Purification, biochemical and biophysical characterization of an acidic α-galactosidase from the seeds of Annona squamosa (custard apple).

Alpha galactosidase is an exoglycosidase that cleaves α-D-galactose and has numerous applications in medicine, biotechnology, food and pharma industries. In this study, a low molecular weight acidic α-galactosidase was identified from the seeds of custard apple. The purification of α-galactosidase from the crude extract of defatted seeds was achieved by employing ammonium sulphate fractionation, hydrophobic interaction and gel filtration chromatographic techniques. The purified custard apple α-galactosidase (CaG) migrated as a single band in native PAGE corresponding to molecular weight of ~67 kDa and cleaved chromogenic, fluorogenic and natural substrates. CaG was found to be a heterodimer with subunit masses of 40 and 30 kDa. The kinetic parameters such as KM and Vmax were found to be 0.67 mM and 1.5 U/mg respectively with p-nitrophenyl α-D-galactopyranoside. Galactose, methyl α-D-galactopyranoside and D-galacturonic acid inhibited CaG activity in mixed mode. The CD spectral analysis at far UV region showed that purified CaG exists predominantly as helix (35%), beta sheets (16.3%) and random coils (32.3%) in its secondary structure. These biochemical and biophysical properties of CaG provide leads to understand its primary sequence and glycan structures which will eventually define its novel physiological roles in plants and potential industrial applications.

Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

Purification, biochemical and biophysical characterization of a zinc dependent α-mannosidase isoform III from Custard Apple (Annona squamosa) seeds.

In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p-nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0-5.0 respectively and CAM was stable up to 60-70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC50 value of 1.5 µM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters KM and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190-300 nm) shows that purified CAM exists as helix (30.4%), ß turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N-Bromosuccinimide revealed the presence of tryptophan in its active site.

Soluble glycoproteins of the lacrimal sac: role in defense with special reference to prolactin-inducible protein (PIP).

Purpose: Glycoproteins play an important role in human mucosal defenses and immunity-related cell-to-cell interactions. The aim of the present study is to investigate the presence and patterns of lacrimal sac glycoproteins involved in defense mechanisms with a special reference to prolactin-inducible protein (PIP). Methods: The study was performed on healthy lacrimal sacs obtained from exenteration samples immediately after surgery and frozen at -80 degrees for subsequent analysis. Four lectins namely Concanavalin A (Con A), Dolichos lablab lectin (DLL), Wheat Germ agglutinin (WGA), and Momordica charantia lectin (MCL) were purified by affinity chromatography. Soluble proteins extract of the lacrimal sac was subjected to chromatography on lectin-affigel columns. Eluted samples from each of the lectin coupled-affigels were analyzed by 10% SDS-PAGE under reducing conditions and the protein bands were visualized using Coomassie blue stain. The protein gel bands were further subjected to mass spectrometry for glycoprotein analysis. Results: Mass spectrometry identified several glycoproteins from the lacrimal sac extracts, with known roles in defense mechanisms. The number of such glycoproteins identified were 9 each from Con A and DLL-I affinity eluted gel bands and 8 and 14 from MCL and WGA affinity eluted gel bands, respectively. Interestingly, PIP was detected in significant proportions in all the eluted gel bands with WGA showing the highest expression. Conclusions: This study is the first step towards the lacrimal sac glycoprotein profiling. PIP could be a major lead for further work on the etiopathogenesis of lacrimal drainage obstructions.

Lysosomal enzymes and mannose 6-phosphate receptors in the lacrimal drainage system: Evidence and its potential implications.

PURPOSE: To investigate the presence and patterns of lysosomal enzymes and mannose 6-phosophate receptor (MPRs) in human lacrimal drainage system. METHODS: The study was performed on healthy lacrimal sacs and nasolacrimal ducts obtained from exenteration samples immediately after surgery and frozen at -80°C for subsequent analysis. Soluble proteins' extract was used for enzyme assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), native PAGE, activity staining, and western blot analysis. Membrane proteins were separately assessed for detection of mannose 6-phosphate receptors, MPR 46. Sepharose gels, 4-methylumbelliferyl substrates, and antibodies against common lysosomal enzymes and MPRs were used. Enzyme assays were carried out in triplicate to ascertain the results. RESULTS: Differential lysosomal enzyme activities were documented, and among them acid phosphatase and ß-hexosaminidase were found to be high. Western blot analysis using enzyme antibodies and subsequent activity staining confirmed strong signals for moderately expressed enzymes such as fucosidase, glucuronidase, and mannosidase. Membrane extracts demonstrated the presence of MPR 46, which indicates the possible roles of cation-dependent MPRs in lysosomal targeting in human lacrimal drainage system. CONCLUSION: This study provides a proof of principle for the presence of differential lysosomal activity and mannose 6-phosphate ligand transport receptors in human lacrimal drainage system and hypothesizes the potential implications of their dysfunctions.

Biochemical characterization of cathepsin D from the mussel Lamellidens corrianus.

A lysosomal cathepsin D (EC 3.4.23.5) was purified to homogeneity from the soft tissues of the fresh water mussel (Lamellidens corrianus) by pepstatin A affinity chromatography. The purified enzyme is a glycoprotein and migrates as a single protein species in native PAGE and shows a single band in SDS-PAGE corresponding to a molecular mass of ~43 kDa. Under both these conditions cathepsin D hydrolyzes hemoglobin as shown by zymogram analysis. The purified enzyme cross-reacts with an antiserum to purified starfish (Asterias rubens) cathepsin D. Additionally, the enzyme was recognized by the starfish lysosomal enzyme targeting receptors (mannose 6-phosphate receptors: MPR 300 and 46) in ligand blot analysis. The KM value of the purified enzyme with hemoglobin is 1.5mM. pH and temperature optimum for the enzyme are 3.5 and 60 °C respectively.

Isolation, purification, and biochemical characterization of two forms of lysosomal ß-N-acetylhexosaminidase from the invertebrate Unio.

Lysosomal hexosaminidases are glycosyl hydrolases that remove the terminal hexosamine residues of glycoconjugates. Though mammalian hexosaminidases are well characterized, the biochemical nature of these enzymes among invertebrates remains elusive. In this study, we purified two thermostable N-acetyl ß-D-hexosaminidases (hex A and B) to homogeneity from soluble extracts of whole Unio animal tissue by a combination of chromatographic procedures. Purified hex A and hex B migrated as a single protein species on native PAGE and exhibited enzyme activity. However on SDS-PAGE, hex A dissociated into two subunits of molecular masses about 75 kDa and 30 kDa respectively, while hex B showed a molecular mass of 40 kDa. Hex A and B were recognized by the affinity purified mannose 6-phosphate receptor 46 on ligand blot analysis. This specific interaction was similar to what is known for the vertebrate receptors and lysosomal enzymes. The enzymes showed different K(M) values with respect to the substrates p-nitrophenyl N-acetyl-ß-D-glucosaminide and p-nitrophenyl N-acetyl-ß-D-galactosaminide. The enzymes were thermally stable up to 80 °C and showed pH optima between 5.0 and 6.0. This is the first report on the purification of two forms of hexosaminidases from Unio.