Purification and characterization of myo-inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg²⁺ and Mn²⁺. The K_m value for myo-inositol hexaphosphate has been found to be 0.41 × 10^?4m and V is 90.0 nmol of P_i transferred per milligram of protein per 20 min. K_m for ADP is 0.88 × 10^-4m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).     

The binding of physiologically significant protons to 2,3-diphosphoglycerate

The intrinsic pKa values of protons of 2,3-diphosphoglycerate (DPG) which titrate in the physiologically significant range (i.e., pH 6.8-7.8) have been determined by measuring the changes in chemical shifts of the two phosphate resonances of the molecule as a function of pH using 31P-NMR spectroscopy. While conventional acid-base titration techniques resulted in apparent pKa values of 6.39 and 7.39 for these protons, analysis of the 31P-NMR data by statistical thermodynamic methods yielded intrinsic pKa values of 6.99 +/- 0.07 and 7.28 +/- 0.04, for protons associated with the phosphates bound to carbon-3 (C-3) and carbon-2 (C-2), respectively, with an interaction energy of +0.77 kcal/mol. The free energies for the binding of protons to the C-2 and C-3 phosphates and the associated interaction energies determined by 31P-NMR were used to generate a theoretical titration curve which was essentially identical to that determined by conventional acid-base titration. The physiological implications of this work are briefly discussed.     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

The conformation of abscisic acid by n.m.r. and a revision of the proposed mechanism for cyclization during its biosynthesis.

The n.m.r. spectrum of abscisic acid (ABA) formed from [1,2-13C2]acetate by the fungus Cercospora rosicola shows 13C-13C coupling between C-6' (41.7 p.p.m.; 36 Hz) and the downfield 6'-methyl group (6'-Me) (24.3 p.p.m, 36 Hz). This 6'-Me, therefore, is derived from C-3' of mevalonate [Bennett, Norman & Maier (1981) Phytochemistry 20, 2343-2344]. An i.n.e.p.t. (insensitive nuclei enhanced by polarization transfer) pulse sequence demonstrated that the downfield 13C signal is produced by the 6'-Me that gives rise to the upfield 1H 6'-Me signal (23.1 d). The absolute configuration of this, the equatorial 6'-Me group, was determined as 6'-pro-R by decoupling and n.O.e. (nuclear-Overhauser-enhancement) experiments at 300 MHz using ABA, ABA in which the axial 6'-pro-S 5'-hydrogen atom had been exchanged with 2H in NaO2H and the 1',4'-cis- and 1',4'-trans-diols formed from these samples. The configuration at C-1' and at C-6' are now compatible with a chair-folded intermediate during cyclization, as proposed for beta- and epsilon-rings of carotenoids. ABA in solution exists, as in the crystalline form, with the ring in a pseudo-chair conformation. The side chain is axial and the C-3 Me and the C-5 hydrogen atoms are predominantly cis(Z).     

Synthesis and structural study of 5-nitro-6-(penta-O-acetylpentitol-1-yl)norbornenes

Uncatalyzed reaction between cyclopentadiene and (E)-3,4,5,6,7-pentaacetoxy-1-nitrohept-1-enes having the -manno, -galacto, and -gluco configurations at C-3—C-7 led, in each case, to the four stereoisomeric 5-nitro-6-(1,2,3,4,5-penta-O-acetylpentitol-1-yl)bicyclo[2.2.1]hept-2-enes. Face selectivity is discussed in terms of the sugar-chain configuration. The structures assigned the adducts are based on their n.m.r. spectra, and, in the case of the -manno compounds, on X-ray data. Also described are the ¹³C-n.m.r. spectra of the starting nitroalkenes. The crystal structures of (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-exo-nitrobicyclo[2.2.1]hept-2-en-6-endo-yl- -manno-pentitol (3a) and (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-endo-nitrobicyclo[2.2.1]hept-2-en-6-exo-yl- -manno-pentitol (5a) were determined from three-dimensional, X-ray data. Crystals of 3a are monoclinic, space group P2₁, with two molecules in a cell of dimensions a = 9.054(3), b = 15.580(11), c = 10.138(4) Å, β = 116.27(3)°. The structure was refined to an R-factor of 0.050 on the basis of 1485 observations. Crystals of 5a are triclinic, space group P1, with one molecule in a cell of dimensions a = 8.680(4), b = 9.760(4), c = 8.695(7) Å, = 98.69(5), β = 103.13(5), γ = 112.09(3)°. The structure was refined to an R-factor of 0.074 based on 970 observations.     

Physical studies on phosphonium phosphatidylcholine. A unique [31P]phosphorus nuclear-magnetic-resonance probe for model and biological membranes.

1. Distearoyl phosphatidylcholine and the phosphonium analogue, in which the nitrogen atom is replaced by phosphorus, show similar gel-liquid crystalline transition temperatures as detected by differential scanning calorimetry. 2. The temperature-dependence of the 31P n.m.r. (nuclear-magnetic-resonance) linewidths of the phosphate resonances of sonicated vesicles of distearoyl phosphatidylcholine and the phosphonium analogue are similar. Below the phase-transition temperature the linewidths decrease as the temperature is raised. Above the phase-transition temperature the phosphate resonances are relatively temperature-independent. The phosphonium 31P n.m.r. signal exhibits the same pattern of temperature-dependence. 3. The 31P n.m.r. phosphonium resonance is sensitive to the paramagnetic shift reagent, K3Fe(CN)6. Use of K3Fe(CN)6, together with Nd(NO3)3, enabled the determination of the trans-bilayer distribution of egg-yolk phosphatidylcholine and its phosphonium analogue in co-sonicated vesicles. Both are distributed comparably across the bilayer of the vesicles. 4. The phosphonium 31P n.m.r. signal is much sharper than the corresponding phosphate resonance in both sonicated and unsonicated dispersions of the phosphatidylcholine analogue. 5. The properties of the phosphonium analogue of phosphatidylcholine are discussed in terms of its suitability as a probe of membrane structure.     

Dietary myo-inositol hexaphosphate prevents dystrophic calcifications in soft tissues: a pilot study in Wistar rats

Myo-inositol hexaphosphate (InsP6) is an abundant component of plant seeds. It is also found in significant levels in blood and mammalian tissues, but they are totally dependent on their dietary intake. In the present paper, we describe studies on the effect of InsP6 on a model of dystrophic calcification, which was chemically induced by subcutaneous injection of a 0.1% KMnO4 solution. Male Wistar rats were randomly divided into four groups for treatment over 31 days. A: animals consuming a purified diet in which InsP6 was absent but to which 1% of InsP6 (as sodium salt) was added. In this group, the InsP6 plasma levels (0.393 +/- 0.013 microM) were similar to those observed in rats consuming a standard diet. B: animals consuming only the purified diet in which InsP6 was absent. In this case the InsP6 plasma levels decreased (0.026 +/- 0.006 microM); C: animals consuming the same purified diet as group B but received daily subcutaneous injections of 50 microg kg(-1) etidronate during the last 14 days. In this case the InsP6 plasma levels were also very low (0.025 +/- 0.007 microM); D: animals consuming the same diet as group B but a 6% of carob germ (InsP6 rich product) was added. The InsP6 plasma levels (0.363 +/- 0.035 microM) were also similar to those observed in rats consuming a standard diet. After 21 days plaque formation was induced. Calcification plaques were allowed to proceed for 10 days, after which the plaque material present was excised, dried and weighed. It was found that the presence of myo-inositol hexaphosphate (phytate) in plasma at normal concentrations (0.3-0.4 microM) clearly inhibited the development of dystrophic calcifications in soft tissues. These results demonstrates that myo-inositol hexaphosphate acts as an inhibitor of calcium salt crystallization.     

Active-site serine phosphate and histidine residues of phosphoglucomutase: pH titration studies monitored by 1H and 31P NMR spectroscopy

1H and 31P NMR pH titrations were conducted to monitor changes in the environment and protonation state of the histidine residues and phosphoserine group of rabbit muscle phosphoglucomutase on binding of metal ions at the activating site and of substrate (glucose phosphate) at the catalytic site. Imidazole C epsilon-H signals from 8 of the 10 histidines present in the free enzyme were observed in 1H NMR spectra obtained by a spin-echo pulse sequence at 470 MHz; their pH (uncorrected pH meter reading of a 2H2O solution measured with a glass electrode standardized with H2O buffer) titration properties (in 99% 2H2O) were determined. Three of these histidine residues, which have pKa values ranging from 6.5 to 7.9, exhibited an atypical pH-dependent perturbation of their chemical shifts with a pHmid of 5.8 and a Hill coefficient of about 2. Since none of the observed histidines has a pKa near 5.8, it appears that these three histidines interact with a cluster consisting of two or more groups which become protonated cooperatively at this pH. Binding of Cd2+ at the activating site of the enzyme abolishes the pH-dependent transition of these histidines; hence, the putative anion cluster may constitute the metal ion binding site, or part of it. Two separate 31P NMR peaks from phosphoserine-116 of the phosphoenzyme were observed between pH 6 and 9. Apparently, the metal-free enzyme exists as a pH-dependent mixture of conformers that provide two different environments, I and II, for the enzymic phosphate group; the transition of the phosphate group between these two environments is slow on the NMR time scale.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear relaxation studies on human methemoglobin. Observation of cooperativity and alkaline Bohr effect with inositol hexaphosphate.

Ehanced spin-lattice relaxation (1/t1) of water protons induced by the heme iron of human aquomethemoglobin is exchanged-limited (koff = 1.4 times 10-4 per s at 30 degrees, H+ =7.5 Cal per mol) as indicated by the temperature and frequencey dependencies. A comparison of deuteron and proton relaxation rates revealed an order of magnitude primary isotope effect and a small inverse secondary isotope effect on the escape rate of protons from the heme iron into bulk water establishing the exchange of protons and not the exchange of the entire water molecule to be the chemical mechanism of the entire water molecule to be the chemical mechanism of the exchange process. With fluoromethemoglobin, the relaxation rate is in the fast exchange region. The results can be understood in terms of a water molecule interacting with the heme iron at an iron to proton distance less than 3.4 A in aquomethemoglobin and a single proton at a distance of 4.11 A assignable to the NH proton of the distal histidine imidazole group in fluoromethemoglobin. The relaxation rates are pH-dependent and normal titrations with Hill coefficients n = 1 are observed. The pKa is less than or equal to 6. 7 with aquomethemoglobin and 8.5 with fluoromethemoglobin at 30 degrees C. The binding of inositol hexaphosphate in stoichiometric amounts has no significant effect on the magnetic susceptibility of solutions of aquomethemoglobin and fluoromethemoglobin, but in the former case it increases koff to 3.8 times 10-4 per s by lowering the H+ barrier to 6.8 Cal per mol. In fluoromethemoglobin, inositol hexaphosphate decreases the iron to distal histidine NH distance by 0.17 A and the electron relaxation time taus by 10% as determined by the frequency dependence of 1/T1. In the aquomethemoglobin system, inositol hexaphosphate induces a Bohr effect, raising the pKa of the ionization responsible for the 1/T1 titration to 7.2, and induces cooperativity in the pH titration with a Hill coeffocoemt n = 2.8 plus or minus 0.1. With fluoromethemoglobin, the normal pH titration curve is unaffected by inositol hexaphosphate (n approximately equal to 1). Further, relaxivity titrations with varying amounts of azide and fluoride near neutral pH show normal behavior (n = 1) with and without inositol hexaphosphate. These results indicated that inositol hexaphosphate alters the quaternary structure of methemoglobin to the deoxy conformation without causing a change in the spin state of the heme iron...     

Uridine diphosphate galactose 4-epimerase. pH dependence of the reduction of NAD+ by a substrate analog

Neutron activation analysis of UDP-galactose 4-epimerase from Escherichia coli for 53 metals shows that the enzyme does not contain any of these metals at significant levels. The substrate analog P1-5'-uridine-P2-glucose-6-yl pyrophosphate (UGP), a structural isomer of UDP-glucose with the sugar linked to UDP through the C-6 hydroxyl group, is an inactivator that irreversibly reduces epimerase.NAD+ to epimerase.NADH. The pH dependence of kobs reveals the essential involvement of an acidic group, kinetically measured pKa = 5.48 +/- 0.08, in unprotonated form and two weakly acidic or basic groups, apparent pKa values of 10.03 +/- 0.43, in protonated forms. Measurements of kobs as a function of [UGP] show that it is given by kobs = k[UGP]/(K + [UGP]) at a given pH, where K = 0.19 +/- 0.04 mM throughout the pH range 4.8-10.4. The pH-dependent first order rate constants range from 0.28 to 1.94 s-1, with the maximum value at pH 7.6 and decreasing at acidic and basic pH values. Reaction of [glucose-1-2H]UGP proceeds with kinetic isotope effects of 5.0, 2.1, 2.0, 1.9, and 3.5 at pH values 5.0, 6.2, 7.6, 9.0, and 10.0, respectively. Therefore, hydride transfer becomes rate-limiting at pH extremes but is not limiting at neutral pH, although deuteride transfer is significantly limiting at all pH values. The isotope effects facilitated correction of the kinetic pK values to the thermodynamic values 6.1-6.2 on the acid side and 9.0-9.6 on the alkaline side. We postulate that the group with pK1 = 5.5 (6.1-6.2 corrected) functions as an enzymic general base that abstracts the glucosyl C-1 hydroxyl proton in concert with transfer of the C-1 hydrogen and two electrons to NAD+. The pH dependence on the alkaline side may be related to the uridine nucleotide-dependent conformational transition that is an essential step in the reduction of epimerase.NAD+ to epimerase.NADH by sugars.     

Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.     

N.m.r. studies of red cells

Recent n.m.r. studies of intact red cells are described. With 1H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90-tau-180 degree spin echo pulse sequence is used, however, many resonances can all be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13C and 31P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1H spin echo n.m.r. This method has also been used to measure isotope exchange (1H-2H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13C/31P spectrometer and [13C-1] glucose, the 13C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31P n.m.r.     

Synthesis of 5-beta-D-ribofuranosylcytosine (pseudo-cytidine) and its alpha isomer

The dilithio derivative of 2,4-di-O,N-trimethylsilylcytosine was condensed with 2,4:3,5-di-O-benzylidene-D-ribose to give a mixture of the protected, epimer at C-1′ pentitols 5 and 6; in addition, a compound substituted at N-3 or N-4, whose structure was not elucidated, was also obtained. The epimers were treated with acid to give 4-amino-2-hydroxy-5-(β-and α-D-ribofuranosyl)pyrimidine (10 and 12). The n.m.r. spectrum of 10 corresponds predominantly to the C-2′ endo structure. On the other hand, the n.m.r. spectrum of 12 presents couplings identical with those of the “α pseudo-uridine”. On nitric deamination, each isomer gave in a highly preponderant yield the corresponding pseudo-uridine at C-1′.     

植酸酶作用机理的初探

电喷雾电离－质谱联用仪分析结果表明,植酸酶水解植酸是以分步方式进行的,植酸酶能将植酸水解成植酸五磷酸酯至植酸一磷酸酯不同的中间产物。但最终产物主要为二磷酸肌醇,与一些同时形成的无机磷分子能与未水解的植酸以“－Ｏ－Ｏ”或“－Ｏ－”键形成多磷酸肌醇的更复杂的分子形式。     

Potentiometric and ³¹P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and 31P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

Structural requirements for the action of steroids as quenchers of albumin fluorescence.

1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant (Kf) and free energy of formation (delta G 0) can be determined by fluorescence titration. The fluorimetrically determined delta G 0 values range from -6.5 to -7.5 kcal/mol. 2. 5 alpha-Androstane and 5 alpha-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroid-albumin interaction. Introduction of hydroxy or oxo groups in 5 alpha-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5 alpha-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. Comparison of delta G 0 values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3 alpha-hydroxyl (with 5 beta-pregnane derivatives; not with 5 alpha-androstane derivatives); (d) 17 beta-acetyl or 17 beta-hydroxyethyl residues; (e) acetylated or propionated 17 beta-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) delta 5 and delta 6 double bonds; and (h) the 19 beta-methyl group. The maximal variation of delta G 0 determined by affinity-enhancing groups is -0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17 alpha-ethynyl and 17 alpha-acetoxyl residues; (k) benzoylated or hexahydro-benzoylated beta-hydroxy groups at C-17; (l) acetylated and benzoylated hydroxy groups at C-3; and delta 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16 alpha, 17 alpha-epoxy group are more effective than the corresponding alpha-hydroxyl in decreasing affinity, while at C-11 and C-17, the alpha-hydroxyl is more effective than the beta-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (alpha) rear surface of the B-, C- and D-ring, and possibly, the 17 beta-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.     

Studies on nucleotide analogs. I. Synthesis of two 9-alpha-D-mannofuranosyladenine phosphates, and their inhibition of adenylate kinase

9-α-D-Mannofuranosyladenine (1) was quantitatively phosphorylated at O-5 by phosphoryl chloride in the presence of triethyl phosphate, giving phosphate 2. Treatment of 9-(2,3-O-isopropylidene-α-D-mannofuranosyl)adenine (3) with phosphoryl chloride-trimethyl phosphate, followed by hydrolysis at pH 1.5 to remove the protecting group, yielded mononucleotides 2 and 4 having the phosphate group at C-5′ and C-6′, respectively. These mononucleotides, chromatographically homogeneous in six solvent systems, were further characterized by their patterns of chromatography on Dowex ion-exchange resin, by their mass spectra, and by phosphorus n.m.r. spectroscopy. Both the 5′- and 6′-phosphates are noncompetitive inhibitors of adenylate kinase (for which a sensitive, accurate, and inexpensive, assay-system was developed). Of the two, the 6′-mononucleotide was the more potent inhibitor of adenylate kinase.     

Studies on anhydro-osazones. Structure and anomeric configuration of the 3,6-anhydro-osazone derivatives obtained from D-galacto-2-heptulose phenylosazone

Dehydration of D-galacto-2-heptulose phenylosazone with methanolic sulfuric acid afforded two 3,6-anhydro-osazone derivatives (2 and 3). Compound 2 was obtained as the preponderant isomer, without inversion at C-1 (C-3 of the starting osazone), and 3 was obtained with inversion. The anomeric configurations of 2 and 3 were determined by n.m.r. spectroscopy. Refluxing of 2 and 3 with copper sulfate afforded two C-nucleoside analogs, namely, 4-β- and 4-α- D-lyxofuranosyl-2-phenyl-1,2,3-triazole, 4 and 5, respectively. The anomeric configurations of 4 and 5 were determined by n.m.r and c.d. spectroscopy. Acetylation of 4 and 5 afforded the tri-O-acetyl derivatives. The mass spectra of these compounds were discussed.     

Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae.

From the lipopolysaccharide of the deep rough mutant I-69 Rd--/b+ of Haemophilus influenzae two oligosaccharides were obtained after de-O-acylation and separation by high-performance anion exchange chromatography. Their chemical structures were determined by one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy as alphaKdo-4P-(2-->6)-betaGlcN-4P-(1-->6)-alphaGlcN-1P and alphaKdo-5P-(2-->6)-betaGlcN-4P-(1-->6)-alphaGlcN-1P. The specificity of mAbs S42-21 and S42-16 specific for Kdo-4P or Kdo-5P, respectively [Rozalski, A., Brade L., Kosma P., Moxon R., Kusumoto S., & Brade H. (1997). Mol. Microbiol. 23, 569--577] was confirmed with neoglycoconjugates obtained by conjugation of the isolated oligosaccharides to BSA. In addition, a mAb S42-10-8 with unknown epitope specificity could be assigned using the neoglycoconjugates described herein. This mAb binds to an epitope composed of the bisphosphorylated glucosamine backbone of lipid A and Kdo-4P, whereby the latter determines the specificity strictly by the position of the phosphate group.