A novel optical tracer for VMAT2 applied to live cell measurements of vesicle maturation in cultured human β-cells

Abstract The islet β-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating β-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live β-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger β-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In β-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living β-cells within complex mixtures for further manipulation and characterisation

Тбилисская Неделя N42

ყოველკვირეული გაზეთი სრული ტელეპროგრამით; Еженедельная газета с полной телепрограммо

Stabilization of a-glucosidase in organic solvents by immobilization on macroporous poly(GMA-co-EGDMA) with different surface characteristics

a-Glucosidase from baker’s yeast was immobilized on macroporous copolymers of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), with various surface characteristics and pore sizes ranging from 44 nmto 270 nm. Immobilization was done by glutaraldehyde on the copolymer previously modified with 1,2-diaminoethane. The specific activity of the obtained immobilized enzyme varied from 27 to 81 U/g, depending on the employed copolymer. The half lives of the immobilized enzyme in cosolvents were influenced by the surface characteristics of the copolymer, ranging from 60 to 150 min in 35 % methanol and from 10 to 44 min in 45 % dimethyl sulphoxide (DMSO). The best stabilities were obtained when the enzyme was immobilized onto a copolymer having a pore size of 48 nm in methanol and 270 nm in DMSO

Preparation and studies on immobilized α-glucosidase from baker’s yeast Saccharomyces cerevisiae

α-Glucosidase from S. cerevisiae was covalently immobilized onto Sepabeads EC–EA by the glutaraldehyde method. An analysis of the variables controlling the immobilization process is first presented and it is shown that the highest coupling of α-glucosidase occurred within 24 h. Also, a loading of 30 mg/g support proved to be effective, resulting in a rather high activity of around 45 U g–1 with a satisfactory degree of enzyme fixed. Both free and immobilized enzymes were then characterized by determining the activity profile as a function of pH, temperature and thermal stability. The obtained immobilized preparation showed the same optimum pH, but a higher optimum temperature compared with the soluble one. In addition, the immobilized enzyme treated at 45 ºC for 1 h still retained an activity of around 20 %, whereas the free enzyme completely lost its original activity under this condition. In conclusion, the developed immobilization procedure is quite simple, easily reproducible and provides a promising solution for the application of immobilized α-glucosidase

The specificity of alpha-glucosidase from Saccharomyces cerevisiae differs depending on the type of reaction: hydrolysis versus transglucosylation

Our investigation of the catalytic properties of Saccharomyces cerevisiae alpha-glucosidase (AGL) using hydroxybenzyl alcohol (HBA) isomers as transglucosylation substrates and their glucosides in hydrolytic reactions demonstrated interesting findings pertaining to the aglycon specificity of this important enzyme. AGL specificity increased from the para(p)- to the ortho(o)-HBA isomer in transglucosylation, whereas such AGL aglycon specificity was not seen in hydrolysis, thus indicating that the second step of the reaction (i.e., binding of the glucosyl acceptor) is rate-determining. To study the influence of substitution pattern on AGL kinetics, we compared AGL specificity, inferred from kinetic constants, for HBA isomers and other aglycon substrates. The demonstrated inhibitory effects of HBA isomers and their corresponding glucosides on AGL-catalyzed hydrolysis of p-nitrophenyl a-glucoside (PNPG) suggest that HBA glucosides act as competitive, whereas HBA isomers are noncompetitive, inhibitors. As such, we postulate that aromatic moieties cannot bind to an active site unless an enzyme-glucosyl complex has already formed, but they can interact with other regions of the enzyme molecule resulting in inhibition