Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.     

Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galbeta1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galbeta1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of beta1,3-glucuronosyltransferases.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, beta-1,4-galactosylated and alpha-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (-2-1-+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites -4 and -3 of Chit42, and at the subsite -4 of Chit33. Fucose units can be accepted as a branch at the putative -3 and -4 sites of Chit33 and as a branch point at -3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.     

Anomeric specificity of glucose uptake systems in Lactococcus cremoris, Escherichia coli, and Saccharomyces cerevisiae: mechanism, kinetics, and implications

The mechanism and kinetics of the glucose uptake systems of three representative microorganisms are studied during cultivation in a chemostat. The three microorganisms are Lactococcus cremoris, Escherichia coli, and Saccharomyces cervisiae. Two models describing respectively competitive and independent uptake of the two glucose anomers are tested on experimental data where alpha- and beta-glucose are determined by flow injection analysis after pulse addition of the pure anomers to a chemostat. The very accurate experimental results are used to give a convincingly clear model discrimination for all three microorganisms. The uptake of glucose by S. cervisiae occurs by a competitive mechanism with preference for alpha-glucose (K(alpha) = 32 mg/L and K(beta) = 48 mg/L). Surprisingly, the glucose uptake by the two bacteria is shown to be mediated by anomer specific transport systems with no competitive inhibition from the other glucose anomer. This novel finding has not been described in the literature on the phosphotransferase system. In L. cremoris the relative uptake rates of the glucose anomers match the equilibrium composition exactly (36% alpha-glucose). In E. coli the relative uptake rate of alpha-glucose at glucose unlimited growth is 26%, which means preference for beta-glucose. However, the saturation constants of the two sites in E. coli are K(alpha) = 2 mg/L and K(alpha) = 15 mg/L, and a preference for alpha-glucose is exhibited at very low glucose concentrations. The results are of considerable improtance in relation to enzyme based on-line measurements during fermentations as well as to the modeling of glucose limited growth and product formation.     

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Crystal structure of Vibrionaceae Photobacterium sp. JT-ISH-224 alpha2,6-sialyltransferase in a ternary complex with donor product CMP and acceptor substrate lactose: catalytic mechanism and substrate recognition

Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose. Delta16psp26ST has three structural domains. Domain 1 belongs to the immunoglobulin-like beta-sandwich fold, and domains 2 and 3 form the glycosyltransferase-B structure. The CMP and lactose were bound in the deep cleft between domains 2 and 3. In the structure, only Asp232 was within hydrogen-binding distance of the acceptor O6 carbon of the galactose residue in lactose, and His405 was within hydrogen-binding distance of the phosphate oxygen of CMP. Mutation of these residues greatly decreased the activity of the enzyme. These structural and mutational results indicated that Asp232 might act as a catalytic base for deprotonation of the acceptor substrate, and His405 might act as a catalytic acid for protonation of the donor substrate. These findings are consistent with an in-line-displacement reaction mechanism in which Delta16psp26ST catalyzes the inverting transfer reaction. Unlike the case with multifunctional sialyltransferase (Delta24PmST1) complexed with CMP and lactose, the crystal structure of which was recently reported, the alpha2,6 reaction specificity of Delta16psp26ST is likely to be determined by His123.     

Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins

The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass ∼41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% α-helix, 26.5% β-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Cα subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl₂ and 50 μM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein α crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.     

Staphylorchis cymatodes (Gorgoderidae: Anaporrhutinae) from carcharhiniform, orectolobiform and myliobatiform elasmobranchs of Australasia: Low host specificity, wide distribution and morphological plasticity

Anaporrhutine gorgoderids (Digenea: Gorgoderidae: Anaporrhutinae) found in the body cavity of six species of elasmobranchs from the orders Carcharhiniformes, Myliobatiformes and Orectolobiformes from Australian waters were found to belong to the genus Staphylorchis. Although these specimens were morphologically variable, sequences of ITS2 and 28S ribosomal DNA from specimens from three host families and two host orders were identical. Based on morphological and molecular data these specimens were identified as the type-species of the genus, Staphylorchis cymatodes. New measurements are provided for S. cymatodes, and for the first time genetic data are presented for this species. In addition to providing new morphological and molecular data for S. cymatodes, the previously described species S. gigas, S. parisi and S. scoliodonii, are here synonymised with S. cymatodes. This implies that S. cymatodes, as conceived here, has remarkably low host-specificity, being recorded from eight elasmobranch species from four families and three orders, has a wide geographical distribution in the Indo-west Pacific from off India, in the Bay of Bengal, to Moreton Bay in the Coral Sea, and is morphologically plastic, with body size, size of specific organs and body shape differing dramatically between specimens from different host species. The genus Staphylorchis now contains only two valid species, S. cymatodes and S. pacifica.     

Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications

In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non- cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed – that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fold recognition,homology modeling,docking simulations,kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii

ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.     

Conformation of peptides constructed from achiral amino acid residues Aib and DeltaZPhe: computational study of the effect of L/D- Leu at terminal positions

Conformational studies of the peptides constructed from achiral amino acid residues Aib and Delta(Z)Phe (I) Ac-Aib-Delta(Z)Phe-NHMe (II), and Ac-(Aib-Delta(Z)Phe)(3)-NHMe; peptides III-VI having L-Leu or D-Leu at either the N- or the C-terminal position and of peptides VII-X having Leu residues in different enantiomeric combinations at both the N- and the C-terminal positions in peptide II have been studied to design the peptide with the required helical sense. Peptide II, as expected, adopts degenerate left- and right-handed helical structures. It has been shown that the peptides IV and VI having D-Leu at either the N or the C terminus can be realized in the right-handed helical structure with the phi,psi values of -20 degrees and -60 degrees for the Aib/Delta(Z)Phe residues. L-Leu and D- Leu at both the terminals in peptides VII and VIII, respectively, have hardly any effect as both the left- and the right-handed structures are found to be degenerate. Peptides III and IX can be realized in right- and left-handed helical structures, respectively, in solvents of low polarity whereas peptides V and X are predicted to be in the right-handed helical structures stabilized by carbonyl-carbonyl interactions without the formation of hydrogen bonds. The conformational states with the phi,psi values of 0 degrees and -85 degrees in peptide V are characterized by rise per residue of 2.03 A, rotation per residue of 117.5 degrees , and 3.06 residues per turn. In all peptides having Leu residue at the N terminus, the methyl moiety of the acetyl group is involved in the CH/pi interactions with the Cepsilon--Cdelta edge of the aromatic ring of Delta(Z)Phe (3) and the amino group NH of Delta(Z)Phe is involved in the NH/pi interactions with its own aromatic ring. The CH(3) groups of the Aib residues are also involved in CH/pi interactions with the i + 1th and i + 3th Delta(Z)Phe's aromatic side chains.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.     

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning,expression and substrate specificity analysis

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3′- and 5′-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.     

Investigations on the glucocorticoid-binding protein from rat thymocytes: II. Stability,kinetics and specificity of binding of steroids

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1. The glucocorticoid-binding protein from rat thymocytes was shown to be stabilized by 40% (v/v) glycerol at −5°.