Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

BACKGROUND: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. RESULTS: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3ß inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. CONCLUSIONS: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

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.

Electron microscopic features of the lacrimal sac mucopeptide concretions.

PURPOSE: The aim of this study was to examine the ultrastructural features of the mucopeptide concretions obtained from the lacrimal sac. METHODS: Mucopeptide concretions obtained from the lacrimal sacs of 10 patients during a dacryocystorhinostomy were immediately fixed for electron microscopic analysis. The surfaces were studied separately and longitudinal and transverse ultra-thin sections were obtained at different levels and all were studied using the standard protocols of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: Mucopeptide concretions based on their extent take the shape of the lacrimal sac and nasolacrimal duct. The external surfaces and cut sections show mostly areas of homogenous deposits with occasional intervening heterogenic areas. Two distinct types of craters were noted, mostly in the heterogeneous areas. The core of the concretions was made up of extensive networks of fibril like tangles filled predominantly with granular material and red blood cells with occasional presence of granulocytes and epithelial cells. Numerous vacuoles and fissures appear to be more of artifacts than any metabolic process. No organic fibers of fungal filaments were noted within the concretions. There was no evidence of any bacterial biofilms other than few focal areas of scattered bacteria. Possible events in the development of mucopeptide concretions have been hypothesized based on the ultrastructural findings. CONCLUSION: Ultrastructural features of mucopeptide concretions from the lacrimal sac help in better understanding of their etiopathogenesis and tissue interactions. Further exploration of different stages of a concretion is needed to understand the potential factors that trigger its genesis and evolution.

Expression of Surfactant Proteins in the Human Canaliculus: Evidence and Potential Insights Into the Tear Flow Dynamics.

PURPOSE: To investigate the presence and distribution patterns of 6 surfactant proteins (SPs) in the human lacrimal canaliculus. METHODS: The study was performed on fresh frozen cadaveric samples of canaliculi. Immunohistochemical labeling was performed for assessing the presence and distribution of SP: SP-A, SP-B, SP-C, SP-D, SP-G/SFTA2, and SP-H/SFTA3. Immunofluorescence double staining was performed using the respective fluorescein-conjugated antibodies and the results were scored as positive or negative and the distribution pattern within the canalicular system was assessed. Western blot analysis was performed on the protein content which was resolved by reducing 15% sodium dodecyl sulfate-polyacrylamide electrophoresis and bands were studied following staining with primary and secondary antibodies. Human lung tissues were used as controls. RESULTS: Fluorescence double staining with 4,6-diamidino 2-pheynlindole and SPs showed strong immunostaining for SP-A, SP-B, SP-C, SP-D, and SP-H/SFTA3. The positive immunofluorescence was noticed across all the layers of the epithelium but not the subepithelial structures. The expression was noted on the surfaces and superficial cytoplasm of the superficial and deep epithelial cells. There was no expression of SP-G/SFTA2 across the canalicular system. Western blot analysis of the proteins confirmed and concurred with the immunofluorescence findings. CONCLUSIONS: This study provides a proof of principle for the presence of SPs known from lungs in the canalicular system and hypothesizes their possible functions and also their potential role in the tear flow dynamics between the ocular surface and the lacrimal drainage system.

Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels.

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (ß-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff's staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0-7.0. The enzyme was inhibited by EDTA, Hg(2+), Ag(2+), and Cu(2+). The activity lost by EDTA was completely regained by addition of Zn(2+). The purified enzyme was characterized in terms of the kinetic parameters K m (1.6 mM) and V max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 µM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn(2+) as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Isolation of Momordica charantia seed lectin and glycosidases from the protein bodies: Lectin-glycosidase (ß-hexosaminidase) protein body membrane interaction reveals possible physiological function of the lectin.

Momordica charantia seeds are known to contain a galactose specific lectin that has been well characterized. Seed extracts also contain glycosidases such as the ß-hexosaminidase, α-mannosidase and α-galactosidase. In the present study, lectin was affinity purified from the seed extracts and protein bodies isolated by sucrose density gradient centrifugation. From the protein bodies, lectin was identified and ß-hexosaminidase was isolated by lectin affinity chromatography and subsequently separated from other glycosidases by gel filtration. In the native PAGE, the purified ß-hexosaminidase migrated as a single band with a molecular weight of ∼235 kDa and by zymogram analysis using 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide substrate it was confirmed as ß-hexosaminidase. Under reducing conditions in SDS-PAGE, the purified enzyme dissociated into three bands (Mr 33, 20 and 15 kDa). The prominent bands (20 and 15 kDa) showed immunological cross-reactivity with the human Hexosaminidase B antibody in a western blot experiment. In gel digestion of the purified enzyme, followed by proteomic analysis using tandom MS/MS revealed sequence identity as compared to the genomic sequence of the Momordica charantia with a score of 57 (24% sequence coverage). Additionally, by CD analysis the purified ß-hexosaminidase showed 39.1% of α-helix. Furthermore, secondary structure variations were observed in presence of substrate, lectin and at different pH values. Protein body membrane prepared from the isolated protein bodies showed a pH dependent interaction with the purified lectin and mixture of glycosidases.

Momordica charantia seed proteins - Purification, biochemical characterization of a class II α-mannosidase isoenzyme and its interaction with the lectin and protein body membrane.

Momordica charantia seeds contain a galactose specific lectin and mixture of glycosidases. These bind to lectin-affigel at pH 5.0 and are all eluted at pH 8.0. From the mixture, α-mannosidase was separated by gel filtration (purified enzyme Mr âˆ¼ 238 kDa). In native PAGE (silver staining) it showed three bands that stained with methylumbelliferyl substrate (possible isoforms). Ion exchange chromatography separated two isoforms in 0.5 M eluates and one isoform in 1.0 M eluate. In SDS-PAGE it dissociated to Mr ∼70 and 45 kDa subunits, showing antigenic similarity to jack bean enzyme. MALDI analysis confirmed the 70 kDa band to be α-mannosidase with sequence identity to the genomic sequence of Momordica charantia enzyme (score 83, 29 % sequence coverage). The pH, temperature optima were 5.0 and 60o C respectively. Kinetic parameters KM and Vmax estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC50 value was 50 nM). Secondary structural analysis at far UV (190-300 nm) showed 11.6 % α-helix and 36.5 % ß-sheets. 2.197 mg of the enzyme was found to interact with 3.75 mg of protein body membrane at pH 5.0 and not at pH 8.0 suggesting a pH dependent interaction.

Characterization and modeling of the in-plane collagen fiber distribution in the porcine dermis.

The structural arrangement of collagen fibers in the plane of the dermis layer plays a critical role in accurately predicting the mechanical behavior of skin tissues. This paper combines a histological analysis with statistical modeling to characterize and model the in-plane collagen fiber distribution in the porcine dermis. The histology data reveals that the fiber distribution in the plane of the porcine dermis is non-symmetric. The histology data forms the basis of our model, which employs a combination of two π-periodic von-Mises distribution density functions to create a non-symmetric distribution. We demonstrate that a non-symmetric in-plane fiber distribution is a significant improvement over a symmetric distribution.

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.

Role of cation independent mannose 6-phosphate receptor protein in sorting and intracellular trafficking of lysosomal enzymes in chicken embryonic fibroblast (CEF) cells.

Delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptors (MPRs) in mammals. However, in non-mammalian cells the role of MPR300 in sorting and trafficking of acid hydrolases to lysosomes is not fully understood till now. In this paper, we tested the role of MPR300 in sorting and trafficking of lysosomal enzymes in CEF cells using a small interfering RNA (siRNA) technology. Inactivation of MPR300 resulted in the secretion of large amounts of newly synthesized hydrolases into the medium and also inhibited the endocytosis of mannose 6-phospharylated ligands. Knockdown of MPR300 in CEF cells results in missorting of fucosidase and arylsulfatse A enzymes into the medium. The results indicated that the MPR300 in CEF cells plays a key role in sorting and trafficking of these soluble hydrolases.

Purification, biochemical and biophysical characterization of lysosomal ß-D-glucuronidase from an edible freshwater mussel, Lamellidens corrianus.

A lysosomal glycosidase, ß-glucuronidase, has been purified to homogeneity, from the soluble extracts of a freshwater mussel, L. corrianus, by a series of chromatography techniques involving phenyl-Sepharose, ion exchange, affinity and gel filtration chromatography. In native PAGE, ß-glucuronidase resolved into a single band and the molecular mass determined by gel filtration chromatography was found to be 250 kDa. Zymogram analysis with 4-methyl umbelliferyl ß-glucuronide substrate validated the purified enzyme as ß-glucuronidase. In SDS-PAGE, the purified enzyme was resolved into four sub-units with molecular weights around 90, 75, 65, and 50 kDa, respectively, and two of the subunits (90 and 50 kDa) cross-reacted with human ß-glucuronidase antiserum. The optimum pH and temperature of the purified glycosidase were 5.0 and 70 °C, respectively. The enzyme kinetics parameters, substrate affinity (KM) and maximum velocity (Vmax) of the purified protein estimated with p-nitrophenyl ß-D-glucuronide were 0.457 mM and 0.11867 µmol-1 min-1 mL-1, respectively. The secondary structure of ß-glucuronidase was determined in the far-UV range (190 nm to 230 nm) using CD spectroscopy. Heat denaturation plots determined by CD spectroscopy showed that the purified enzyme was stable up to 70 °C.

Comparative analysis of ß-hexosaminidase and acid phosphatase from Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1: Localization studies of acid phosphatase and ß-hexosaminidase from H. vulgaris Ind-Pune.

The present report describes a comprehensive study on comparative biochemical characterization of two lysosomal enzymes, acid phosphatase and ß-hexosaminidase in three different strains of Hydra; Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1 (self-feeder-1). Since morphology and habitat of Hydra effect lysosomal enzymes and their response to environmental pollutants, it would be interesting to identify them in different Hydra strains so as to use them as toxicity testing. Preliminary studies revealed a differential expression of acid phosphatase, ß-hexosaminidase and ß-glucuronidase in three Hydra strains. Expression of all three lysosomal enzymes in H. vulgaris Ind-Pune was low in comparison to H. vulgaris Naukuchiatal and H. magnipapillata sf-1, while their expression is comparable in H. vulgaris Naukuchiatal and H. magnipapillata sf-1. The Michaelis-Menten (KM) values for lysosomal ß-hexosaminidase using 4-nitrophenyl N-acetyl-ß-D-glucosaminide as substrate were found to be 1.3 mM, 1.1 mM and 0.8 mM, respectively for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. For acid phosphatase using 4-nitrophenyl-phosphate as substrate, the KM values were 0.38 mM, 1.2 mM and 0.52 mM respectively, for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and sf-1 strains. The optimum temperature for ß-hexosaminidase was 60 °C for H. vulgaris Ind-Pune, while 50 °C was observed for H. vulgaris Naukuchiatal and sf-1 strains. The optimum pH for ß-hexosaminidase was found to be 6.0 for H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal, and 5.0 for sf-1. The optimum temperature and pH of acid phosphatase was similar in all three strains, viz., 40 °C and 3.0, respectively. Preliminary localization studies using whole mount in situ hybridization revealed predominant endodermal expression of three enzymes in H. vulgaris Ind-Pune. Our results thus support the conservation of lysosomal hydrolases in Hydra.

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.

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of Dolichos lablab.

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits alpha and beta. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (alpha) and 28.815 kDa (beta) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(alpha) and 263(beta) amino acids respectively. The beta subunit differed from the alpha subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the beta subunit is derived from the alpha subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation.

C(1)-/C(2)-aromatic-imino-glyco-conjugates: experimental and computational studies of binding, inhibition and docking aspects towards glycosidases isolated from soybean and jack bean.

Several C(1)-imino conjugates of D: -galactose, D: -lactose and D: -ribose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Similar C(2)-imino conjugates of D: -glucose have also been synthesized. All the glyco-imino-conjugates, which are transition state analogues, exhibited 100% inhibition of the activity towards glycosidases extracted from soybean and jack bean meal. Among these, a galactosyl-napthyl-imine-conjugate (1c) showed 50% inhibition of the activity of pure alpha-mannosidase from jack bean at 22 +/- 2.5 microM, and a ribosyl-naphthyl-imine-conjugate (3c) showed at 31 +/- 5.5 microM and hence these conjugates are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition by these conjugates. The studies are also suggestive of the involvement of aromatic, imine and carbohydrate moieties of the glyco-imino-conjugates in the effective inhibition. The binding of glyco-imino-conjugate has been established by extensive studies carried out using fluorescence emission and isothermal titration calorimetry. The conformational changes resulted in the enzyme upon interaction of these derivatives has been established by studying the fluorescence quench of the enzyme by KI as well as from the secondary structural changes noticed in CD spectra. All these studies revealed the difference in the binding strengths of the naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential inhibition of these glyco-conjugates has been addressed by quantifying the specific interactions present between the glyco-conjugates and the enzyme by using rigid docking studies.

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.

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate Asterias rubens (Echinodermate): biochemical and functional characterization of MPR 46 protein.

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr((-/-)) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab.

The affinity purified galactose-specific seed lectin from Dolichos lablab, designated as DLL-II, is a tetrameric protein with an apparent native molecular mass of 120 kDa that is composed of two non-identical subunits of 31 and 29 kDa, respectively, associated non-covalently. The stems and leaves of the D. lablab plant also contain a galactose-specific lectin that cross-reacts with the seed lectin antiserum (antiserum raised against the 31 kDa subunit of DLL-II). Anti-lectin antibodies have been purified from this antiserum using a gel containing purified DLL-II lectin. Lectin specific antibodies have been used to develop simple and efficient immuno-affinity matrix, which allowed the purification of the lectin from stems and leaves of the D. lablab. The vegetative lectin (DLL-VL) exhibits similar electrophoretic properties as the seed lectin. Using these antibodies, an ELISA method was developed that allowed quantification of the lectin in the vegetative tissues (stems, leaves and roots) at concentrations of 0.5-50 ng. MS and database analysis of the tryptic peptides of the purified subunits of the DLL-VL suggested the purified protein to be a lectin.