Formation of soluble oligomers and amyloid fibrils with physical properties of the scrapie isoform of the prion protein from the C-terminal domain of recombinant murine prion protein mPrP-(121-231)

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.     

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.     

Pilot-scale semisolid fermentation of straw.

Semisolid fermentation of ryegrass straw to increase its animal feed value was successfully performed on a pilot scale. The pilot plant, which could handle 100 kg of straw per batch, was designed so that all major operations could take place in one vessel. The straw was hydrolyzed at 121 degrees C for 30 min with 0.5 N H2SO4 (7:3 liquid:solid), treated with ammonia to raise the pH to 5.0, inoculated with Candida utilis, and fermented in a semisolid state (70% moisture). During fermentation the straw was held stationary with air blown up through it. Batch fermentation times were 12 to 29 h. Semisolid fermentation did not require agitation and supported abundant growth at 20 to 40 degrees C even at near zero oxygen tensions. Fermentation increased the protein content, crude fat content, and in vitro rumen digestibility of the straw.     

Pilot-scale semisolid fermentation of straw

Semisolid fermentation of ryegrass straw to increase its animal feed value was successfully performed on a pilot scale. The pilot plant, which could handle 100 kg of straw per batch, was designed so that all major operations could take place in one vessel. The straw was hydrolyzed at 121 degrees C for 30 min with 0.5 N H2SO4 (7:3 liquid:solid), treated with ammonia to raise the pH to 5.0, inoculated with Candida utilis, and fermented in a semisolid state (70% moisture). During fermentation the straw was held stationary with air blown up through it. Batch fermentation times were 12 to 29 h. Semisolid fermentation did not require agitation and supported abundant growth at 20 to 40 degrees C even at near zero oxygen tensions. Fermentation increased the protein content, crude fat content, and in vitro rumen digestibility of the straw.     

Rapid purification of inositol monophosphate phosphatase from beef brain

A procedure is described for preparation of homogeneous inositol monophosphate phosphatase (EC 3.1.3.25) from beef brain in less than 2 days with an overall recovery of 15-25%. This enzyme, an essential part of the inositol phospholipid cycle in brain, is a proposed site of action of lithium ions in manic-depressive disorders. The major purification steps are: a) removal of most interfering protein by heat denaturation at 75 degrees C for 1 h, b) separation by anion exchange at a pH (6.0) near the enzyme's pI (4.9), and c) adsorption of most remaining impurities on a Procion Red affinity column.     

Resolution and characterization of two forms of phosphoinositide-specific phospholipase C from bovine rod outer segments.

Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes.     

Purification and characterization of a novel fungal alpha-glucosidase from Mortierella alliacea with high starch-hydrolytic activity

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable alpha-amylase activity. Here, a alpha-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal alpha-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55 degrees C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-alpha-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50 degrees C.     

Interaction of the cellular prion protein with raft-like lipid membranes

Conversion of the cellular isoform of the prion protein (PrP(C)) into the disease-associated isoform (PrP(Sc)) plays a key role in the development of prion diseases. Within its cellular pathway, PrP(C) undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrP(C) with model membranes, we purified posttranslationally modified PrP(C) from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrP(C) free in solution were analyzed by analytical ultracentrifugation. The interaction of PrP(C) with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrP(C) association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrP(C) released from the membrane into aqueous solution was estimated at between 10(-9) and 10(-7) M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrP(C) to PrP(Sc) are discussed.     

Determination of hydroxyproline by high pressure liquid chromatography.

A rapid, precise, and simple HPLC method provides an assay of hydroxyproline from tissue extracts or solutions of collagen. Samples are hydrolyzed with 6 N HCl, derivatized with phenyl isothiocyanate, and chromatographed on a small, C18 reverse-phase HPLC column. Hydroxyproline (Hyp) is separated from other amino acids and detected by absorption at 254 nm. The method detects 0.40 to 36 micrograms of Hyp with a linear response. Separation requires a total of 6 min, including column cleanup and reequilibration. All components are commercially available, making this a convenient method for routine measurement of collagen concentration.     

Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates.

The kinetics of folding of mPrP(121-231), the structured 111-residue domain of the murine cellular prion protein PrP(C), were investigated by stopped-flow fluorescence using the variant F175W, which has the same overall structure and stability as wild-type mPrP(121-231) but shows a strong fluorescence change upon unfolding. At 22 degrees C and pH 7.0, folding of mPrP(121-231)-F175W is too fast to be observable by stopped-flow techniques. Folding at 4 degrees C occurs with a deduced half-life of approximately 170 micros without detectable intermediates, possibly the fastest protein-folding reaction known so far. Thus, propagation of the abnormal, oligomeric prion protein PrP(Sc), which is supposed to be the causative agent of transmissible spongiform encephalopathies, is unlikely to follow a mechanism where kinetic folding intermediates of PrP(C) are a source of PrP(Sc) subunits.     

Purification and mode of action of an alkali-resistant endo-1, 4-beta-glucanase from Bacillus pumilus

Alkaline endo-1,4-beta-d-glucanase was secreted by Bacillus pumilus grown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pI values of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0-8.0 and 60 degrees C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30 degrees C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-beta-d-glucoside, 4-nitrophenyl-beta-d-cellobioside, and 4-nitrophenyl-beta-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.     

Histidines in the octapeptide repeat of PrPC react with PrPSc at an acidic pH

Cellular PrP is actively cycled between the cell surface and the endosomal pathway. The exact site and mechanism of conversion from PrP(C) to PrP(Sc) remain unknown. We have previously used recombinant antibodies containing grafts of PrP sequence to identify three regions of PrP(C) (aa23-27, 98-110, and 136-158) that react with PrP(Sc) at neutral pH. To determine if any regions of PrP(C) react with PrP(Sc) at an acidic pH similar to that of an endosomal compartment, we tested our panel of grafted antibodies for the ability to precipitate PrP(Sc) in a range of pH conditions. At pH near or lower than 6, PrP-grafted antibodies representing the octapeptide repeat react strongly with PrP(Sc) but not PrP(C). Modified grafts in which the histidines of the octarepeat were replaced with alanines did not react with PrP(Sc). PrP(Sc) precipitated by the octapeptide at pH 5.7 was able to seed conversion of normal PrP to PrP(Sc) in vitro. However, modified PrP containing histidine to alanine substitutions within the octapeptide repeats was still converted to PrP(Sc) in N2a cells. These results suggest that once PrP has entered the endosomal pathway, the acidic environment facilitates the binding of PrP(Sc) to the octarepeat of PrP(C) by the change in charge of the histidines within the octarepeat.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes

5-Methyltryptamine, through a GTP-dependent mechanism, stimulated breakdown of endogenous [3H]inositol-labeled phosphoinositides in membranes prepared from blowfly salivary gland homogenates through a phospholipase C exhibiting a pH optimum of approximately 7.0. Unlabeled membranes, prepared from salivary gland homogenates, hydrolyzed exogenous [3H]phosphatidylinositol 4,5-bisphosphate substrate with generation of labeled inositol phosphates. Inositol trisphosphate formation was increased approximately 200% by 10 microM guanosine 5'-(O-thio)-trisphosphate (GTP gamma S) within 30 s. 5-Methyltryptamine, in the presence of 10 microM GTP gamma S, increased the rate of inositol trisphosphate formation by approximately 500% within 30 s. Half-maximal activation of hormone-stimulated breakdown of exogenous substrate required approximately 0.05 microM GTP gamma S. [3H]Phosphatidylinositol was also hydrolyzed during incubation with membranes, resulting in the generation of inositol, glycerol phosphoinositol, and inositol monophosphate. Formation of inositol monophosphate was stimulated approximately 30% by 10 microM GTP gamma S and 10 microM 5-methyltryptamine. Neither inositol nor glycerol phosphoinositol formation was affected by hormone. These results indicate that in a cell-free system from blowfly salivary glands, 5-methyltryptamine, through a GTP-dependent mechanism, directly activates a phospholipase C which mediates phosphoinositide hydrolysis.     

Immobilization of the serine protease from Thermomonospora fusca YX on porous glass

As much as 84% of the thermostable serine protease from Thermomonospora fusca strain YX was covalently attached to silanized glass using glutaraldehyde. The immobilized protease exhibited a higher temperature optimum (86 degrees C) and pH optimum (9.4) for activity compared to soluble YX-protease (80 degrees C and pH 9.0, respectively). Immobilization improved enzyme thermo-stability above 90 degrees C and reduced inactivation during prolonged storage (9% loss of activity after 90 days at 12 degrees C). A continuous-flow column reactor packed with immobilized protease readily hydrolyzed casein over broad ranges of temperature and pH.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.     

Scrapie PrP 27-30 is a sialoglycoprotein.

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.     

植酸酶产生菌的选育及固态产酶条件研究

植酸酶催化植酸,并将其盐类水解成肌醇和磷酸,因此植酸酶的使用可以提高植酸磷的吸收利用率,降低饲料成本,同时还可保护生态环境.经分离和亚硝基胍诱变选育,得到一株植酸酶高产菌株绿色木霉LH374,并对该菌株固态发酵产植酸酶的条件和扩大生产进行了研究.结果表明,固态发酵的最佳条件:稻草和米糠的比例为8:2,培养基起始pH为6.5,最适温度为30℃,最适培养时间为96 h,含水量为60%,硫酸铵的流加量为2%.绿色木霉LH37在上述最适条件下生产植酸酶平均可达1 580 U·g^-1.     

Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.