo-2-naphthyl β-D-galactopyranoside) to produce β-D-galactose and 6-bromo-2-naphthol, which after simultaneous azocoupling with hexazotized p-rosaniline, or basic fuchsine, produce a reddish brown compound, nearly insoluble in water (a diazonium salt). The colouring under the callus culture and around it, as well as the colouring of the roots of the sprouting plants, is a manifestation of the activity of extracellular β-galactosidase by the objects under study. The intensity of the colouring is a measure of their enzymatic activity. The share of intracellularly localized enzyme represents the majority and the share of extracellular β-galactosidase the minority.
A new enzymatic method for the synthesis of β-galactosides of nucleosides and acyclic nucleoside analogues has been developed, using β-galactosidase from Escherichia coli as a catalyst and lactose as a sugar donor. The method is very rapid, feasible and last but not least inexpensive. Its applicability has been proven for a broad variety of possible substrates with respect to its scaling up for preparative use. Five new compounds from a series of nucleoside and acyclic nucleoside analogues have been prepared on a scale of several hundred milligrams, in all cases revealing very good results of the method concerning the reproducibility of the reaction yields and simplicity of the purification process.
Ultrastructural and histochemical studies of bioptic and postmortem tissue samples from ten Fabry hemizygotes showed lysosomal storage in adipocytes as a constant feature of the classic phenotype of α-galactosidase (GLA) deficiency. The storage was represented by a crescent-shaped line of storage lysosomes of varying thicknesses restricted to the perinuclear subplasmalemmal area. The ultrastructure of the storage lysosomes was dominated by concentric lipid membranes modified by simultaneous deposition of autofluorescent ceroid. Storage was widely expressed in adipose tissue. The number of storage lysosomes was increased, and the lysosomes were more clustered in adipocytes with less voluminous lipid content. The findings should attract interest to studies of adipose tissue biology in Fabry disease, a topic that has not been studied so far. In terms of cell biology, the observations represent indirect evidence of significant lysosomal turnover of α-galactose lipid conjugates in adipocytes demasked by GLA deficiency. The results extend the thus far limited information on the adipocyte lysosomal system and its participation in lysosomal storage disorders.
- MeSH
- alfa-galaktosidasa genetika metabolismus MeSH
- biopsie MeSH
- Fabryho nemoc enzymologie genetika patologie MeSH
- fenotyp MeSH
- genetická predispozice k nemoci MeSH
- glykogen metabolismus MeSH
- hemizygot MeSH
- lidé MeSH
- lyzozomy enzymologie ultrastruktura MeSH
- mutace MeSH
- pitva MeSH
- tukové buňky enzymologie ultrastruktura MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
