Analysis of oligomannosyl cores of cellular glycopeptides by digestion with endo-beta-N-acetylglucosaminidases.

Most of mannose-labeled glycopeptides from SV-40 transformed fibroblasts were hydrolyzed either by endo-β-N-acetylglucosaminidase D in the presence of β-galactosidase, β-N-acetylglucosaminidase and neuraminidase or by endo-β-N-acetylglucosaminidase H. The products were oligosaccharides with the probable structure of Man_nGlcNAc (n=3,5,6,….). The D enzyme preferentially released smaller oligosaccharides, while the H enzyme released larger oligosaccharides. The results indicate the structural homology between oligomannosyl cores of the cellular glycopeptides and those of non-membrane glycopeptides.     

Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.     

Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi

The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear beta-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA(3)Xyl(3) (aldotetraouronic acid), MeGlcA(3)Xyl(4) (aldopentaouronic acid) and MeGlcA(3)Xyl(5) (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA(2)Xyl(2), MeGlcA(2)Xyl(3) and MeGlcA(2)Xyl(4). As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3-14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with beta-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA(2)Xyl(2). In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.     

Biochemical Studies on “Bakanae” Fungus. Part 68. Synthesis of Substances related to Gibberellins

Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-β-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-β-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.     

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

Endo-β-N-acetylglucosaminidase H (endo H) is an enzyme which acts on asparagine- and lipid-linked oligosaccharides containing five or more mannose residues. Complex oligosaccharides and glycopeptides are completely resistant to the action of the enzyme. We have carried out pulse-chase experiments with ³⁵S-methionine and ³H-mannose in uninfected cells and in cells infected with Sindbis virus and vesicular stomatitis virus (VSV). In each case, the labeled materials were analyzed for sensitivity to endo H by polyacrylamide gel electrophoresis and gel filtration. We find that endo H releases all the labeled mannose from pulse-labeled proteins. Initially, the released material is nearly identical in size to the endo H cleavage product derived from lipid-linked oligosaccharides present in the same cells. During chase periods, ³⁵S-methionine and ³H-mannose protein becomes increasingly resistant to the enzyme. Moreover, the ³H-mannose-labeled material released from the protein during chase periods is smaller in size than the oligosaccharide from the lipid.On the basis of these results and results from other laboratories, we propose that during glycosylation of asparagine residues, a common oligosaccharide is transferred from the lipid carrier to protein and is subsequently processed to yield the so-called “high mannose” and “complex” oligosaccharides. Since, on the basis of present evidence, the lipid-linked oligosaccharide contains two N-acetylglucosamine, 8–12 mannose and 1–2 glucose molecules, it seems probable that the carbohydrate-processing systems remove half or more of the mannose and all of the glucose residues at sites destined to become complex glycopeptides. Removal of mannose and glucose residues may also occur at sites destined to become mature high mannose glycopeptides.     

Enzymatic prebleaching of sulphite pulps

The ability of a crude enzyme preparation ofAureobasidium pullulans containing xylanase [61 units (U)/ml] and xylosidase (3 U/ml) activity to remove pentosans from unbleached sulphite pulps was investigated. Greater amounts of pentosans and reducing sugars were released from the pulp when the enzyme dosages and incubation times were increased. A combination of enzyme hydrolysis and alkali extraction resulted in a greater removal of pentosans than using the enzyme preparation alone. By treatment with a xylanase loading of 450 U/g pulp for 24 h followed by alkaline extraction, 35% of the pentosans were removed. The kappa number decreased up to 30% whereas viscosity was only slightly affected by these treatments. Enzymatic hydrolysis released mainly xylose whereas xylobiose was the main product liberated by alkaline extraction. Scanning electron micrographs indicated improved fibrillation and flexibility of the fibre structure by enzyme treatment.     

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.     

A Novel Fungal Endo-β-N-acetylglucosaminidase that Specifically Acts on Plant Glycoproteins

An endo-β-N-acetylglucosaminidase specific for plant glycoprotein oligosaccharides was purified from the culture fluid of a fungus. The Mr of the purified enzyme was 89,000. This enzyme was stable at pH 5.5-7.0, up to 30°C, and showed the highest activity at pH 6.0. Among sugar chains tested, xylose-containing sugar chains (M3X, M3FX, and M2FX) were the most favored substrates. Oligomannose type (M3, M5, and M9) and hybrid type (GNM3) sugar chains were hydrolyzed much more slowly than xylose-containing sugar chains, and a complex type sugar chain (GN2M3) was not hydrolyzed at all by the enzyme. Moreover, the enzyme released sugar chains from native horseradish peroxidase and stem bromelain, which were not hydrolyzed by other endo-β-N-acetylglucosaminidases (Endo H, D, and F). The enzyme could transfer the xylose-containing sugar chain from bromelain to DNS-Asn-GlcNAc-Fuc.     

Continuous photometric determination of endo-1,4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate

A very simple and sensitive procedure for the determination of the activity of highly purified endo-1,4-beta-glucanase from the microscopic fungus Trichoderma reesei using 4-methylumbelliferyl-beta-D-cellobioside has been developed. The HPLC study has shown that this substrate is cleaved by endo-1,4-beta-glucanase to form predominantly free 4-methylumbelliferone, Km and kcat being 1.25 mM and 7.9 s-1, respectively (30 degrees C, pH 5.0). The possibility of continuous photometric determination of the enzyme using the difference absorptivity coefficient of 1600 M-1 cm-1 at 350 nm has been demonstrated.     

Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides.

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.     

Apple-fruit xyloglucans: a comparative study of enzyme digests of whole cell walls and of alkali-extracted xyloglucans.

Apple cell walls or alkali-extracted xyloglucans were digested with an endo-glucanase from Trichoderma viride and the resulting oligosaccharides were isolated by chromatography on Bio-Gel P-4. Three main oligosaccharides were present in similar proportions, and their structures were shown to be [Xyl(Glc)]3-Glc, [Xyl(Glc)]2-(FucGalXyl)Glc-Glc, and XylGlc-(GalXyP)Glc-(FucGalXyl)Glc-Glc. Each non-reducing-end Glc was 6-linked, each reducing-end Glc was 4-substituted, and each other Glc was 4,6-disubstituted. The Xyl was either terminal or 2-substituted, the Fuc was terminal, and the Gal was either terminal or 2-substituted. The 1H-NMR spectra of the oligosaccharides extracted directly from the cell wall showed that they are not acetylated. Other oligosaccharides, notably GalXyl3Glc4, Xyl2Glc4, and Xyl2Glc3, were present in smaller proportions in the digest of the cell walls.     

Induction of cellulose- and xylan-degrading enzyme systems in Aspergillus terreus by homo- and heterodisaccharides composed of glucose and xylose

Synthetic heterodisaccharides composed of glucose and xylose were tested as inducers of cellulose- and xylan-degrading enzymes in Aspergillus terreus, and the inducing abilities were compared with those of sophorose and xylobiose or their positional isomers. Measurement of secreted and cell-associated enzyme activities revealed that the heterodisaccharides induced the synthesis of the cellulolytic and xylanolytic enzymes, 2-O-beta-D-glucopyranosyl D-xylose (Glcbeta 1-2Xyl) being the most powerful inducer. Sophorose and 2-O-beta-D-xylopyranosyl D-Xylose (Xylbeta 1-2Xyl), or their positional isomers, selectively induced the synthesis of cellulases and beta-xylanases, respectively. An analysis of the extracellular enzymes (which were separated by isoelectric focusing followed by detection using chromogenic and fluorogenic substrates) showed that Glcbeta 1-2Xyl initiated the synthesis of specific endo-1,4-beta-glucanases and specific endo-1,4-beta-xylanases identical to those produced separately in response to sophorose or Xylbeta 1-2Xyl. Glcbeta 1-2Xyl also induced specific endo-1,4-beta-glucanases that hydrolysed 4-methylumbelliferyl beta-lactoside at the agluconic bond. The results strengthen the concept of separate regulatory control of the synthesis of cullulases and beta-xylanases. The results also suggest that mixed disaccharides, composed of glucose and xylose moieties, which may occur in nature, could play an important role in regulating the synthesis of wood-degrading enzymes.     

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Two endo-(1→4)-β-d-xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii, were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l-arabinose and d-xylose. Both xylanases were free from cellulase activity.     

A comparison of the substrate specificities of endo-β-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae

The substrate specificities of the endo-β-N-acetylglucosaminidases from $\text{Diplococcus pneumoniae}$ and $\text{Streptomyces griseus}$ were compared and found to differ considerably. The enzyme from $\text{D. pneumoniae}$ released Asn-GlcNAc-Fuc-containing glycopeptides from exoglycosidase-treated acidic IgM glycopeptides but was limited in its capacity to hydrolyze ovalbumin glycopeptides larger than Asn(GlcNAc)₂(Man)₅. In contrast, the enzyme from $\text{S. griseus}$ hydrolyzed this and larger neutral oligosaccharides but could not hydrolyze the above fucose-containing IgM glycopeptides. Removal of the fucose residue, however, converted the latter to an active substrate for the $\text{S. griseus}$ enzyme, thus broadening its substrate range to encompass most of those substrates hydrolyzed by the $\text{D. pneumoniae}$ endoglycosidase.     

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.     

Changes in N-Linked Oligosaccharides during Seed Development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-β-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans.

The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc_2~0Man₃Xyl₁Fuc_1~0GlcNAc₂) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc₂Man₃Xyl₁Fuc₁GlcNAc₂ and GlcNAc₁Man₃Xyl₁Fuc₁GlcNAc₂ decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages.

Concerning the endogenous substrates for plant endo-β-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen-1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO₂), were isolated from the basified biological matrix using a liquid–liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate–hexane–triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.     

Localization of phytase and acid phosphatase isoenzymes in aleurone layers of barley

The localization of acid phosphatase (EC 3.1.3.2) in aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) grains was studied. Phosphatase (EC 3.1.3.26) activity, assayed with phytic acid as the substrate, is present in the dry grain at low leveis and increases during incubation in H₂O at 25°C for three days. When aleurone layers are isolated from imbibed grain and incubated for 18 h in buffer with or without 50 μM gibberellic acid (GA₃), the level of extractable phosphatase activity increases two- to threefold, and phosphatase is released into the medium. GA, promotes the release of phosphatase activity: aleurone layers incubated in GA, release twice as much phosphatase as layers incubated in buffer. Nine isoenzymes of phosphatase are found in aleurone layers of barley by non-denaturing polyacrvlamide gel electropho-resis. Six of these forms, isoenzymes 1,2,3,5,6 and 8, can be extracted from dry tissue, and after three days of imbibition in H₂O an additional isoenzyme, isoenzyme 9, is found in aleurone extracts. When isolated aleurone layers are incubated for a further 22 h in buffer with or without GA₃, isoenzyme 7 is found and yet another form, isoenzyme 4, is found in layers incubated in GA₃. Eight isoenzymes are released from aleurone layers into the incubation medium. Isoenzymes 5 and 6 are released in buffer both with and without GA₃, even when cycloheximide is present; cycloheximide inhibits the release of the other isoenzymes. Isoenzymes 1-4, 7 and 8, on the other hand, are secreted into the incubation medium only when GA₃, is present. Isoenzyme 9 is not released into the incubation medium. Acid phosphatase activity was localized in aleurone tissue using cytochemical, cell fractionation, and enzymatic methods. Cytochemical localization of ATPase (EC 3.6.1.8) in aleurone tissue showed the presence of enzyme activity in cell wall, protein bodies, endoplasmic reticulum, Golgi apparatus, and mitochondria. Analysis of organelle fractions isolated by density gradient centrifugation showed that the activity of acid phosphatase isoenzymes 1, 2 and 3 was prominently associated with the phytin globoid of protein bodies, and analysis of the activity released from the cell wall by enzymatic digestion showed that it was almost exclusively isoenzymes 5 and 6.