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This study aimed to isolate and comprehensively characterize chitinase-producing bacterial isolates collected from diverse aquatic stations in the Red Sea. Out of twenty nine isolates, chitinase-producing marine Enterococcus hirae (OR064172) and Enterococcus faecalis (OR053873) were biochemically and genetically identified. The two isolates have primary specific activity of 10.7 and 11.5U/ mg for E. hirae and E. faecalis, respectively. To enhance the purity of the chitinase enzymes, ammonium sulfate precipitation was employed, yielding 12.9 and 13.47U/ mg for 70% fractions of E. hirae and E. faecalis, respectively. Afterward, DEAEcellulose chromatography was utilized for the purification process, resulting in two peaks for E. hirae and one peak for E. faecalis. The results of chitinase characterization indicated optimal conditions with a temperature of 37°C, a pH of 6.5, and a salt tolerance of up to 1% for both strains. The indepth kinetic chitinase analysis unveiled the optimum Km (0.03 and 0.07) and Vmax (0.124 and 0.0003) conditions for chitinase activity for both E. hirae and E. faecalis, respectively. The application of the previous strains at the economic level involves the production of chitinase using marine shrimp waste in an attempt to extract beneficial products. The solid state fermentation process of this waste for both isolates was also tested, with results indicating that the specific activity of the produced enzyme was 8.48 and 11.38U/ mg, respectively, achieving the highest production after 48 hours. This study adeptly accomplished the isolation and identification of chitinase-producing marine bacterial isolates. The rigorous purification and comprehensive characterization of chitinase enzymes offer valuable insights into their production, refinement, and utilization across diverse industries, particularly within aquaculture and waste management. [ABSTRACT FROM AUTHOR]

Arginase was purified from human serum of healthy and patient with type II Diabetes mellitus subjects. The purification steps included, DEAE-cellulose chromatography and gel filtration on Sepharoe6B.The molecular weight of native arginase was find to be (94 Kd), by gel filtration. The purified human serum arginase is characterized by optimum pH of (9.5), optimum pH of stability is between (9-10), optimum temperature (350C), temperature of stability is between (30-500C) in both healthy and patient subjects. [ABSTRACT FROM AUTHOR]

Thermostable α-amylase from germinating Sword bean ( Canavalia gladiata (Jacq.) DC.) seeds has been successfully immobilized on DEAE-cellulose (ICgAmy1) and chitosan bead (ICgAmy2) support materials. Optimum conditions of immobilization for DEAE-cellulose and chitosan bead revealed 97% and 96% immobilization yield, respectively. The optimum pH and temperature of both DEAE-cellulose and chitosan bead immobilized α-amylases were pH 7 and 70°C. Both ICgAmy1 and ICgAmy2 were high stability over a wide pH range of pH 5-9 and a temperature range of 70-90°C. In addition, ICgAmy1 and ICgAmy2 led to an operationally stable biocatalyst with above 74% and 76% residual activity after 10 reuses, respectively. Immobilized α-amylases showed high storage stability with 81% (ICgAmy1) and 85% (ICgAmy2) residual activity after 120 days of storage. The easy immobilization process on low-cost, biodegradable, and renewable support materials exhibited an increase in the enzyme operation range and storage stability which reduces production costs. This makes immobilized amylases an effective biocatalyst in various industrial applications especially a potential candidate for bioethanol production, a key renewable energy source., Competing Interests: Conflict of interestThe authors declare no conflicts of interest., (© 2024 Japanese Society for Plant Biotechnology.)

Since around 100 years ago, the best treatment for millions of global snakebite victims has been polyclonal antivenoms. However, common antivenoms need continuous improvement to reduce rare, their side effects and get better performance. In the present study, Fab antivenom was produced through papain digestion of anti-cobra venom plasma, multi-step purification, and optimization, including ammonium sulfate precipitation and DEAE-cellulose column chromatography. Then, the existence of the corresponding Fab fragment antibody was seen and confirmed by SDS-PAGE method and double immunodifusion (Ouchterlony) test. In addition, the potency test in NIH laboratory mice revealed that each milliliter of the new Fab antivenom was able to neutralize 624 micrograms and (80LD 50 ) of cobra venom, which is about 15% more efficient than the primary plasma of the same concentration, and 1.57 times more effective than the cobra antivenom found in commercial hexavalent antivenom of Razi Institute. According to the findings, it seems that this new Fab antivenom can be used as a new candidate for treatment of cobra snakebite victims., Competing Interests: The authors declare that they have no conflict of interest.

The use of a modified, four-stage purification scheme allowed us to obtain electrophoretically homogeneous preparations of constitutive forms of succinate dehydrogenase from maize (Zea mays L.) scutellum in the late stages of seed germination. It has been established that the isoenzymes differed significantly in their quaternary structure. Thus, succinate dehydrogenase 1 turned out to be a heterotetramer, and succinate dehydrogenase 2 turned out to be a heteroterodimer. It was found that their main catalytic and kinetic characteristics also differed, in particular, by their affinity to the substrate (succinate) and the value of pH optimum of the catalyzed reaction. Succinate dehydrogenase 2 was characterized by enhanced resistance to a specific inhibitor—malonate. [ABSTRACT FROM AUTHOR]

L-Asparaginase (L-asparagine amido-hydrolase, EC 3.5.1.1) is an industrially and pharmaceutically important enzyme that catalyses the hydrolysis of asparagine into aspartic acid and ammonia. In this study, L-asparaginase was produced from Fusarium sp. LCJ273. The crude L-asparaginase produced was precipitated using ammonium sulphate precipitation followed by purification using ion exchange chromatography. The molecular weight of L-asparaginase was found to be approximately 66 kDa by SDS-PAGE. The culture filtrate, ammonium sulphate precipitate and partially purified Lasparaginase exhibited optimum activity in pH 7.0 at 30°C. The partially purified L-asparaginase from Fusarium sp. LCJ273 showed 100% relative activity towards the natural substrate L-asparagine. Further, the kinetics studies of partially purified L-asparaginase revealed that Km value was 24.2 mM and Vmax value was 125 mM min-1. The in vitro anticancer activity of partially purified L-asparaginase was investigated. Different concentration of partially purified enzyme was tested on three different cell lines namely MCF-7 (breast cancer cell line), Hep-2 (human laryngeal carcinoma cells) and vero cell lines by MTT assay (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide). Purified L-asparaginase reduced cell viability of MCF-7 (16.61%), Hep-2 (8.12%) and vero cell line (88.73%) after 72 hours with IC50 value of 33.21, 9.28 and 622.99 µg/mL respectively and showed better toxicity on the cell lines at a concentration of 120 µg/mL. [ABSTRACT FROM AUTHOR]

5-Hydroxy-isoxazolidinic heterocycles can be easily prepared from hydroxamic acids and suitable α,β- unsaturated aldehydes or ketones in good yields from solid state synthesis, selectively promoted by DEAE-C. The synthesized products were efficiently derivatized with representative heterobases for the preparation of nucleoside analogues suitable for biological evaluation and SAR analysis. [ABSTRACT FROM AUTHOR]

Regularities in the concentration and distribution of basic forms of phosphorus such as mineral, organic, suspended and bound to iron in complexes with humic substances were determined for diverse Karelian water bodies. Seasonal variations of the concentrations of phosphorus forms in Karelian water bodies of different trophic status were found.

We characterized polysaccharide complexes from Centaurea scabiosa L. and Centaurea pseudomaculosа Dobrocz. We proposed the technique of sequential selection of water-soluble polysaccharides and pectin substances from the aerial parts of studied objects. We have discovered that the content of water-soluble polysaccharides in the aerial parts of C. scabiosa was 2.8 times higher (2.7 ± 0.3%, n = 3) than in C. pseudomaculosа (0.97 ± 0.50%, n = 3). The content of pectin substances in the aerial parts of C. scabiosa was 2 times higher (7.6 ± 0.4%, n = 3) than in C. pseudomaculosа (3.9 ± 0.3%, n = 3). The residues of D-galacturonic acid, L-rhamnose, D-xylose, D-mannose, D-glucose, and D-galactose are the monomeric units of polysaccharide complexes from C. scabiosa and C. pseudomaculosa. Using ion-exchange chromatography, three polysaccharide fractions (molecular weights 667, 722, and 1027 kDa), whose monomer units are D-galacturonic acid, L-rhamnose, D-galactose, D-xylose, and D-glucose were isolated from the water-soluble polysaccharides of C. scabiosa. [ABSTRACT FROM AUTHOR]

The results of the study of the physicochemical properties of the high-molecular-weight soluble and insoluble components of nectarine cell walls obtained by fruit treatment under conditions that modulate of gastric digestion are presented. Homogenized nectarine fruits were sequentially treated by natural saliva and simulated gastric fluid (SGF) at pH 1.8 and 3.0. The isolated polysaccharides were compared with polysaccharides obtained by sequential extraction of nectarine fruit with cold, hot, and acidified water, solutions of ammonium oxalate and sodium carbonate. As a result, high-molecular-weight water-soluble pectic polysaccharides, weakly bound in the cell wall, were dissolved in the simulated gastric fluid, regardless of pH. Homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) were identified in all pectins. It was shown that their quantity and ability to form highly viscous solutions determine high values of the rheological characteristics of the nectarine mixture formed under simulated gastric conditions. The modifications occurring with the insoluble components under the influence of acidity of SGF were importance. They determined difference in the physicochemical properties of both the insoluble fibres and the nectarine mixtures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This work investigates the adsorption behavior of Diethylaminoethyl-cellulose-thiourea-glutaraldehyde (DEAE-C/TU/GA) sorbent, which was synthesized through the reaction of crosslinking of glutaraldehyde using thiourea for platinum (Pt(IV)) and palladium (Pd(II)) ions from leaching solutions. The effects of pH and adsorption parameters, such as the sorbent dosage, contact time, temperature, and adsorption isotherms, were studied. Furthermore, chemical adsorption kinetic studies for adsorptions of these metal ions were carried out. The adsorption equilibrium data were fit better using the Langmuir model than the Freundlich model. The activation energies (Ea) of Pt(IV) and Pd(II) were 77.70 and 23.03 kJ/mol, respectively. The best desorption reagent was 1.0 M HCl–1.0 M TU solution for both Pd(II) and Pt(IV), which could remove more than 85% of the Pt(IV) and Pd(II) in the fourth adsorption–desorption cycle. [ABSTRACT FROM PUBLISHER]

Chemical characteristics of dissolved organic matter (DOM) extracted from an ombrotrophic bog in northern Minnesota by two methods – XAD-8 and DEAE-cellulose – were compared using 13 C NMR spectroscopy, excitation–emission matrix fluorescence spectroscopy (EEMS), and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). DOM extracted with XAD-8, a relatively hydrophobic sorbent, had a larger 13 C NMR signal in the aromatic carbon region, but DOM extracted by DEAE-cellulose, a weak anion exchanger, had a larger signal in the carboxylic carbon region. DOM extracts prepared by the two methods were similar in their overall chemical characteristics that serve as proxies for organic matter sources. For example, only small differences were observed in the fluorescence index (FI) values of the extracts, and all values were within the range expected for terrestrially-derived DOM. EEMS spectra of both extracts had “humic-like” peak A areas smaller than that of standard reference Suwannee River fulvic acid. Of 2801 distinct chemical formulas assigned during FT-ICR MS analysis of the two extracts, 66% were present in both; 15% were unique to the XAD-8 extract, and 19% were unique to the DEAE-cellulose extract. Van Krevelen plots showed that the DEAE-cellulose extract had more tannin-like and condensed aromatic entities and formulas with higher O/C ratios, whereas the XAD-8 extract had more lignin-like material and formulas with higher H/C ratios. Overall, differences in chemical characteristics of the extracts reflect the mechanisms by which the extractants operate. [ABSTRACT FROM AUTHOR]

Highlights: [•] Using Box–Behnken technique, wheat α-amylase was optimally immobilized onto DEAE-cellulose. [•] This immobilization resulted from combination of covalent attachment and ionic adsorption. [•] The immobilized enzyme showed better thermostability and storage stability over free enzyme. [Copyright &y& Elsevier]

An antifungal protein with a molecular mass of 14.3 kDa was isolated from the seeds of butterfly pea ( Clitoria ternatea) and designated as Ct protein. The antifungal protein was purified using different methods including ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-50 column. Ct protein formed a single colourless rod-shaped crystal by hanging drop method after 7 days of sample loading. The protein showed lytic activity against Micrococcus luteus and broad-spectrum, fungicidal activity, particularly against the most clinically relevant yeasts, such as Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus laurentii, Candida albicans and Candida parapsilosis. It also exerted an inhibitory activity on mycelial growth in several mould species including Curvularia sp., Alternaria sp., Cladosporium sp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus sp., and Sclerotium sp. The present study adds to the literature on novel seed proteins with antifungal activity. [ABSTRACT FROM AUTHOR]

Cladosporium parasphaerospermum, Cladosporium chlamydosporigenum, and Cladosporium compactisporum have all been discovered and characterized as new Cladosporium species. The three new species seemed to generate cold-active pectinases with high activity at pH 6.0 and 10 °C, pH 6.0 and 15 °C, and pH 5.0 and 15 °C, respectively, with the most active being C. parasphaerospermum pectinase. In submerged fermentation (SmF), C. parasphaerospermum produced the most cold-active pectinase with the highest activity and specific activity (28.84 U/mL and 3797 U/mg) after 8 days. C. parasphaerospermum cold-active pectinase was isolated using DEAE-Cellulose anion exchange resin and a Sephadex G 100 gel filtration column. The enzyme was purified 214.4-fold and 406.4-fold greater than the fermentation medium using DEAE-cellulose and Sephadex G 100, respectively. At pH 7.0 and 10 °C, pure pectinase had the highest activity (6684 U/mg), with K m and V max determined to be 26.625 mg/mL and 312.5 U/min, respectively. At 5 mM/mL, EDTA, MgCl 2 , and SDS inhibited the activity of pure pectinase by 99.21, 96.03, and 94.45%, respectively. The addition of 10 U/mL pure pectinase enhanced the yield of apple, orange, apricot, and peach juice by 17, 20, 13, and 24%, respectively, and improved the clarity and colour of orange juice by 194 and 339%, respectively. We can now add cold-active pectinase production to the long list of Cladosporium species that have been identified. We also report three new species that can be used in biotechnological solutions as active microbial pectinase producers. Although further research is needed, these distinct species might be used to decompose difficult and resistant pertinacious wastes as well as clear fruit juices., (© 2022. The Author(s).)

Five fractions from crude Hericium erinaceus polysaccharides (HEPs), including HEP-1, HEP-2, HEP-3, HEP-4 and HEP-5, were obtained through column chromatography with a DEAE Cellulose-52 column and Sephadex G-100 column. The contents of total carbohydrates and uronic acid in HEPs were 53.36% and 32.56%, respectively. HEPs were mainly composed of Fuc, Gal and Glu in a molar ratio of 7.9:68.4:23.7. Its chemical structure was characterized by sugar and methylation analysis, along with 1 H and 13 C NMR spectroscopy. HEP-1 contains the backbone composed of (1→6)-linked-galactose with branches attached to O-2 of some glucose. The immunological activity assay indicated that HEP-1 significantly promoted the production of nitric oxide, interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α and the phosphorylation of signaling molecules. Collectively, these results suggested that HEP-1 could improve immunity via NF-κB, MAPK and PI3K/Akt pathways. Hericium erinaceus polysaccharides might be explored as an immunomodulatory agent for use in dietary supplements.

β-D-fructofuranosidase, (Ec 3.2.1.26) was immobilized on DEAE-cellulose, polyacrylamide and hen-egg white. storage at 4°C for 5 months indicated that the polyacrylamide and DEAE-cellulose preparations were more stable. The effect of lyophilization on immobilized invertase activity was also studied. It was found that lyophilization of the enzyme attached to DEAE-cellulose caused a decrease in its activity as compared to the other two enzymes. All lyophilized preparations could be stored for 4-5 months with a slight decrease in their activities. Batch processes were used to trace the activity of immobilized invertase. The results showed that the enzyme preparations could be re-used for eight cycles with slight effect on their activities. The inversion degree of sucrose at different concentrations and low rates using the polyacrylamide-enzyme gel ranged between 52 to 99%. A polyacrylamide-invertase gel was operated continuously for 5 days with a very low decrease in its retained activity (3%). After storage for one month the polyacrylamide-enzyme matrix conserves 83% of its initial velocity. [ABSTRACT FROM AUTHOR]

Abstract: The present work investigates gold recovery using DEAE-cellulose, a common biopolymer derivative, from synthetically prepared diluted gold-bearing solutions of 50 ppm. The effects of different recovery parameters on gold recovery efficiency were studied in detail. It was demonstrated that gold recovery efficiency increased with an increasing amount of sorbent, as well as increasing contact time. A gold recovery efficiency of 99% was attained under conditions of 20–40 g DEAE-cellulose per liter at a shaking rate of 130 rpm for 30 min at room temperature. On the other hand, with smaller amounts of sorbent (6 g/l), it was also possible to recover gold from the solution with 99% efficiency when the reaction temperature was increased to 60 °C. The shaking rate and temperature were demonstrated to play a vital role in the recovery process. It was also found that gold recovery by DEAE-cellulose is an intermediate-controlled process with an activation energy of 37.11 kJ/mol. The XRD pattern and SEM images revealed that the recovered gold was in the metallic form. [Copyright &y& Elsevier]

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I-IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques.

Cytotoxic activity (lymphotoxin (LT) associated with the 50,000 dalton beta-LT class of human lymphotoxins has been resolved into multiple components by chromatography on DEAE-cellulose or electrophoresis on polyacrylamide gels. The two components, termed beta-LT, and beta-LT2, are separable from the 90,000 m.w. alpha-LT, since neither component is neutralized by incubation with heterologous rabbit anti-alpha-LT antisera. However, the two components differ in their overall charge and relative heat stability with the betaLT1 component being more positively charged than the beat-LT2 molecule and extremely heat labile.

Lymphotoxin (LT), and proliferation inhibition factor (PIF) activities found in 5-day supernatants of mitogen-activated human lymphocytes (SAL) were further compared. In agreement with previous results, the activities could not be distinguished functionally. Quantitative differences in the amount of activity detected in the SAL could be accounted for on the basis of target cell differences, concentration of the lymphocyte effector molecules in the supernatant, and the parameter employed to assess cell function. Growth inhibitory activity detected at high supernatant dilutions was completely reversible, whereas the cytotoxic activity detected at low supernatant dilutions was irreversible. When the active medium was fractionated on DEAE, two peaks of inhibitory activity were detected. Depending upon the amount of activity and target cell, both peaks of activity were growth inhibitory or cytotoxic. Since both peaks of material affected HeLa and L-929 cells, the materials were not species specific. Thus, it appears that cloning inhibitor factor, LT, and PIF activities may actually be measures of the same stable materials found in 5-day activated lymphocyte supernatants.

Molecules with LT activity which can be identified in supernatants from PHA stimulated human lymphocytes by lysis of murine L-929 cell in vitro are heterogeneous. They can be separated by MW on sephadex or ultrogel columns into four separate classes: (a) complex (>200,000 daltons); (b) α (70-90,000 d); (c) β (25-50,000 d); and (d) γ (10-20,000 d). The amount of activity in a supernatant due to each class varies but is approximately: Cx-5 to 20%, α-40 to 60%, β-20 to 40%, and γ-0 to 10%. These classes differ one from another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. Each class, except γ, can be further separated into subclasses by ion exchange chromatography and polyacrylamide gel electrophoresis. Alpha class can be separated on DEAE into the three subclasses, termed, α1 (rf 0.25), α2 (rf 0.37), and α3 (rf 0.50). Alpha2 subclasses can be further separated on phosphocellulose at pH 6.6 into α2a (rf 0.37) and α2b (rf 0.30). However, α2 contains additional subclasses, which were resolved on PC columns at pH 5.5. Beta class activity can be resolved by DEAE and PAGE into two subclasses, termed β1 (rf 0.28) and β2 (rf 0.49). Gamma class activity was not studied, because of its instability. The complex class of LT activity is a macromolecular aggregate greater than 200,000 daltons which appears to contain smaller MW LT class(es). This study demonstrates that materials with LT activity in supernatants from PHA activated human lymphocytes in vitro are very heterogeneous. © 1978.

Human lymphoid cells, when stimulated in vitro with Con-A† or PHA-P, release a group of cytolytically active molecules, termed lymphotoxins (LT). These can be physically separated into various classes and sub-classes by means of molecular sieving columns and ion exchange resins. These studies examine whether these molecules contain carbohydrate. The use of lectin-Sepharose affinity chromatography reveals that human LT molecules in the unfractionated supernatant, and in separated classes and sub-classes, contain at least six different heterogeneously distributed saccharide units. We find that some of the charge heterogeneity associated with α2 sub-class of these molecules appears to be due to the presence of sialic acid, since neuraminidase treatment alters the charge on approximately 20% of this material by ion-exchange analysis. However, this enzyme treatment has no effect on the antigenicity of these molecules. In addition, the carbohydrate moieties do not appear to be necessary for the in vitro lytic activity measured on L-929 cells, since removal with neuraminidase or the presence of saccharides in the lytic reaction has no effect. These studies indicate that LT molecules are glycoproteins. © 1979.

The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E. coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. The fpg gene is different from the xth, nth, and nfo genes of E. coli, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity. The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved beta elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coli DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coli exonuclease III. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.

A new rapid assay for phosphatidylserine decarboxylase, which is sensitive in the nanomolar range, is described. Synthesis of radiolabeled phosphatidylserine for use as a substrate is not required, since the assay, unlike previous ones, is based on the detection of CO(2) liberated from unlabeled phosphatidylserine. The assay employs a gas chromatographic procedure for the analysis of methane formed by catalytic conversion of the CO(2) produced as a product of the enzymatic reaction.