Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Characterization of recombinant yeast exo-beta-1,3-glucanase (Exg 1p) expressed in Escherichia coli cells

Yeast exo-beta-1,3-glucanase gene (EXG1) was expressed in Escherichia coli and the recombinant enzyme (Exg1p) was characterized. The recombinant Exglp had an apparent molecular mass of 45 kDa by SDS-PAGE and the enzyme has a broad specificity for beta-1,3-linkages as well as beta-1,6-linkages, and also for other beta-glucosidic linked substrates, such as cellobiose and pNPG. Kinetic analyses indicate that the enzyme prefers small substrates such as laminaribiose, gentiobiose, and pNPG rather than polysaccharide substrates, such as laminaran or pustulan. With a high concentration of laminaribiose, the enzyme catalyzed transglucosidation forming laminarioligosaccharides. The enzyme was strongly inhibited with high concentrations of laminaran.     

Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.     

Improved laminaribiose phosphorylase production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in a bioreactor: A comparative study of different cultivation methods

Laminaribiose phosphorylase (EC 2.4.1.31) catalyzes a reversible phosphorolysis reaction in which laminaribiose, a very high value sugar is produced. This enzyme is not being produced commercially therefore, to realize the most effective method for producing laminaribiose phosphorylase and obtaining as much activity units as possible per liter of culture, different cultivation methods of Euglena gracilis were compared. Heterotrophic and mixotrophic cultivations of Euglena gracilis in two different pHs, in flask and bioreactor were performed. The reverse phosphorolysis activity of laminaribiose phosphorylase produced under different cultivation methods was measured. The heterotrophic approach showed to be the more effective cultivation method as 47.6 IU/L was obtained compared to 27 IU/L in the mixotrophic one. The heterotrophic cultivation then was further investigated under two different pH values of the culture media. The culture at pH 6.8 resulted in 7.94 IU/L/day whereas only 4.06 was obtained for the culture at pH 4. Cultivation in a bioreactor resulted in a distinctive amount of 191.5 IU/L and an activity yield of 9.7 IU/g glucose compared to 5.4 in flask cultivation. Heterotrophic cultivation of Euglena gracilis in a bioreactor containing a culture media at pH 6.8 and controlled operation conditions showed enhanced laminaribiose phosphorylase activity production per liter and day of cultivation.     

Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.     

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBP_Bl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k _cat/K _m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBP_Bl showed similar k _cat/K _m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.     

Chitosan-based hybrid immobilization in bienzymatic reactions and its application to the production of laminaribiose

A hybrid-immobilization method was developed to improve the long-term stability of laminaribiose phosphorylase immobilized on epoxy supports Sepabeads EC-EP/S. Entrapment in chitosan retained all of the enzyme activity depending on the amount of entrapped solid materials and increased half-life by a factor of 10–94.4 h. No enzyme activity loss was determined during 12 times reuse. The immobilization method is also applicable to sucrose phosphorylase immobilized on Sepabeads EC-EP/S. Up to 31.9 g/L laminaribiose were produced in bienzymatic batch experiments with reaction-integrated product separation by adsorption on zeolites.

     

Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations

The production of Leuconostoc mesenteroides sucrose phosphorylase has been studied in 10- and 20-L batch fermentations. A fermentation medium was devised combining rapid growth, high cell yield, and high enzyme levels. Overall fermentation dynamics and enzyme fermentation patterns are elucidated here in detail. Sucrose is phosphorolyzed into fructose and glucose-1-phosphate (G-1-P) with G-1-P preferentially utilized (thus saving ATP). Subsequently, fructose is gradually metabolized and is also converted to mannitol. Invertase activity is absent. Sucrose phosphorylase is formed transitorily with peak levels toward the end of active growth; a sharp decline in enzyme activity occurs upon further fermentation. The moment of cell (enzyme) harvest is thus critical in view of obtaining active cell or enzyme preparations for sucrose phosphorolysis. Microaerophilic and strictly anaerobic fermentations displayed no appreciable difference in sucrose phosphorylase formation profile. The enzyme is intracellularly located. It is constitutively formed in the absence of sucrose, contrary to that of Pseudomonas species; other disaccharide phosphorylases are not formed.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Isolation and characterization of two types of β-1,3-glucanases from the common sea hare Aplysia kurodai

Two types of β-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward β-1,3-glucans with a few β-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched β-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan β-1,3-glucanases.     

Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.     

Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).     

The structure of a GH149 β-(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-β-(1 → 3)-glucose (laminaribiose) phosphorylase

Glycoside phosphorylases (GPs) with specificity for β-(1 → 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 → 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.     

A homogeneous nucleic acid hybridization assay based on strand displacement.

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement.     

Identification,cloning, and characterization of β-glucosidase from <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.     

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycoplasma contamination alters 2'-deoxyadenosine metabolism in deoxycoformycin-treated mouse leukemia cells

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.     

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.