13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.     

The use of dietary loading of 133Cs as a potassium substitute in NMR studies of tissues

133Cs NMR chemical shifts and relaxation times have been measured for tissue samples in vitro and in vivo from rats which have been fed on a high cesium, low potassium diet, which leads to a predominantly intracellular distribution of this ion, similar to that of K+. The high sensitivity, large chemical shift range, and narrow linewidths of 133Cs, compared with 39K, allow chemical shift differences to be observed between tissues, and in subcellular organelles such as mitochondria. For example, in vitro tissue chemical shifts, relative to 150 mM CsCl, are 1.06 +/- 0.11 ppm for liver, 0.02 +/- 0.05 ppm for brain, 1.76 +/- 0.20 ppm for erythrocytes, and -0.13 +/- 0.02 ppm for plasma. T1 and spin-echo T2 values range from 1.26 +/- 0.05 s (T1), and 0.028 +/- 0.006 s (T2) for liver, to 6.49 +/- 0.19 s and 1.12 +/- 0.03 s for plasma. 133Cs relaxation times show the same relative trends between tissues as are observed in 39K tissue studies.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

Patterns of occurrence of second primary non-mammary malignancies in breast cancer patients: results from 1382 consecutive autopsies

In a previous study, we demonstrated the existence of a 3.2 +/- 0.2 ppm peak in the 1H NMR spectrum at 60 MHz from human pancreatic adenocarcinomas (Capan-1 cell) heterotransplanted into nude mice. This peak, which is not present in normal human pancreas, was attributed to enhanced membrane fluidity and/or or an increase in phospholipid turnover. The present study was designed to identify this signal by comparing the 1H NMR spectra recorded in vivo at 100 MHz from Capan-1 tumors, after suppression of the tissular water proton peak, to those recorded from normal pancreatic tissue, and to those recorded at 300 MHz from lipid extracts. The 1H NMR spectra at 100 MHz of the Capan-1 tumors in vivo exhibited three main peaks in the 3.2 +/- 0.2 ppm region: 1. A peak at 2.8 +/- 0.1 ppm from CH2 protons of the acyl chains of unsaturated phospholipids; 2. A peak at 3.2 +/- 0.1 ppm from the protons of the N(CH3)3 group of choline; and 3 A peak at 3.5 +/- 0.1 ppm attributed to GPC. The NMR 1H 300 MHz spectrum of phospholipid extracts of Capan-1 tumors displayed 12 principal resonances, of which only the N(CH3)3 peak of PC had a similar chemical shift to that observed at low resolution (3.2 +/- 0.2 ppm). This peak had a higher intensity in the xenografts than in normal human pancreatic tissue. HPLC analysis of the same lipid extracts from Capan-1 cells in culture, of tumors derived from these cells and from normal pancreas showed: 1. Identical concentrations of the different phospholipids from cancerous human pancreatic cells in vivo and in culture; and 2. A significantly higher level of PC in the extracts of normal human pancreatic tissue. The increase in intensity of the N(CH3)3 peak of PC in the Capan-1 tumors was not thought to be caused by an increase in PC concentration, but to a difference in conformation or mobility of the PC protons in the xenografts. The increase in relaxation time in cancerous tissue (from 60 to 125 ms) was also taken to be evidence in favor of a high mobility of protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR studies of the Escherichia coli Trp repressor.trpRs operator complex

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.     

1H and 15N nuclear magnetic resonance assignments, secondary structure in solution, and solvent exchange properties of azurin from Alcaligenes denitrificans

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14,000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32 degrees C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in NH chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C alpha protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3JHN alpha values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N epsilon 2 of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.     

Mortality and cancer incidence following radiotherapy for seminoma of the testis

The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pKa-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm.     

Structure of gene 5 protein-oligodeoxynucleotide complexes as determined by 1H, 19F, and 31P nuclear magnetic resonance

1H nuclear magnetic resonance (NMR) spectra at 270 MHz of gene 5 protein from bacteriophage fd and its complexes with tetra- and octadeoxynucleotides show that approximately 12 of the 37 aromatic protons of the protein undergo upfield shifts upon nucleotide binding. In the complex with d(pT)8, the upfield shifts of the aromatic protons average approximately 0.3 ppm, while in the d(pA)8 complex the same resonances (assigned to tyrosyl protons) shift upfield approximately 0.8 ppm. These are interpreted as ring current shifts induced by stacking of the phenyl rings of three of the five tyrosyl residues with the bases of the nucleotides. 19FNMR of m-fluorotyrosyl gene 5 protein shows five separate resonances: two downfield from m-fluorotyrosine corresponding to "buried" tyrosyls and three near m-fluorotyrosine corresponding to "surface" tyrosyls. The latter (assigned to Tyr-26, -41, and -56, shown by chemical modification to be exposed to solvent) move upfield on nucleotide binding. The downfield 19F resonances are unaffected. Thus the aromatic protons shifted upfield on nucleotide binding appear to be those of Tyr-26, -41, and -56. In contrast to tetra-, octanucleotide binding to gene 5 protein induces large changes in the 1H resonances of the -CH3 groups of the Val, Leu, and Ile side chains. These may reflect conformational changes induced by protein-protein interactions between two monomers bound to the octanucleotide. 1H resonances of the epsilon-CH2 groups of the lysyl residues in the protein and the complexes with nucleotides are narrow with long T2 values, suggesting considerable rotational motion. Thus epilson-NH3+-phosphate interactions, if they occur, are on the surface of the complex and allow the epsilon-CH2 groups to retain considerable rotational freedom. 31P NMR of the bound nucleotides shows large decreases in T1 for the 3'-5' diesters, but little chemical shift suggesting no unusual distortion of the nucleotide backbone on binding to gene 5 protein. A three-dimensional model of a gene 5 protein-octanucleotide complex has been built based on predictions of the secondary structure from the amino acid sequence (87 AA) and tertiary folding dictated by known chemical and NMR features of the complex.     

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.     

Identification of group VS116 strains among Borrelia burgdorferi sensu lato grown from the hard tick, Ixodes ricinus (Linnaeus, 1758) by off-coupled restriction fragment length polymorphism analysis

The degree of chemical shift similarity for homologous proteins has been determined from a chemical shift database of over 50 proteins representing a variety of families and folds, and spanning a wide range of sequence homologies. After sequence alignment, the similarity of the secondary chemical shifts of C alpha protons was examined as a function of amino acid sequence identity for 37 pairs of structurally homologous proteins. A correlation between sequence identity and secondary chemical shift rmsd was observed. Important insights are provided by examining the sequence identity of homologous proteins versus percentage of secondary chemical shifts that fall within 0.1 and 0.3 ppm thresholds. These results begin to establish practical guidelines for the extent of chemical shift similarity to expect among structurally homologous proteins.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 microM cyclic AMP (cAMP), 1 mM calcium and 1 microM calmodulin (Ca2+/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 microM phorbol 12,13-dibutyrate (Ca2+/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca2+/calmodulin, respectively, but was unaffected in the presence of Ca2+/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 microM okadaic acid and 0.01 microM microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Modulation of neuropeptide FF receptors by guanine nucleotides and cations in membranes of rat brain and spinal cord

Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF (NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I-YLFQPQRFamide (125I-Y8Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine nucleotides dose-dependently inhibited specific 125I-Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 +/- 6 and 71 +/- 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP > or = GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 microM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-O-(3-thiotriphosphate). Our data suggest that 125I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.     

Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit

The isolated hemeprotein subunit of sulfite reductase (SiR-HP) from Escherichia coli consists of a high spin ferric isobacteriochlorin (siroheme) coupled to a diamagnetic [4Fe-4S]2+ cluster. When supplied with an artificial electron donor, such as methyl viologen cation radical, SiR-HP can catalyze the six electron reductions of sulfite to sulfide and nitrite to ammonia. Thus, the hemeprotein subunit appears to represent the minimal protein structure required for multielectron reductase activity. Proton magnetic resonance spectra are reported for the first time on unligated SiR-HP at 300 MHz in all three redox states. The NMR spectrum of high spin ferric siroheme at pH 6.0 was obtained for the purpose of comparing its spectrum with that of oxidized SiR-HP. On the basis of line widths, T1 measurements, and 1D NOE experiments, preliminary assignments have been made for the oxidized enzyme in solution. The pH profile of oxidized SiR-HP is unusual in that a single resonance shows a 9 ppm shift over a range of only 3 pH units with an apparent pK = 6.7 +/- 0.2. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned using deuterium substitution for all redox states. One beta-CH2 resonance has been tentatively assigned to the bridging cysteine on the basis of chemical shift, T1, line width, and the presence of NOEs to protons from the siroheme ring. The observed pattern of hyperfine shifts can be used as a probe to measure the degree of coupling between siroheme and cluster in solution. The cluster iron sites of the resting (oxidized) enzyme are found to possess both positive and negative spin density which is in good agreement with Mossbauer results on frozen enzyme. The NMR spectrum of the 1-electron reduced form of SiR-HP is consistent with an intermediate spin (S = 1) siroheme. Intermediate spin Fe(II) hemes have only been previously observed in 4-coordinate model compounds. However, the amount of electron density transferred to the cluster, as measured by the isotropic shift of beta-CH2 resonances, is comparable to that present in the fully oxidized enzyme despite diminution of the total amount of unpaired spin density available. Addition of a second electron to SiR-HP, besides generating a reduced S = 1/2 cluster with both upfield and downfield shifted cysteine resonances, converts siroheme to the high spin (S = 2) ferrous state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic basis of the MbrC "ploidy" phenotype in Escherichia coli

We measured the spin-lattice relaxation times (T1) of water protons and intermolecular cross-relaxation times (T(IS)) from irradiated protein protons (f2-irradiation at 1.95 or -4.00 ppm) of rabbit normal and monoiodoacetate-induced degenerated knee articular cartilages to observed water protons. The mean values of T1 (T1) for control and degenerated rabbit knee cartilages were 1.87+/-0.15 (mean+/-SD, n=29) and 1.82+/-0.13 s (n=34), respectively. The mean values of water content (W(H2O)) for control and degenerated rabbit knee cartilages were 82.9+/-2.09 (n=26) and 83.1+/-2.57% (n=28), respectively. These values were not statistically different from each other. However, the mean values of T(IS) (T(IS)) for normal knee articular cartilage were significantly different from those for degenerated cartilage: (normal), T(IS) (f2=1.95 ppm)=2.46+/-0.62 s (n=28), T(IS) (f2=-4.00 ppm)=4.25+/-1.26 s (n=26); (degenerated), T(IS) (f2=1.95 ppm)=1.99+/-0.76s (n=34), T(IS) (f2=-4.00 ppm)=3.33+/-0.76 s (n=31). Obtained results may be attributed to the reported switchover from type II to type I collagen syntheses in osteoarthritic cartilage, resulting in broad collagen fibers. This specificity of cross-relaxation effect may prove useful in the noninvasive and pathophysiological evaluation of cartilage tissues in vivo.     

Glucose-induced thermal stabilization of the native conformation of GLUT 1

The glucose transporter, GLUT 1, was purified from erythrocyte membranes and incorporated into vesicles of erythrocyte lipids. These protein-containing vesicles were studied with differential scanning calorimetry. It was found that the protein underwent an irreversible denaturation at 68.5 +/- 0.2 degreesC (at a scan rate of 0.25 degreesC/min) which was shifted to 72.6 +/- 0.2 degreesC in the presence of 500 mM D-glucose, while 500 mM L-glucose or 10 microM cytochalasin B did not produce a significant shift. The calorimetric enthalpy was found to be 150 kcal/mol, independent of the presence of D-glucose. On a weight basis this value is lower than that for soluble proteins, but it is comparable to values obtained with other integral membrane proteins. The van't Hoff enthalpy is similar to the calorimetric enthalpy, within the experimental error, indicating that the transition is not likely to be cooperative. The activation energy is estimated from both the scan rate dependence of the transition temperature and from the shape of the DSC curve. The presence of 500 mM D-glucose slightly decreases the activation energy. It is concluded that the shift to a higher denaturation transition temperature in the presence of D-glucose is not a result of increased kinetic stability of GLUT 1.     

Transferrins. Hen ovo-transferrin, interaction with bicarbonate and iron uptake

Fe(III) uptake by the iron-delivery and iron-scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo-ovotransferrin loses two protons with K1a = 50 +/- 1 nM and K2a = 4.0 +/- 0.1 nM. These acid-base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 +/- 0.15 mM and KN = 110 +/- 10 mM. FeNAc3 exchanges its Fe(III) with the C-site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 +/- 25 M-1 s-1, reverse rate constant k-1 = (6.0 +/- 0.1) x 10(3) M-1 s-1 and equilibrium constant K1 = 0.11 +/- 0.01. This iron-protein intermediate loses then a single proton, K3a = 3.50 +/- 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 +/- 0.01 s-1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 +/- 0.05) x 10(-2) s-1. These modifications in the monoferric protein conformation are essential for iron uptake by the N-site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15,000 s. The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics?     

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.     

Differential effects of thiocyanate on the binding thermodynamics of bicuculline methiodide versus SR95531 (gabazine) to the gamma-aminobutyric acid receptor-ionophore complex

The temperature dependence of the binding of [3H]SR 95531 (Gabazine), an antagonist of gamma-aminobutyric acid (GABA(A)) receptors, was studied in synaptosomal membranes of rat brain in the presence of 50 mM KSCN. The displacing potencies of the antagonists bicuculline methiodide and Gabazine were determined at five temperatures between 0 degrees and 37 degrees. Van't Hoff plots of the displacing potencies were analyzed by linear regression in the presence and absence of thiocyanate. Thiocyanate hardly affected the exothermic ionic binding interaction of gabazine. In contrast, thiocyanate strongly potentiated the binding of bicuculline methiodide and deprived it of its exothermic nature. The enhanced binding of bicuculline methiodide in the presence of chaotropic SCN- ions might be reconciled with "entropic trapping" in a sterically constrained hydrophobic binding pocket.