Three-step Purification Scheme Research Articles

Characterization of glucose Oxidase from Penicillium chrysogenum MDC 8358: Prospects for application in food industry

Backgrounds: Food enzymes play a crucial role in enhancing specific food attributes, such as improving texture, eliminating toxins and allergens, producing carbohydrates, and enhancing flavor and appearance characteristics. Glucose oxidase (GOX) catalyzes the oxidation of glucose to produce gluconic acid and hydrogen peroxide, making it applicable in various practical scenarios. This enzyme could be utilized to eliminate glucose, thereby facilitating the creation of functional food suitable for individuals with diabetes and glucose intolerance.Our main goal was to characterize GOX from a Penicillium chrysogenum strain isolated from Armenian ecosystems.Methods:GOX of P. chrysogenum MDC 8358 was obtained by surface fermentation using glucose as a carbon source. The enzyme was purified 230-fold to homogeneity using a three-step purification scheme consisting of ion exchange, adsorption, and gel filtration chromatography. Results:The specific activity of the purified enzyme reached 271.2 U/mg. GOX from P. chrysogenum MDC 8358 has shown to be dimeric with a molecular weight of 135.5 kDa, consisting of two equal subunits with a molecular weight of 70 kDa. Isoelectric focusing has revealed a pH of 4.6. The enzyme has displayed a temperature optimum of 40 °C, and a pH optimum of 6.8, being more thermostable at acidic pH. Characterized GOX was highly specific for β-D-glucose and has shown only minor activities to mannose, xylose, and galactose. Keywords:Penicillium chrysogenum MDC 8358, surface fermentation, glucose oxidase, purification, characterization, functional food preservation

SUMO-fusion and autoinduction-based combinatorial approach for enhanced production of bioactive human interleukin-24 in Escherichia coli.

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at Go/G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications. KEY POINTS: • Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli. • Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media. • Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL. • Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features. Graphical abstract.

Multi-epitope insert modulates solubility-based and chromatographic purification of human papilloma virus 16 L1-based vaccine without inhibiting virus-like particle assembly

The separation of heterogeneous protein mixtures has always been characterized by a trade-off between purity and yield. One way this issue has been addressed in the past is by recombinantly modifying protein to improve separations. Such modifications are mostly employed in the form of tags used specifically for affinity chromatography, though it is also possible to make changes to a protein that will have a sizeable impact on its hydrophobicity and charge/charge distribution. As such, it should also be possible to use protein tags to modulate phase separations and protein-resin binding kinetics when performing ion exchange chromatography. Here, we employed a three-step purification scheme on E. coli expressed, His-tagged, human papilloma virus 16 L1-based recombinant proteins (rHPV 16 L1) that consisted of an inclusion body (IB) wash step, a diethylaminoethyl (DEAE) anion exchange chromatography (AEX) step, and an immobilized metal affinity chromatography (IMAC) polishing step. Purification of the wild type rHPV 16 L1 protein (WT) was characterized by substantial losses during the IB wash but relatively high yield over the DEAE column. In contrast, purification of modified rHPV 16 L1, a chimeric version of the WT protein that had the last 34 amino acids replaced with an MHC class II multi-epitope insert derived from tetanus toxin and diphtheria toxin (WTΔC34-2TEp), was characterized by little to no losses in the IB wash but had a relatively low yield over the DEAE column. Since the fate of these proteins was to be used in vaccine formulations, it is important to note that the modifications made to the WTΔC34-2TEp protein had little to no effect on its ability to assemble into virus-like particles (VLPs). These results demonstrate that modifications of the WT protein via the recombinant insertion of immunofunctional polypeptides can modulate both phase-based separation and charge-based chromatographic processes. Additionally, incorporation of the specific, multi-epitope tag used in this study may prove to be beneficial in recombinant HPV vaccine development due to its potential to improve phase separation yield and vaccine immunogenicity without inhibiting VLP formation.

Extinction coefficient per CdE (E = Se or S) unit for zinc-blende CdE nanocrystals

The extinction coefficient of semiconductor nanocrystals is a key parameter for understanding both the quantum confinement and applications of the nanocrystals. The existing extinction coefficients of CdE (E = Se, S) nanocrystals were found to have an unacceptable deviation for the zinc-blende CdE quantum dots (QDs). The analysis reveals that, in addition to the interference of impurities, the commonly applied extinction coefficient per CdE nanocrystal is sensitive to the size, shape, and density of the surface ligands of nanocrystals. The extinction coefficient per CdE unit does not depend on accurate information of the size, shape, and number of surface ligands of the nanocrystals. A new three-step purification scheme was developed to investigate three classes of possible impurities for accurate determination of the extinction coefficient per CdE unit, including CdE clusters not considered previously. Given that the sole ligands of zinc-blende CdE nanocrystals are cadmium fatty acid salts (CdFa2), a universal formula for the nanocrystals can be written as (CdE)n(CdFa2)m. The n:m ratio was accurately determined for purified nanocrystals. The resulting extinction coefficients per unit for both CdSe and CdS QDs were found to decrease exponentially as the size of the QDs increases, with the corresponding bulk value as the large-size limit.

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification.

A protein with high purity has become an essential pre-requisite for investigating its bioactivity, molecular structure and characteristics. Therefore, the development of technologies for efficient purification of protein is urgently necessary. The objective of this study was to establish a purification protocol for a recombinant protein rG17PE38. Different forms of chromatography such as hydrophobic interaction and ion exchange chromatography were chosen as the core purification steps. The performance of each technique was optimized to meet the requirements and the purification steps were arranged in a logical way of facilitating to operate in next step. In addition, some characteristics of the protein such as stability, bioactivity and cellular location were determined. Finally, whether the protein could induce cell apoptosis was also explored. The results showed the protein purified via the suggested three-step purification scheme could obtain a purity of 95%, and its bioactivity in the form of IC₅₀ was 17.6 ng/mL, furthermore it could keep stable at 4 °C for at least 10 days. The protein could bind on its target cell membrane specifically, and inducing cell apoptosis was demonstrated to be one of the cytotoxicity mechanisms of the protein. Results obtained in our study may provide useful information on strategies of protein purification and lay a substantial foundation for the followed animal or clinical experiments on rG17PE38.

Detergent-compatible, organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization

ABSTRACTProteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Km and Vmax values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca2+, Mg2+, Mn2+); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.

Isolation and characterization of wild-type lipoxygenase LOX(Psa)1 from Pleurotus sapidus.

The lipoxygenase LOX(Psa) 1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOX(Psa) 1 were identified by de novo sequencing, and the postulated molecular mass of 72.5 kDa was confirmed. With linoleic acid as the substrate, the LOX(Psa)1 showed a specific activity of 113 U mg(-1) and maximal activity at pH 7.0 and 30 degrees C, respectively.

Purification and characterization of xylanase from Aspergillus fumigatus isolated from soil

The objectives of the present study were to purify and characterize xylanase enzyme from the fungus obtained from soil. A total of 40 fungi were isolated from 25 soil samples collected after primary screening on Potato Dextrose Agar. In the secondary screening (malt extract agar, 0.5% birch wood xylan), based on the diameter of the clear zone, the fungus was identified as Aspergillus fumigatus by microbial type culture collection (MTCC), Chandigarh, India and was selected for xylanase enzyme production in solid state fermentation using wheat bran. Xylanase was subjected to a three-step purification scheme involving ammonium sulphate precipitation, gel filtration chromatography and anion exchange chromatography. Purity was verified by running the extracted protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single band was observed. When compared with the standard wide range protein molecular markers on SDS-PAGE, it was found to have a molecular weight of 43 KDa. The Km and Vmax value of xylanase was 3.12 mg/ml and 2857 μmol/min/mg protein as obtained from a Lineweaver-Burk plot. The optimal temperature and pH was found to be 30 and 10°C, respectively. After 4 h of incubation, enzyme retained 100% activity at 30°C. Xylanase was incubated at various pH levels (2 to 12) for 4 h at 30°C, and the residual activity was measured. More than 65% of the original activity was retained at pH ranging from 4 to 10 after 4 h.

An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation

The preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus. The overall downstream processing scheme gave 80% infectious particle yield. The contamination ratio of helper virus in the helper-dependent virus preparation are reduced from 2.57 to 0.03% corresponding to a reduction of helper virus by factors of 85 by two iodixanol purification steps. It was also demonstrated that size-exclusion chromatography is an excellent step for the removal of iodixanol and polishing of the final helper-dependent virus preparation.

Purification and Biochemical Characterization of Two Xylanases from Aspergillus sydowii SBS 45

Two xylanases were isolated and purified from crude culture filtrate of Aspergillus sydowii SBS 45 after 9 days of growth on wheat bran containing 0.5% (w/v) birch wood xylan as the carbon source under solid-state fermentation. After a three-step purification scheme involving ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200), and anion exchange chromatography (DEAE-Sephadex A-50), xylanase I was purified 93.41 times, and xylanase II was purified 77.40 times with yields of 4.49 and 10.46, respectively. Molecular weights of xylanase I and II were 20.1 and 43 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature was 50 degrees C, and optimum pH was 10.0 for both xylanase I and II. The Km value of xylanase I for birch wood xylan was 3.18 mg ml(-1) and for oat spelt xylan 6.45 mg ml(-1), while the Km value of xylanase II for birch wood xylan was 6.51 mg ml(-1) and for oat spelt xylan 7.69 mg ml(-1). Metal ions like Al3+, Ba2+, Ca2+, Na+, and Zn2+ enhanced the activity of xylanase I and II at 10 mM concentration. Among the additives, L-tryptophan enhanced the activity of xylanase I and II at 10-, 20-, and 30-mM concentrations. Both xylanases appeared to be glycoproteins.

Phytase activity in tobacco ( Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco ( Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg −1 and an overall yield of 3%. SDS–PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0–5.5 and 45 °C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate ( pNPP; K m = 13.9 μM) and phytate ( K m = 14.7 μM), but a higher kcat for pNPP (2,056 s −1) than phytate (908 s −1). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.

Properties of beta-propeller phytase expressed in transgenic tobacco

Phytases are enzymes that liberate inorganic phosphates from phytate. In a previous study, a beta-propeller phytase ( 168phyA) from Bacillus subtilis was introduced into transgenic tobacco, which resulted in certain phenotypic changes. In the study described herein, the recombinant phytase (t168phyA) was purified from transgenic tobacco to near homogeneity by a three-step purification scheme. The biochemical properties and kinetic parameters of t168phyA were compared with those of its counterpart from B. subtilis. t168phyA was glycosylated, and it showed a 4 kDa increase in molecular size in SDS–PAGE (44 kDa vs. 40 kDa). Although its thermostability remained unchanged, its temperature optimum shifted from 60 °C to 45–50 °C and its pH optimum shifted from pH 5.5 to 6.0. Kinetic data showed that the t168phyA had a lower K cat, but a higher K m than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants.

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme β-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid–water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni–NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni–NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective 15N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial 15N, 1H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography.

In this study we describe a novel method for purification of Arabidopsis thaliana dehydrins overproduced in Escherichia coli. The cDNAs corresponding to the four dehydrin genes RAB18, LTI29, LTI30, and COR47 were inserted into a bacterial expression vector under an isopropyl beta-d-thiogalactopyranoside (IPTG) inducible bacterial promoter. After IPTG induction all four proteins accumulated in high amounts. The recombinant proteins were efficiently purified to over 95% purity with a three-step purification scheme: heat fractionation, immobilized metal ion affinity chromatography (IMAC), and ion exchange chromatography. In this study we introduce the novel use of IMAC as an efficient purification method for native dehydrins. Characterization of the purified proteins was done by Edman degradation, mass spectrometry, reverse-phase chromatography, and analytical gel filtration under native and denaturing conditions. Yields of purified proteins were between 2.8 and 12.5 mg per liter of bacterial culture, sufficient for further biochemical studies.

Selenium-containing xanthine dehydrogenase from Eubacterium barkeri.

A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent specific activity of 164 micromol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH-dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum-complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.

High-level secretory production of intact, biologically active staphylokinase fromBacillus subtilis

Staphylokinase is a promising blood-clot dissolving agent for the treatment of patients suffering from a heart attack. It would be desirable to produce this protein in large quantities for biochemical characterization and clinical trials. Production of intact, biologically active staphylokinase from bacterial expression systems has been a challenge because of N-terminal microheterogeneity, plasmid instability, or low-production yield. By using a seven-extracellular-protease deficient Bacillus subtilis strain, WB700, intact staphylokinase can be produced via secretion. However, native staphylokinase gene (sak) in a high-copy number plasmid was found to be unstable in B. subtilis. To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of sak were replaced by B. subtilis promoters (P43, a constitutively expressed promoter; Pamy, a stationary-phase promoter; and PsacB, a sucrose-inducible promoter) and the levansucrase-signal sequence, respectively. This overcame the plasmid instability problem. To enhance transcription from the sacB promoter, degQ encoding a transcriptional activator for sacB and other protease genes was also installed in the expression vector. The use of WB700 as the expression host allowed enhanced production of staphylokinase from the sucrose-inducible plasmid without causing any obvious degradation of staphylokinase. Both the P43 and PsacB (with DegQ) promoters worked well. Over 90% of staphylokinase synthesized can be secreted effectively. With the optimization of both the culture media and the fermentation conditions, production of staphylokinase reached a level of 337 mg/L, and staphylokinase could be purified to homogeneity by a simple three-step purification scheme. Secreted staphylokinase did not show any N-terminal heterogeneity. This presents an attractive system for the production of staphylokinase in both high quality and quantity.

Stress induction of the mammalian GRP78/BiP protein gene: in vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element.

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.

Stress Induction of the Mammalian GRP78/BiP Protein Gene: In Vivo Genomic Footprinting and Identification of p70CORE from Human Nuclear Extract as a DNA-Binding Component Specific to the Stress Regulatory Element

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.

Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g(-1) tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P2) was increased (Km = 6.88 versus 2.44 μmol 1-(-1) as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P2 (mixed-type inhibitor, Ki = 0.201 μmol 1(-1), pH 7.0) and AMP (non-competitive inhibitor, Ki = 0.438 μmol 1(-1), pH 7.0). Inhibition by F-2,6-P2 was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I50 increased from 0.14 to 0.32 μmol 1(-1)). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH4 (+) and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.

Purification and Characterization of Heat-Stable Proteases from Bacillus stearothermophilus RM-67

The extracellular proteases of Bacillus stearothermophilus RM-67 were purified by ammonium sulfate fractionation (40 to 70% saturation), gel filtration through Sephadex G-100, and diethylaminoethyl-Sephadex A-50 ion-exchange chromatography. Gel filtration resulted in separation of the enzyme preparation into one minor (protease I) and one major (protease II) peak. The three-step purification scheme resulted in 39.5-fold purification and an overall recovery of 8.1% of protease I and 87.8-fold purification and 59.7% recovery of protease II.Purified proteases had pH and temperature optima of 8.0 and 70°C. Protease I and II, when together, retained 100% activity at 60°C for 30min. Manganese imparted 100% stability to the pooled proteases at 65°C for 30min. Amino acid analysis of the major peak (protease II) revealed the absence of half cystine and methionine. Protease I and II had molecular weights of 67,610 and 19,950 and Michaelis-Menten constants (casein) of 1.33 and 2.0 mg/ml. Energy of activation was 14,300 cal/mol for protease I and 11,150 cal/mol for protease II. Corresponding heat of activation was 13,620 and 10,470 cal/mol.