Specific Activity Of Β-glucosidase Research Articles

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes. N-acetylglucosaminidase, β-glucosidase, α-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of β-glucosidase, α-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and α-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.

Acid Glycosidase Isoenzymes and Activities in Developing Hulls and Seeds of Pisum sativum L.

Summary The changes in β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC 3.2.1.24) and α-glucosidase (EC 3.2.1.20) activities that occur during development of the pea seed have been measured. The highest tissue specific activity of β-glucosidase is found in the seedcoats, whereas the highest specific activities of the other enzymes occur in the cotyledons. Glycosidase isoenzymes were separated by ammonium sulphate fractionation followed by anion exchange chromatography and gel filtration. The α-mannosidase and β-galactosidase activities of the cotyledons are attributed mainly to single forms of each specific enzyme of MW 70,000 and 46,000 daltons respectively. By comparison, a wider spectrum of isoenzymes was observed in the hull and seedcoats. For seedcoats, two forms of β-glucosidase, three forms of β-galactosidase and four forms of α-mannosidase were distinguished. The main form of β-glucosidase from the seedcoats, isoenzyme 2, had a MW estimated to be 65,000 daltons.

The role and expression of β-glucosidase during spore germination of mutant and wild-type Dictyostelium discoideum

Dormant spores of Dictyostelium discoideum are contained in a viscous matrix of extracellular material at the top of sorocarps. Analysis of crude extracts of the sorus demonstrates that there is a higher specific activity of β-glucosidase in the matrix than in the dormant spores. Polyacrylamide gel electrophoresis of the matrix material reveals the presence of β-glucosidase 2 with trace amounts of β-glucosidase 1. In the dormant spores only β-glucosidase 2 is detected. Upon heat activation of the spores, β-glucosidase 2 activity decreases to approximately 25% of the activity found in the preheated spores. The enzyme appears to have a surface location in dormant spores, since trypsin treatment will destroy all of the β-glucosidase 2 activity. The normal germination of dormant spores after trypsin treatment suggests that β-glucosidase 2 activity is not required for activation, swelling, or emergence processes.During the emergence of myxamoebae, β-glucosidase 2 is replaced by a newly synthesized enzyme, β-glucosidase 1. The formation of this new enzyme requires both RNA and protein syntheses. The complete germination of a β-glucosidase 1 minus mutant, however, casts doubt on the necessity for β-glucosidase1 activity during emergence. This is further supported by the normal germination of strain SG1 which contains four- to five-fold higher β-glucosidase activity than that found in wild-type amoebae at the end of the germination period. In addition, strain SG1 may be activated with a number of techniques which results in a separation of the time of β-glucosidase synthesis from the time of myxamoebae emergence. Furthermore, spores (which have been blocked from emerging by cycloheximide treatment) will slowly release spheroplasts when treated with only pronase and D. discoideum cellulase. However, addition of D. discoideum β-glucosidase 1 to the cellulose–pronase-treated spores does accelerate the rate of spheroplast formation. Thus, β-glucosidase 1 activity may only play a minor and dispensable role in D. discoideum spore germination.

Heterozygote detection of Type I Gaucher disease using blood platelets

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of β-glucosidase and 4-methylumbelliferyl-β- d-glucoside as substrate. Platelet lysates have at least two identifiable β-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the β-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of β-hexosaminidase to βglucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of β-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of β-glucosidase at pH 5.6 in the presence of sodium taurocholate with the. ratio of β-hexosaminidase to β-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

The cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (Cx)-and β-glucosidase (βG)-activities. They were characterised further by measuring their Cx and βG profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific β-glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of β-glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher β-glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.

Effect of culture conditions on enzyme activities in cultivated human fibroblasts

Human diploid cell cultures were used to study the effects of three variables of tissue culture, type of media, frequency of media change and the degree of confluency, upon the specific activity of three lysosomal enzymes, β-glucosidase, aryl sulfatase, and acid phosphatase. Cells were grown in either Ham's Nutrient F-10 or Minimum Essential Medium (Earle salts) supplemented with 15% fetal calf serum. The medium was either changed daily or left unchanged for the 5 days of the study, and the cells were harvested at 24 h intervals and assayed for each of the enzymes mentioned. The specific activity of β-glucosidase and aryl sulfatase was significantly higher when cells were grown in MEM, when either medium was unchanged and at confluency. These variables had no significant affect on acid phosphatase activity.

Lysosomes and disuse atrophy of skeletal muscle

The specific activities of the acid hydrolases β-glucosidase, β-galactosidase, β-N-acetylhexosaminidase, arylsulfatase, and acid phosphatase have been measured during the time course of disuse atrophy of rat gastrocnemius muscles. These enzymes are considered to be lysosomal in origin. The specific activity of β-glucosidase increased to 180% of the control level by the second day following the production of disuse, and to 400% of the control level by the 12th day. The specific activities of β-galactosidase, β-N-acetylhexosaminidase, arylsulfatase, and acid phosphatase were increased markedly by the 12th day. All enzyme activities returned to control levels by the 15th day. The early increase in the activity of β-glucosidase indicates that lysosomal enzymes may an important role in the etiology of disuse atrophy.

Comparison of the catalytic and immunological properties of beta-glucosidases from three strains of Saccharomyces lactis.

beta-Glucosidase from Saccharomyces lactis strains Y-123 (B(h)), Y-14 (B(m)), and Y-1057A (B(1)) was partially purified. The pH optima, Michaelis constants, and activation energies were determined for the hydrolysis of p-nitro-phenyl-beta-d-glucoside by each of the enzymes. Differences among these constants were not enough to account for the low specific activity of beta-glucosidase in strains Y-14 and Y-1057A. Enzyme-inhibitor constants were measured for a series of alkyl and aryl glucosides. In general, the three enzymes are arylglucosidases. Tris(hydroxymethyl)-aminomethane inhibited all three enzymes in an uncompetitive fashion. The inhibition was antagonized by Mg(++). An antiserum was prepared to the highly purified (200-fold) beta-glucosidase from strain Y-123. The nature and degree of cross-reaction between the three beta-glucosidases was investigated by double diffusion in agar and neutralization tests. Spur formation in the immunodiffusion tests and similar equivalence points in the neutralization tests indicated a strong degree of cross-reaction between the three enzymes. The ratio of enzyme activity to antigenicity was used to compare the relative molecular activity of beta-glucosidase in the three strains. Each strain produced the same amount of beta-glucosidase per milligram of cell protein. The results are consistent either with a lower turnover number for the beta-glucosidase in strains Y-14 and Y-1057A or with the production of beta-glucosidase with a "normal" turnover number and enough cross-reacting material to effectively reduce the specific activity to the observed levels.