31P NMR of alkaline phosphatase. Saturation transfer and metal-phosphorus coupling.

The rate constants which characterize the formation and breakdown of the noncovalent (E.P) and covalent (E-P) enzyme-phosphate intermediates on the alkaline phosphatase reaction pathway are known to be sensitive to the nature of the metal ion bound to the enzyme. 31P NMR saturation transfer has been demonstrated to provide a simple and sensitive method for measuring the metal ion dependence of these rates under equilibrium conditions. When the native Zn2+ was replaced by Cd2+, the 31P NMR spectrum at high pH revealed a new resonance at 12.6 ppm which has been assigned to the noncovalent enzyme.phosphate complex. Reconstituting the enzyme with enriched 113Cd2+ caused this unusually downfield-shifted resonance to appear as a doublet due to 113Cd-31P spin coupling (2J31P-O-113Cd = 30 Hz). This result provides the first unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase.     

Alkaline phosphatase inhibitors as labels of DNA probes

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.     

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.     

Understanding the radical mechanism of lipoxygenases using 31P NMR spin trapping

In this paper, we use our quantitative (31)P NMR spin trapping methods, already developed for simple oxygen- and carbon-centered radicals, to understand the radical intermediates generated by enzymatic systems and more specifically lipoxygenases. Our methodology rests on the fact that free radicals react with the nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using (31)P NMR in the presence of a phosphorus containing internal standard. This system was thus applied to better understand the mechanism of enzymatic oxidation of linoleic acid by soybean lipoxygenases-1 (LOX). The total amount of radicals trapped by DIPPMPO was detected by (31)P NMR at different experimental conditions. In particular the effect of dioxygen concentration on the amount of radicals being trapped was studied. At low dioxygen concentration, a huge increase of radicals trapped was observed with respect to the amount of radicals being trapped at normal dioxygen concentrations.     

Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline phosphatase in the thymus.

(1) Fetal thymuses, organs from patients who died from diseases that are not clinically known to be associated with concomitant lymphoid tissue involvement, as well as thymuses from patients dying from diseases which effect the lymphatic complex of the body, one way or another, have been investigated for their alkaline phosphatase activity, using Gomori technique and applying four different phosphate esters as substrates. (2) Three substrates (beta-glycerophophate, riboflavin 5-phosphate and adenosine triphosphate) showed essentially the same pattern of activity in which the cortex and Hassall's corpuscles were reactive, while the medulla was negative. A reversal of this pattern was demonstrated with 5-monophosphoric acid. (3) Before the age of 32-36 weeks of intra-uterine life there is no alkaline phosphatase activity in the thymus; therafter, the enzyme begins to make its first appearance. (4) There is a definite increase in the intensity of the reaction with advance of intra-uterine life. This increase in phosphatase content is continued postnatally, to reach its maximum at about the age of 10 years: after that, the enzyme activity gradually subsides. (5) There is a tremendous augmentation of phosphatase activity in the case of disease which are known to affect the lymphoid complex. (6) The phosphatase activity of the thymus has been discussed in relation to the prevailing concepts about the function of the thymus, with special emphasis on a possible association with 'lymphocyte-stimulating factor' production and/or secretion.     

31P NMR spin-transfer in the phosphoglyceromutase reaction

The rate of exchange of phosphoryl groups between 2- and 3-phosphoglycerate catalysed by (a) high concentrations (approximately equal to 5.0 mg protein ml-1) of rabbit muscle phosphoglyceromutase and (b) lysed human erythrocytes was measured using saturation and inversion transfer techniques with 31P-NMR spectroscopy. This is the first reported application of these techniques to a study of this particular enzymic reaction either in vitro or in situ in a cell cytosol. Selective irradiation of resonances was achieved by the DANTE pulse sequence which had not previously been used for spin-transfer studies. New equilibrium exchange theory was developed for the simplest model of a two-reactant enzyme-catalysed reaction and this was used to calculate turnover rates for the enzymes. There was a close similarity between the turnover rates calculated from the spin-transfer data obtained from the systems in vitro and in situ and those obtained by conventional enzymic assays, at low enzyme concentrations. This suggested an absence of any homogeneous enzyme-enzyme interactions which modify the kinetics at high protein concentrations either in lysates or in the system in vitro.     

Metabolic control principles and 31P NMR

31P NMR is a unique research tool for studying metabolism during exercise. This communication gives the underlying theory and experimental data for obtaining the transfer characteristic relating work and NMR-determined metabolic paramenters, particularly the ratio of free inorganic phosphate to phosphocreatine (Pi/PCr). Furthermore, illustrations of the types of transfer characteristics observed in different individuals and different training regimens can be obtained, including both hyperbolic (Michaelis-Menten) and sigmoid transfer characteristics. With a hyperbolic transfer characteristic, oxygen delivery appears to be adequate to support the exercise regimen over the range studied (up to 50% of Vmax), whereas with a sigmoid transfer characteristic, there appears an imprint of limited oxygen delivery on the kinetics from rest up to 50% of Vmax. These two types of transfer functions are appropriate to explain the transition to anaerobic metabolism (anaerobic threshold), with a hyperbolic transfer characteristic representing a graded transition; and a sigmoid transfer characteristic representing an abrupt transition. A sigmoid transfer characteristic may involve greater lactate accumulation at submaximal exercise levels, and in both types of transfer characteristic sufficient aerobic lactate is formed to meet the metabolic demand for pyruvate. The results suggest an important role for 31 P NMR in studies of energy-related metabolites as a means of determining exercise performance in terms of regulation of tissue oxidative metabolism and oxygen delivery to tissue.     

31P NMR Spectra of Alkalophilic Bacillus No. A-59

Some advanced cancer patients suffer from pungent sulfury malodor. To determine the chemical identity of the odorant, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles from fungating cancer wounds. We identified the source of the characteristic smell as dimethyl trisulfide, a compound that is known to be emitted from some vegetables and microorganisms. Controlling the production of dimethyl trisulfide should improve quality of life of patients.     

31P NMR Investigation of vanadium-treated chick muscle

The phosphorus NMR profile of normal and vanadium-treated chick muscle was obtained in vivo. The data show that the differentiation of breast and thigh muscles in terms of pH, lipid related metabolites, and bioenergetic parameters can be readily followed. Although the vanadium-treated chicks showed substantial retardation of growth, the only NMR parameter that was significanty affected by dietary vanadium was the pH of breast muscle, which was substantially more acidic in the vanadium-treated animals.     

31P NMR studies on the interaction of deoxyuridylate with thymidylate synthase.

The 31P nuclear magnetic resonance signal of deoxyuridylate was studied in the presence and absence of thymidlate synthase. In the absence of enzyme the chemical shift of deoxyuridylate is pH dependent with a pKa of 6.25. In the presence of enzyme, a peak corresponding to the dianioinc form of deoxyuridylate is observed which is independent of pH between pH 5.7 and pH 7.4. The pKa of the phosphate in the deoxyuridylate-thymidylate synthase complex is therefore less than 5. The release of inorganic phosphate from deoxyuridylate catalyzed by contaminating phosphatase was also observed.     

NMR relaxation processes of 31P in macromolecules

³¹P-Nmr relaxation parameters (spin-lattice relaxation time, linewidth, and nuclear Overhauser effect) were obtained at three different frequencies for poly(U) and a well-defined (145 ± 3 base-pair) fragment of DNA in solution. Data sets for the two samples were analyzed by theories which included relaxation by the mechanisms of ³¹P chemical shift anisotropy as well as by ¹H-³¹P dipole–dipole interaction. Neither data set could be satisfactorily described by a single correlation time. A model of a rigid rotor most nearly fits the data for the DNA molecule. Parameters obtained from the least-square fit indicate (1) that the DNA undergoes anisotropic reorientation with a correlation time τ₀ = 6.5 × 10^?7 sec for the end-to-end motion, (2) the ratio of diffusion constants D_∥/D_⊥ is 91, and (3) that the linewidth is due to chemical shift dispersion to the extent of 0.5 ppm. Some deviations of the calculated from the observed values suggested that significant torsional and bending motions may also take place for this DNA. Another model which contains isotropic motion but with a broad distribution of correlation times was required to fit the data for poly(U). A log ? χ² distribution function of correlation times [Scheafer, J. (1973) Macromolecules 6 , 881–888] described well the motion of poly(U) with the average correlation time τ = 3.3 × 10^?9 sec and a distribution parameter p = 14.     

High resolution 31P NMR of extracted phospholipids

The common phospholipids from biological sources were quantitated using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy in conjunction with an analytical reagent composed of two parts: 1) 2 ml of reagent chloroform in which was dissolved 0.01-100 mg of crude tissue lipid extracted from tissue sources using chloroform-methanol 2:1, the extract having been washed with 0.2 vol. of 0.1 M KCl; 2) 1 ml of an aqueous methanol reagent composed of one part 0.2 M (ethylenedinitrilo)-tetraacetic acid in D2O titrated to pH 6 with CsOH and four parts of reagent methanol. In a magnetic field of 11.75 Tesla, the extracted phospholipids yield narrow signals (1.8-3.2 Hz at half-height), corresponding to each generic species, e.g., phosphatidylcholines, phosphatidylethanolamines, etc., permitting resolution among the various phospholipid families and their lyso and plasmalogen derivatives. The reagent permits assays of high precision and accuracy using a modest amount of NMR spectrometer time (ca. 15 min/assay). The procedures described, which are compared to high-performance liquid chromatography, are convenient for the routine analysis of phospholipids from biological sources.     

31P NMR of phospholipid glycerol phosphodiester residues

Saponification of extracted tissue phospholipids yields a set of isolated glycerol 3-phosphoryl phospholipid polar headgroups from which semi-quantitative 31P NMR spectra can be obtained. The resonance signals from these molecules, which frequently have been reported as uncharacterized phosphate signals observed in perchloric acid extracts of tissue, can be used as an aid in the characterization of isolated phospholipids and of tissue phospholipid 31P NMR profiles. 31P NMR chemical-shift values of the resonances at pH 7 in water and relative to 85% phosphoric acid are: glycerol 3-phosphocholine (-0.13 delta), glycerol 3-phosphoethanolamine (0.42 delta), glycerol 3-phospho(monomethyl)ethanolamine (0.29 delta), glycerol 3-phospho(dimethyl)ethanolamine (0.16 delta), glycerol 3-phosphoserine (0.14 delta), glycerol 3-phosphoinositol (-0.07 delta), glycerol 3-phosphoglycerol (0.92 delta), bis(glycerol 3-phospho)glycerol (0.79 delta), serine ethanolamine phosphodiester (-0.46 delta), glycerol 3-phosphate (0.60 delta; 4.29 delta at pH 10) glycerol 2-phosphate (0.15 delta; 3.92 delta at pH 10). In addition, analysis of extracted cancer tissue phospholipid samples yielded a new and uncharacterized polar headgroup fragment with a chemical-shift value of 0.29 delta that is independent of sample pH.     

Investigation of the mechanism of the synthesis of oligonucleotides. IX. 31P NMR spectra of the active dinucleotide derivatives and their analogs.

The interaction of 3'-O-acetyldithymidilate (pdTpdT(Ac)), thymidine-3',5'-diphosphate (pdTp) and thymidine-3'-phenyl-phosphate-5'-phosphate (pdTpPh) with 2,4,6-triisopropylbenzene sulphonyl chloride (TPS) and N,N'-dicyclohexylcarbodiimide (DCC) in pyridine and dimethylformamide (DMF) was studied by pulsed NMR spectroscopy on phosphorus nuclei. Thymidine cyclic 3',5'-pyrophosphate and dimeric pyrophosphate derivatives were shown to be the main products of the reaction of pdTp with TPS and DCC. The former shows spin AB-system with the unusually large spin-spin coupling constant about 28Hz upfield to the signals of the dimeric pyrophosphates in NMR spectrum. Analogous spin AB-systems with large spin-spin coupling constants (up to 32 Hz) were observed in the spectra of the reaction mixtures of pdTpdT(Ac) with TPS or DCC and of pdTpPh with TPS. These spin AB-systems were ascribed to 3',5'-cyclic pyrophosphate derivatives of pdTpdT(Ac) and pdTpPh.