Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer

When the substrate isocitrate-Mn2+ is present, the fluorescent nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with pig heart NADP+-specific isocitrate dehydrogenase at the coenzyme binding site on one subunit of the dimeric enzyme [Bailey, J. M., & Colman, R. F. (1985) Biochemistry 24, 5367-5377]. The modified enzyme, which retains partial activity, binds 1 mol of NADPH or 1 mol of the coenzyme analogue, reduced thionicotinamide adenine dinucleotide phosphate (TNADPH), per dimer. TNADPH quenches the fluorescence of enzyme-bound 2-BDB-T epsilon A-2',5'-DP with an efficiency of energy transfer of 9.8%. From this value and the spectral properties of the donor and acceptor chromophores, a distance of 32 A was calculated as the average distance between coenzyme sites on the two subunits. Isocitrate dehydrogenase activity requires a divalent metal ion, such as Mn2+, Co2+, or Ni2+. Co2+ and Ni2+ have absorption spectra that overlap the emission spectra of enzyme-bound 2-BDB-T epsilon A-2',5'-DP. In the presence of isocitrate, each of these two metal ions quenches the fluorescence of the enzyme-bound reagent with an efficiency of energy transfer of 28-29%. From this value and the spectral characteristics of the energy donor and acceptors, an average distance of 8.0 A was estimated between the metal-isocitrate site and the labeled coenzyme site. These distances have provided constraints in formulating a model of the spatial arrangement of active-site ligands on isocitrate dehydrogenase.     

Active site ligand stabilization of quaternary structures of glutamine synthetase from Escherichia coli

Auto-inactivated EScherichia coli glutamine synthetase contains 1 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2 eq of Mn2+ tightly bound to the active site of each subunit of the dodecameric enzyme (Maurizi, M. R., and Ginsburg, A. (1982) J. Biol. Chem. 257, 4271-4278). Complete dissociation and unfolding in 6 M guanidine HCl at pH 7.2 and 37 degrees C requires greater than 4 h for the auto-inactivated enzyme complex (less than 1 min for uncomplexed enzyme). Release of ligands and dissociation and unfolding of the protein occur in parallel but follow non-first order kinetics, suggesting stable intermediates and multiple pathways for the dissociation reactions. Treatment of Partially inactivated glutamine synthetase (2-6 autoinactivated subunits/dodecamer) with EDTA and dithiobisnitrobenzoic acid at pH 8 modifies approximately 2 of the 4 sulfhydryl groups of unliganded subunits and causes dissociation of the enzyme to stable oligomeric intermediates with 4, 6, 8, and 10 subunits, containing equal numbers of uncomplexed subunits and autoinactivated subunits. With greater than 70% inactivated enzyme, no dissociation occurs under these conditions. Electron micrographs of oligomers, presented in the appendix (Haschemeyer, R. H., Wall, J. S., Hainfeld, J., and Maurizi, M. R., (1982) J. Biol. Chem. 257, 7252-7253) suggest that dissociation of partially liganded dodecamers occurs by cleavage of intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit interactions are stabilized by active site ligand binding. Isolated tetramers (Mr = 200,000; s20,w = 9.5 S) retain sufficient native structure to express significant enzymatic activity; tetramers reassociate to dodecamers and show a 5-fold increase in activity upon removal of the thionitrobenzoate groups with 2-mercaptoethanol. Thus, the tight binding of ligands to the subunit active site strengthens both intra- and inter-subunit bonding domains in dodecameric glutamine synthetase.     

The apparently symmetrical hexagonal bilayer hemoglobin from Lumbricus terrestris has a large dipole moment

The giant approximately 3.6 MDa hexagonal bilayer hemoglobin (HBL Hb) from Lumbricus terrestris consists of 12 213-kDa dodecamers of four globin chains ([b + a + c]3[d]3) tethered to a central scaffold of approximately 36 non-globin, linker subunits L1-L4 (24-32 kDa). Three-dimensional reconstructions obtained by electron cryomicroscopy showed it to have a D6 point-group symmetry, with the two layers rotated approximately 16 degrees relative to each other. Measurement of the dielectric constants of the Hb and the dodecamer over the frequency range 5-100 kHz indicated relaxation frequencies occurring at 20-40 and 300 kHz, respectively, substantially lower than the 700-800 kHz in HbA. The dipole moments calculated using Oncley's equation were 17,300 +/- 2300 D and 1400 D for the Hb and dodecamer, respectively. The approximately threefold higher dipole moment of the dodecamer relative to HbA is consistent with an asymmetric shape in solution suggested by small-angle X-ray scattering. Although a two-term Debye equation and a prolate ellipsoid of revolution model provided a good fit to the experimental dielectric dispersion of the dodecamer, a three-term Debye equation based on an oblate ellipsoid of revolution model was required to fit the asymmetric dielectric dispersion curve of the Hb: the required additional term may represent either an induced dipole moment or a substructure which rotates independently of the main permanent dipole component of the Hb. The D6 point-group symmetry implies that the dipole moments of the dodecamers cancel out. Thus, in addition to a possible contribution from fluctuations of the proton distribution, the large dipole moment of the Hb may be due to an asymmetric distribution of the heterogeneous linker subunits.     

Allosteric control in Limulus polyphemus hemocyanin: functional relevance of interactions between hexamers

Hemocyanin of the horseshoe crab Limulus polyphemus is composed of 48 oxygen-binding subunits, which are arranged in eight hexameric building blocks. Allosteric interactions in this oligomeric protein have been examined by measurement of high-precision oxygen-equilibrium curves, using an automated Imai cell. Several models were compared in numerical analysis of the data. A number of conclusions can be drawn with confidence. (1) Oxygen binding by Limulus hemocyanin cannot satisfactorily be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118] for allosteric transitions with either the hexamer or dodecamer as the allosteric unit. (2) Of the models tested, the data sets can be best described by an extended MWC model that allows for an equilibrium, within the 48-subunit ensemble, between cooperative hexamers and cooperative dodecamers. The model invokes T and R states for both hexamers (T6 and R6) and dodecamers (T12 and R12). Allosteric effectors modulate oxygen affinity and cooperativity by affecting the R to T equilibria within hexamers and dodecamers and by shifting the equilibria between hexamers and dodecamers. (3) The fitted model parameters show that under most conditions the intersubunit contacts within T-state hexamers are more constrained than those within T-state dodecamers. (4) The oxygen affinities of the hexameric and dodecameric R states are the same, but under all conditions examined the conformation of the fully oxygenated molecule is that of the dodecameric R state. (5) Between pH 7.4 and pH 8.5 the dodecameric T state has a higher affinity for oxygen than the hexameric T state, allowing for "T-state cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 x 10(-2) h(-1) at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 M of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s(-1) and 7.99 s(-1) in the presence of 0.2 M and 1.0 M of KCl, respectively.     

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by L?tscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.     

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Role of N and C-terminal tails in DNA binding and assembly in Dps: structural studies of Mycobacterium smegmatis Dps deletion mutants

Mycobacterium smegmatis Dps degrades spontaneously into a species in which 16 C-terminal residues are cleaved away. A second species, in which all 26 residues constituting the tail were deleted, was cloned, expressed and purified. The first did not bind DNA but formed dodecamers like the native protein, while the second did not bind to DNA and failed to assemble into dodecamers, indicating a role in assembly also for the tail. In the crystal structure of the species without the entire C-terminal tail the molecule has an unusual open decameric structure resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. A Dps dodecamer could assemble with a dimer or one of two trimers (trimer-A and trimer-B) as intermediate. Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of the surface area buried on trimerization indicates that association within trimer-B is weak. It weakens further when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail. A species with nine N-terminal residues deleted formed trimers but not dodecamers in solution, unlike wild-type M. smegmatis Dps, under the same conditions. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighboring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.     

Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence

Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.     

Glycosphingolipids in Plasmodium falciparum. Presence of an active glucosylceramide synthase.

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[(14)C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [(14)C]palmitic acid and [(14)C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.     

Hemocyanin from the Australian freshwater crayfish Cherax destructor. Electron microscopy of native and reassembled molecules.

Examination and measurement of electron micrographs of negatively stained hemocyanin molecules from Cherax destructor show that the predominant aggregated forms, the 16S and 24S components, are typical structures for arthropod hexamers and dodecamers, respectively. In Cherax hemocyanin the hexamers are formed from the monomeric (Mr congruent to 75,000) subunits, M1 and M2, while the dodecamers contain in addition a dimeric (Mr congruent to 150,000) subunit, M3'. Studies of the composition of solutions of the subunits M1 and m2 to which calcium ions have been added at pH 7.8 show that, under these conditions, reassembly occurs to particles indistinguishable from native hexamers. It is noteworthy that dodecamers are not seen since this confirms the previous suggestion that incorporation of the dimeric subunit in the assembly process is necessary for their formation. The results obtained from Cherax hemocyanin are related to those of previous structural studies of arthropod hemocyanins. In particular, the possible controlling role of certain specific subunits in arthropod hemocyanin oligomers containing more than one kind of subunit is illustrated with a model for the Cherax dodecamer, in which the dimeric subunit is shared between the two halves of the molecule.     

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.     

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L ‐lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI‐MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L ‐lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer‐specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L ‐lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structural comparison of the B-DNA dodecamers d(CGCGTTAACGCG) and d(CGCGAATTCGCG) with T2A2 and A2T2 tracts.

The x-ray structure of the deoxy oligonucleotide dodecamer d(CGCGTTAACGCG) recently determined in our laboratory shows that the helical parameters of the central TTAA segment are significantly different compared to the central AATT in d(CGCGAATTCGCG). The roll in the central TA step of the T2A2 dodecamer opens towards the minor groove while the AT step of the A2T2 dodecamer opens towards the major groove. Also, the roll angles at the steps 4 and 8 (GT and AC in T2A2) and (GA and TC in A2T2) are in opposite directions. The high cup and helical twist angles at the central base-pair of T2A2 decreases the base stacking interactions compared to A2T2. Tilt angles within the tetranucleotide segments TTAA and AATT have opposite signs. In spite of the local differences caused by the sequence inversion (TTAA----AATT), the two dodecamers exhibit similar overall bending. The top third is more bent than the bottom third relative to the central segment. This asymmetric bending in the two dodecamers is mainly due to crystal packing interactions.     

Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD<Superscript>+</Superscript>-Reducing Hydrogenase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> H16

Inclusion of an oligomeric enzyme, NAD⁺-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD⁺/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 782–789.Original Russian Text Copyright © 2005 by Tikhonova, Kurkin, Klyachko, Popov.     

Intersubunit fluorescence energy transfer in human factor VIII

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.     

Acceptor requirements for GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase activity solubilized from pea epicotyl membranes.

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[14C]fucose as donor. Native seed XG (Mr greater than 10(6) Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P4. Specific activities (Bq/microgram carbohydrate) of fragments with Mr ranging from 10(6) to 10(4) Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltransferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB3H4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.     

Structure and function of the hemocyanin from a semi-terrestrial crab,Ocypode quadrata

Summary Structural and functional studies of the hemocyanin of the semi-terrestrial ghost crab,Ocypode quadrata, demonstrate a variety of differences in comparison to the hemocyanin of aquatic crabs. These differences may be related to the terrestrial habit of this crab. Unlike aquatic crabs, the major (56%) blood component is the hexamer; the remaining 44% is dodecamer. The hexamers and dodecamers are not in rapid equilibrium. Electrophoretic analysis of the subunit composition indicates three major components referred to as 1, 3, and 4, and one minor component referred to as component 2. These components, although electrophoretically distinct, are alike immunologically. Components 1 and 2 are essentially absent from purified hexamers, whereas they compose 1/3 of the subunits in dodecamers. These results suggest that they are involved in linking hexamers to form dodecamers, and that two, rather than one, subunits are involved in the bridge. Oxygen-binding measurements show a higher degree of cooperativity, and a much reduced allosteric effect ofl-lactate on the dialyzed hemocyanin as compared to the hemocyanin of aquatic crabs. Exercise rapidly, induces a large drop in hemolymph pH (0.5 units) and a corresponding increase in lactate concentrations (to 10 mM).