The fallacy of enzymatic hydrolysis for the determination of bioactive curcumin in plasma samples as an indication of bioavailability: a comparative study.

BACKGROUND: Numerous health benefits have been demonstrated for curcumin which is extracted from turmeric (Curcuma longa L). However, due to its poor absorption in the free form in the gastrointestinal tract and rapid biotransformation, various formulations have been developed to enhance its bioavailability. Previous studies indicate that the free form of curcumin is more bioactive than its conjugated counterparts in target tissues. Most curcumin pharmacokinetics studies in humans designed to assess its absorption and bioavailability have measured and reported total (free plus conjugated) curcumin, but not free, bioactive curcumin in the plasma because enzymatic hydrolysis was employed prior to its extraction and analysis. Therefore, the bioavailability of free curcumin cannot be determined. METHODS: Eight human subjects (4 male, 4 female) consumed a single dose of 400 mg curcumin in an enhanced absorption formulation, and blood samples were collected over 6 h. Plasma was treated either with or without glucuronidase/sulfatase prior to extraction. Curcumin and its major metabolites were analyzed using HPLC-tandem mass spectrometry. In addition, the literature was searched for pharmacokinetic studies involving curcumin using PubMed and Google Scholar, and the reported bioavailability data were compared based on whether hydrolysis of plasma samples was used prior to sample analysis. RESULTS: Hydrolysis of blood plasma samples prior to extraction and reporting the results as "curcumin" obscures the amount of free, bioactive curcumin and total curcuminoids as compared to non-hydrolyzed samples. As a consequence, the data and biological effects reported by most pharmacokinetic studies are not a clear indication of enhanced plasma levels of free bioactive curcumin due to product formulations, leading to a misrepresentation of the results of the studies and the products when enzymatic hydrolysis is employed. CONCLUSIONS: When enzymatic hydrolysis is employed as is the case with most studies involving curcumin products, the amount of free bioactive curcumin is unknown and cannot be determined. Therefore, extreme caution is warranted in interpreting published analytical results from biological samples involving ingestion of curcumin-containing products. TRIAL REGISTRATION: ClinicalTrails.gov, trial identifying number NCT04103788 , September 24, 2019. Retrospectively registered.

Characteristic anti-inflammatory and antioxidative effects of enzymatic- and acidic- hydrolysed mycelium polysaccharides by Oudemansiella radicata on LPS-induced lung injury.

The present work investigated the antioxidative, anti-inflammatory and pulmonary protective effects of enzymatic- and acid- hydrolysed mycelia polysaccharides (En-MPS and Ac-MPS) from Oudemansiella radicata on LPS-induced acute lung injury (ALI) mice. The results demonstrated that both En-MPS and Ac-MPS showed potential pulmonary protective effects by decreasing serum levels of hs-CRP and C3, increasing pulmonary enzyme values of SOD, GSH-Px, CAT and the level of T-AOC; reducing the activity of MPO; and down-regulating the contents of MDA and LPO. In addition, the levels of TNF-ɑ, IL-1ß, and IL-6 in BALF of mice treated with En-MPS at a dosage of 400 mg/kg/d were significantly lower than those in the ALI mice. The in vitro antioxidant effects also showed that the En-MPS was more effective than Ac-MPS. Furthermore, the physical properties of polysaccharides were also investigated by GC, HPGPC, FT-IR and NMR. These results indicated that both En-MPS and Ac-MPS possessed potent antioxidant and anti-inflammatory activities, which could be used as an ingestible drug in preventing lung injury.

Bioinformatics: A rational combine approach used for the identification and in-vitro activity evaluation of potent ß-Glucuronidase inhibitors.

Identification of hotspot drug-receptor interactions through in-silico prediction methods (Pharmacophore mapping, virtual screening, 3DQSAR, etc), is considered as a key approach in drug designing and development process. In the current design study, advanced in-silico based computational techniques were used for the identification of lead-like molecules against the targeted receptor ß-glucuronidase. The binding pattern of a potent inhibitor in the ligand-receptor X-ray co-crystallize complex was used to identify and extract the structure-base Pharmacophore features. Based on these observations; five structure-based pharmacophore models were derived to conduct the virtual screening of ICCBS in-house data-base. Top-ranked identified Hits (33 compounds) were selected to subject for in-vitro biological activity evaluation against ß-glucuronidase enzyme; out of them, twenty compounds (61% of screened compounds) evaluated as actives, however eleven compounds were found to have significantly higher inhibitory activity, including compounds 1, 5-8, 10, 12-13, and 17-19 with IC50 values ranging from 1.2 µM to 34.9 µM. Out of the eleven potent inhibitors, seven compounds 1, 5, 6, 7, 8, 13, and 19 were found new, and evaluated first time for the ß-glucuronidase inhibitory activity. Compounds 1, 5 and 19 exhibited a highly potent inhibition in uM of ß-glucuronidase enzyme with non-cytotoxic behavior against the mouse fibroblast (3T3) cell line. Our combined in-silico and in-vitro results revealed that the binding pattern analysis of the eleven potent inhibitors, showed almost similar non-covalent interactions, as observed in case of our validated pharmacophore model. The obtained results thus demonstrated that the virtual screening minimizes false positives, and provide a template for the identification and development of new and more potent ß-glucuronidase inhibitors with non-toxic effects.

Vitamin D protection from rat diabetic nephropathy is partly mediated through Klotho expression and renin-angiotensin inhibition.

OBJECTIVE: We hypothesised that vitamin D has a beneficial renal protective effect from diabetic nephropathy (DN). METHODS: Four rat groups were included: normal control (control), type 2 diabetes for eight weeks (DM), treated group with angiotensin receptor blocker losartan (DM + L), and vitamin D-treated group started from the onset of diabetes (DM + Vit D). RESULTS: In the both treated groups, we found a significant (p < .05) reduction in the renal pro-inflammatory and profibrotic markers induced by diabetes. Vitamin D caused more reduction in monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGFß-1), and renin-angiotensin levels that gave better kidney function compared to the DM + L group. CONCLUSION: Vitamin D may have a valuable role in the renal protective effect from DN, this may occur via expression of its VDR, Klotho and blocking renin-angiotensin activation, so vitamin D should be considered as a target in renal prophylactic measures against DN.

Identification of isoliquiritigenin as an activator that stimulates the enzymatic production of glycyrrhetinic acid monoglucuronide.

Glycyrrhetinic acid monoglucuronide (GAMG) is a great value-added and has considerable commercial interest due to its strong pharmacological activities and functional low-calorie sweetener. However GAMG is quite rare in natural plants, and it must be prepared from glycyrrhizin (GL) by hydrolysing one terminal glucuronic acid. ß-Glucuronidase is the key enzyme in the biotransformation of GL to GAMG, but its activities need to be enhanced to facilitate the industrial large-scale production of GAMG. In this study, we identified that isoliquiritigenin (ISL), as one of chemical compositions from the total flavonoids glycyrrhiza (TFG), can significantly enhance ß-glucuronidase activity in vitro. Measurements using high-performance liquid chromatography (HPLC) showed that the activity of ß-glucuronidase could be increased by 2.66-fold via the addition of ISL to a ß-glucuronidase solution that contained GL at a 3:10 molar ratio of ISL to GL. ISL was concluded to be an activator because ISL could reduce the Km and Ea of ß-glucuronidase reacting with GL. This study sheds new light on the mechanism of ß-glucuronidase and helps to make industrial production of GAMG through fermentation feasible.

Effects of Snailase Treatment on Wettability of Raw Cotton Yarns in Pre-wetting Process of Foam Sizing.

The main objective of the present study was to meticulously investigate an inclusive set of physicochemical properties of pre-wetted cotton yarns and optimize the snailase treatment for raw cotton yarns. Based on single factor design, effects of snailase treatment on the removal percentage of pectins and cotton waxes, wettability, and weight loss were studied in different snailase concentrations and the optimal concentration range was obtained. The Box-Behnken design was employed to determine the optimal condition of snailase treatment for achieving the maximum wettability of cotton yarns. The maximum wettability was obtained when treated at 16.90 g/L of snailase concentration at 56.85 °C for 27.77 s. The theoretical and observed values were in reasonably good agreement, and the deviation was less than 1%. Results revealed that snailase had a positive effect on pectin and wax removal, had an obvious impact on weight loss, and significantly enhanced the wettability of raw cotton yarns.

In vitro gastrointestinal-resistant pectin hydrogel particles for ß-glucuronidase adsorption.

Pectin hydrogel particles (PHPs) were prepared by ionotropic gelation of low methylesterified pectin of Tanacetum vulgare L. with calcium ions. Wet PHPs prepared from TVF exhibited a smaller diameter and the lower weight as well as exhibited the best textural properties in terms of hardness and elasticity compared to the PHPs prepared from commercial low methylesterified pectin (CU701) used for comparison. Upon air drying, PHPs prepared from CU701 became small and dense microspheres whereas the dry PHPs prepared from TVF exhibited a drop-like shape. The morphology of dry PHPs determined by scanning electron microscopy revealed that the surface of the TVF beads exhibited fibred structures, whereas the PHPs prepared from CU701 exhibited a smooth surface. The characterization of surface roughness using atomic force microscopy indicated less roughness profile of the PHPs prepared from TVF than CU701. PHPs prepared from TVF were found to possess in vitro resistance to successive incubations in simulated gastric (SGF), intestinal (SIF), and colonic fluid (SCF) at 37 °C for 2, 4 and 18 h, respectively. The PHPs prepared from CU701 swelled in SGF and then lost their spherical shape and were fully disintegrated after 4 h of incubation in SIF. The PHPs from TVF, which were subjected to treatment with SGF, SIF and SCF, were found to adsorb microbial ß-glucuronidase (ßG) in vitro. The data obtained offered the prospect for the development of the PHPs from TVF as sorbents of colonic ßG for the inhibition of re-absorption of estrogens.

Structure-Dependent Deconjugation of Flavonoid Glucuronides by Human ß-Glucuronidase - In Vitro and In Silico Analyses.

Flavonoid glycosides are extensively metabolized to glucuronidated compounds after oral intake. Recently, a cleavage of quercetin glucuronides by ß-glucuronidase has been found. To characterize the deglucuronidation reaction and its structural prerequisites among the flavonoid subtypes more precisely, four flavonol glucuronides with varying glucuronidation positions, five flavone 7-O-glucuronides with varying A- and B-ring substitution as well as one flavanone- and one isoflavone-7-O-glucuronide were analyzed in a human monocytic cell line. Investigation of the deglucuronidation rates by HPLC revealed a significant influence of the glucuronidation position on enzyme activity for flavonols. Across the flavonoid subtypes, the C-ring saturation also showed a significant influence on deglucuronidation, whereas A- and B-ring variations within the flavone-7-O-glucuronides did not affect the enzymes' activity. Results were compared to computational binding studies on human ß-glucuronidase. Additionally, molecular modeling and dynamic studies were performed to obtain detailed insight into the binding and cleavage mode of the substrate at the active site of the human ß-glucuronidase.

Discovery of specific inhibitors for intestinal E. coli ß-glucuronidase through in silico virtual screening.

Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli ß-glucuronidase (eßG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eßG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eßG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eßG but not human ßG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eßG activity. Compound 4041 (IC50 = 2.8 µM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 µM) against eßG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eßG via hydrogen-bonding interactions. These novel NYBS-based eßG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eßG to reduce eßG-based carcinogenesis and intestinal injury.

[Cloning and bioinformatic analysis and expression analysis of beta-glucuronidase in Scutellaria baicalensis].

The ß-Glucuronidase gene (sbGUS) cDNA firstly from Scutellari abaicalensis leaf was cloned by RT-PCR, with GenBank accession number KR364726. The full length cDNA of sbGUS was 1 584 bp with an open reading frame (ORF), encoding an unstable protein with 527 amino acids. The bioinformatic analysis showed that the sbGUS encoding protein had isoelectric point (pI) of 5.55 and a calculated molecular weight about 58.724 8 kDa, with a transmembrane regions and signal peptide, had conserved domains of glycoside hydrolase super family and unintegrated trans-glycosidase catalytic structure. In the secondary structure, the percentage of alpha helix, extended strand, ß-extended and random coil were 25.62%, 28.84%, 13.28% and 32.26%, respectively. The homologous analysis indicated the nucleotide sequence 98.93% similarity and the amino acid sequence 98.29% similarity with S. baicalensis (BAA97804.1), in the nine positions were different. The expression level of sGUS was the highest in root based on a real-time PCR analysis, followed by flower and stem, and the lowest was in stem. The results provide a foundation for exploring the molecular function of sbGUS involved in baicalcin biosynthesis based on synthetic biology approach in S. baicalensis plants.

Overestimation of flavonoid aglycones as a result of the ex vivo deconjugation of glucuronides by the tissue ß-glucuronidase.

Flavonoid glucuronides are the main circulating metabolites of flavonoids in humans and animals. There has been a growing interest in the biological function of glucuronides. In order to differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycone and metabolic conjugate in vivo. Many organs and body fluids of humans and animals exhibit ß-glucuronidase against flavonoid glucuronides. Studies have shown that ß-glucuronidase within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycone than actual in vivo concentrations. The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone, a ß-glucuronidase inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides. Our data demonstrate that in mouse liver tissues and human tumor xenografts levels of quercetin and methylated quercetin aglycones could be over-estimated by 7-fold. The inhibition of deconjugation of quercetin and baicalein glucuronides by saccharo-1,4-lactone is dose-dependent. The amount of saccharo-1,4-lactone used to produce optimal inhibition of the enzyme activity is in the range of 15-24µmol per gram of liver tissue. The use of ß-glucuronidase inhibitor blocks the ex vivo deconjugation resulting in an accurate estimation of tissue levels of aglycone and conjugate. Our study described here can be extended to other animal models and human studies with different types of substrates of ß-glucuronidase.

Prebiotics and bioactive natural substances induce changes of composition and metabolic activities of the colonic microflora in cancerous rats.

Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme ß-glucuronidase (P<0.001) and increased activities of ß-galactosidase and ß-glucosidase. Bile acids concentration was significantly decreased (P<0.01) except for the combination of the prebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (P<0.001) and enhanced production of short chain fatty acids. Application of prebiotic and bioactive natural substances significantly reduced number of coliforms (P<0.05). Prebiotic alone significantly increased the count of lactobacilli (P<0.05). These results show that prebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.

Alleviating cancer drug toxicity by inhibiting a bacterial enzyme.

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial ß-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial ß-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial ß-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.

Purification of the crude solution from Helix pomatia for use as beta-glucuronidase and aryl sulfatase in phytoestrogen assays.

Phytoestrogens occur in a variety of foods and are thought to offer a protective effect against a number of complex diseases. Due to the diversity of phytoestrogen conjugates formed in the human body, most assays include an enzymatic hydrolysis step prior to analysis. beta-Glucuronidase from Helix pomatia, which also contains sulfatase activity, is popular for this task but contains appreciable levels of some phytoestrogens and related compounds, which could affect accurate quantification at low concentrations. Use of solid phase extraction on a polymeric resin has been found to remove the majority of these compounds from the enzyme, without affecting the enzyme activity for almost all of the analytes tested.

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma.

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.

Heterodimer formation is essential for heparanase enzymatic activity.

Heparanase is an endo-beta-D-glucuronidase involved in cleavage of heparan sulfate residues and hence participates in extracellular matrix degradation and remodeling. The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a approximately 65 kDa band in SDS-PAGE analysis. The protein undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, Glu(109)-Ser(110) and Gln(157)-Lys(158), yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus, and a 6 kDa linker polypeptide that resides in-between. The active form of heparanase has long been thought to be a 50 kDa polypeptide isolated from cells and tissues. However, attempts to obtain heparanase activity after expression of the 50 kDa polypeptide failed, suggesting that the N-terminal region is important for heparanase enzymatic activity. It has been hypothesized that heterodimer formation between the 8 and 50 kDa heparanase subunits is important for heparanase enzymatic activity. By individually or co-expressing the 8 and 50 kDa heparanase subunits in mammalian cells, we demonstrate specific association between the heparanase subunits by means of co-immunoprecipitation and pull-down experiments. Moreover, a region in the 50 kDa heparanase subunit that mediates interaction with the 8 kDa subunit was identified. Altogether, our results clearly indicate that heterodimer formation is necessary and sufficient for heparanase enzymatic activity in mammalian cells.

Histidine tagged protein recovery from tobacco extract by foam fractionation.

Tobacco plants have the potential to be used for the production of proteins for pharmaceutical applications. This work describes a novel protein recovery strategy where the protein of interest is "tagged" with a histidine sequence, which forms a complex with cobalt ions and surfactant possessing a chelating functionality, such that the protein is recovered in the foamate of a foam fractionation step. His-gus, a histidine-tagged enzyme, was chosen as a model protein to study the feasibility of this strategy. The His-gus is recovered from spiked prefoamed tobacco extract by foam fractionation in the presence of surfactant and cobalt ions with an enrichment of 1.29 and a recovery of 21.5% in terms of an adjusted activity.

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange.

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.

Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence.

Cleavage of heparan sulfate (HS) proteoglycans affects the integrity and function of tissues and thereby fundamental phenomena, involving cell migration and response to changes in the extracellular microenvironment. The role of HS-degrading enzymes, commonly referred to as heparanases, in normal development has not been identified. The present study focuses on cloning, expression, and properties of a chicken heparanase and its distribution in the developing chicken embryo. We have identified a chicken EST, homologous to the recently cloned human heparanase, to clone and express a functional chicken heparanase, 60% homologous to the human enzyme. The full-length chicken heparanase cDNA encodes a 60-kDa proenzyme that is processed at the N terminus into a 45-kDa highly active enzyme. The most prominent difference between the chicken and human enzymes resides in the predicted signal peptide sequence, apparently accounting for the chicken heparanase being readily secreted and localized in close proximity to the cell surface. In contrast, the human enzyme is mostly intracellular, localized in perinuclear granules. Cells transfected with a chimeric construct composed of the chicken signal peptide preceding the human heparanase exhibited cell surface localization and secretion of heparanase, similar to cells transfected with the full-length chicken enzyme. We examined the distribution pattern of the heparanase enzyme in the developing chicken embryo. Both the chicken heparanase mRNA and protein were expressed, as early as 12 h post fertilization, in cells migrating from the epiblast and forming the hypoblast layer. Later on (72 h), the enzyme is preferentially expressed in cells of the developing vascular and nervous systems. Cloning and characterization of heparanase, the first and single functional vertebrate HS-degrading enzyme, may lead to identification of other glycosaminoglycan degrading enzymes, toward elucidation of their significance in normal and pathological processes.

Purification and properties of a plant beta-D-glucuronidase form Scutellaria root.

beta-D-Glucuronidase (baicalinase, GUS [EC 3.2.1.31]) activity in the crude drug, Scutellaria root, was assayed in line with the quality control standards of Kampo (Japanese Herbal) medicines. GUS was purified to homogeneity in the purification steps including DEAE-Sepharose Fast Flow and chromatofocusing used PBETM94 and Polybuffer 74. These results suggest that the Scutellaria GUS is composed of 55kDa active subunits and that the isoelectric point of this enzyme is pH 5.4. Optimal catalytic activity was found at pH 4.7 in the pH range 3.6--6.2 in 50 mM Na-citrate buffer. The purified enzyme hydrolyzed baicalin and wogonin glucuronide, but did not hydrolyze glycyrrhizin or some beta-glucosides found in other crude drugs. GUS activity in several crude drugs is also described.