Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification

Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.     

Enhanced validation of small-molecule ligands and carbohydrates in the Protein Data Bank

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Genetic effects of eelgrass restoration efforts by fishers' seeding to recover seagrass beds as an important natural capital for coastal ecosystem services

The reestablishment of seagrass vegetation is a vital part of recovering coastal marine ecosystem services. Historically the Hinase area was a famous for the fishing by coastal pound netting in eelgrass beds, but this practice was progressively displaced with oyster farming due to an enormous decline in seagrass vegetation. For several decades, the local fishers' cooperative has worked to restore eelgrass beds by a seeding method. Through these efforts, seagrass vegetation in their fishing area has increased to about half of their previous area. This study examined the effect of long-term seeding by fishers on the recovery of eelgrass beds in the Hinase area, based on analysis of eelgrass genetic structure using microsatellite markers. Specimens for the DNA analysis were collected from each of all eelgrass meadows that the fishers conducted sowing eelgrass seeds as well as from the source sites where they collected the seeds. The results found that restored beds in the study area have high genetic diversity comparable to natural ones. The multiple regression analysis revealed that a combined model of seedling intensity and geographic distance (R² = .457) better explained genetic structure across our sampling sites than models of seedling intensity (R² = .092) or geographic distance only (R² = .344). This supports that the eelgrass seeds they sowed did not disturb the genetic structure but rather supplemented natural dispersal, suggesting that the fishers' seeding did not develop nonnatural seagrass meadows but certainly contributed to the recovery of natural seagrass meadows.     

Characterization of glycogen phosphorylase isoenzymes present in cultured skeletal muscle from patients with McArdle's disease.

Muscle biopsy specimens from patients with McArdle's disease lack glycogen phosphorylase activity. Significant phosphorylase activity was detected in cultured muscle cells from these patients. The phosphorylase isoenzymes in the cells were identified electrophoretically and immunochemically. On polyacrylamide disc gel electrophoresis, two types of isoenzymes were separated in about equal amounts. Both differed the muscle type in migration, kinetic, and immunochemical properties. The first type corresponded to a fetal phosphorylase isoenzyme, and the second was a liver-like type which was completely absorbed with antibody against the rat liver isoenzyme. No adult skeletal muscle isoenzyme was detected.     

Catalytic accretion of thermal heterocomplex molecules from amino acids in aqueous milieu

Thermal heterocomplex molecules made by heating amino acids, aspartic acid and proline, exhibit an autocatalytic accretion in their aqueous suspension under appropriate conditions of their temperature and concentration.     

Detection of fluorescamine-labeled amino acids,peptides, and other primary amines on thin-layer chromatograms

Amino acids, peptides, catecholamines, and polyamines were reacted with fluorescamine and subjected to thin-layer chromatography. These fluorescamine derivatives of primary amines were detectable at levels below 100 pmoles. Methods of preparation and chromatography of these fluorophors are presented.     

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes.

Hen liver microsomes contained 0.20 nmol of cytochromeb₅ per mg of protein. Upon addition of NADH about 95% cytochrome b₅ was reduced very fast with a rate constant of 206 s^?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b₅ in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 10⁸m^?1 s^?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b₅. To explain the results, therefore, the reverse electron flow from cytochrome b₅ to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b₅ reductase by NADH is rate-limiting in the NADH-cytochrome b₅ and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm.