Physicochemical characterization of β-crystallins from bovine lenses: Hydrodynamic and aggregation properties

A detailed investigation of the hydrodynamic and aggregation behaviors has been made on the -crystallins of bovine lens. Results from this study indicated that H (high-molecular-weight -crystallin) and L (low-molecular-weight -crystallin) exhibited considerable heterogeneity in their native structures and subunit polypeptides. Low-speed sedimentation equilibrium showed a heterogeneous paucidisperse system in each -crystallin fraction. Viscosity and circular dichroism studies pointed to a compact and globular shape and the presence of -sheet and -turns in these crystallins. Dissociation of H by urea and guanidinium HCl followed by reassociation during gel-filtration chromatography produced an elution pattern with two fractions corresponding to L crystallin and high-molecular-weight aggregates without the formation of native H. By contrast, under similar treatment, about 60% L reassociated into the correct native structure and the rest into high-molecular-weight fractions. Amino acid analyses of H and L and their corresponding subunit polypeptides demonstrated the close similarity of these crystallins. Trace element analyses indicated that both Ca and Mg are present in H and L crystallins and may be involved in maintaining the native quarternary structures of these proteins.     

Thermal stabilization of fungal β-glucosidase through glutaraldehyde crosslinking

Summary The thermal stability of a purifiedAspergillus niger -glucosidase (EC 3.2.1.21) has been dramatically increased by intramolecular crosslinking with glutaraldehyde at pH 9.0, followed by borohydride reduction. The stabilization has been shown to be due to the formation of a distinct subpopulation of enzyme molecules, the activity of which has a half-life at 65°C of approximately 40 hours, which is some 80 times as long as that of the native enzyme at this temperature.     

Visualization of Trichoderma reesei Cellobiohydrolase I and Endoglucanase I on Aspen Cellulose by Using Monoclonal Antibody-Colloidal Gold Conjugates

Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10- and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with cellulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.     

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.     

Decreased brain dopamine synthesis rate and increased [3H]spiroperidol binding in streptozotocin-diabetic rats

The rate of accumulation of 3,4-dihydroxy-phenylalanine following decarboxylase inhibition and of homovanillic acid following probenecid treatment were significantly decreased in streptozotocin-diabetic rats. These changes were observed in both the striatum and limbic forebrain. The Bmax for [3H]spiroperidol receptor binding was significantly increased in both brain regions. All of these neurochemical changes were reversed by insulin replacement therapy. Whether these neurochemical changes are attributable to chronic hyperglycemia or some other aspect of the diabetic state is not known.     

Evidence for alteration of the membrane-bound ribosomes in Micrococcus luteus cells exposed to lead

Micrococcus luteus cells exposed to Pb(NO₃)₂ contained cytosol ribosomal particles and disaggregated membranal ribosomal particles as determined by ultracentrifugation and spectral studies. Approx. 60% of the membrane ribosome fraction from lead exposed cells had a sedimentation value of 8.4S. Cytosol ribosomes from lead exposed cells as well as membranal and cytosol ribosomes from control cells were comparable by their contents of predominantly the 70S type with the 50S and 100S present in relatively small amounts. The lead content of the 8.4S component was more than 200 times higher than the components with higher sedimentation coefficients from lead exposed cells and approx. 650 times more than that of control cell ribosomes. The cells exposed to lead, however, showed no adverse effects from the lead in respect to their growth rates and cellular yields. These results indicate that lead is interacting only at specific sites of the membrane and is inducing events initiated only in strategic cellular regions. These data further substantiate that subtle changes do occur in lead exposed cells that show no obvious effects. It is assumed that these ‘minor’ alterations are, in toto, biologically significant.     

Low molecular weight thermostable β-D-glucosidase from Acidothermus cellulolyticus

Summary Industrial use of cellulase enzymes, including -D-glucosidases, can be greatly improved and expanded by the discovery and characterization of more robust, thermotolerant enzymes. A new thermostable -D-glucosidase was isolated from Acidothermus cellulolyticus culture broth. This enzyme has a molecular weight of 52.5 kDa, an isoelectric pI of 4.1, and a pH activity optimum of 4.5. The optimum temperature for activity on p-nitrophenyl--D-glucoside is 63°C. This value is approximately 20°C higher than temperature optima displayed by most previously characterized -D-glucosidases.     

In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

Background

Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.

Results

Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.

Conclusion

We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.