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Porcine pancreatic colipase possesses a particular three-dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgstrom, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β-sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo-CIDNP) studies of the complexes of colipase with organized bile salt micelles. The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr-I and Tyr-II (Tyr-56 and Tyr-57) and His-II (His-86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo-CIDNP effects of Tyr-I and Tyr-II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo-CIDNP effects observed in the colipase-micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic. A general model for micelle binding to colipase is proposed and involves a two-step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.

A 31P-NMR method for the determination of 13C enrichment in phosphorylated [1-(13)C]glucose metabolites was developed by taking advantage of the 13C satellites detectable for 31P-NMR signals of metabolites such as UDP-hexoses, UDP-N-acetylhexosamines and other phosphorylated compounds generated during glycolysis and subsequent anabolism. HT-29 cells were incubated in culture medium containing 4.5 g/l [1-(13)C]glucose for 24 h prior to cell extraction, and high-resolution 31P-NMR spectra were acquired from perchloric acid extracts. Since glucose and its phosphorylated products are key metabolites for many different metabolic processes, this method may be very helpful for studying specific metabolic pathways involving phosphorylated glucose metabolites.

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by 1H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded beta-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton--proton distance constraints, 14 hydrogen bond distance constraints and 55 phi and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09 +/- 0.02 nm for backbone atoms (residues 11-30, 37-50, 57-69, 83-89). The protein comprises two identical domains, each containing a three-strand beta-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase.

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH= 0.188 mT and g= 2.0054). 1 The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2 The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3 Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4 The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.

The dose-dependent effect of ethanol on the hepatic metabolism of the perfused rat liver has been investigated by (a) 31P-NMR spectroscopy for the follow-up of intracellular phosphorylated metabolites and (b) HPLC for compounds released in the effluents. Perfusion of livers from fed rats with ethanol induced an increase in the level of sn-glycerol 3-phosphate and net accumulations of 3.30 +/- 0.33 and 0.69 +/- 0.15 mumol x g-1 wet liver were reached after 20 min, for 70 mM and 0.5 mM ethanol, respectively. sn-Glycerol-3-phosphate accumulation was fully detected by 31P NMR as indicated by comparing quantitations based on NMR and biochemical assays. Ethanol administration up to a concentration of 10 mM induced a dose-dependent decrease in the release of lactate + pyruvate by the liver. Lactate release decreased from 1129 +/- 39 to 674 +/- 84 nmol x min-1 x g-1, while pyruvate decreased from 230 +/- 9 to 6.2 +/- 0.4 nmol x min-1 x g-1, after 20 min of perfusion with 10 mM ethanol. Nevertheless, the flux through 6-phosphofructo-1-kinase, as measured by both the accumulation of sn-glycerol 3-phosphate and release of lactate + pyruvate, was not affected in the early phase of ethanol oxidation. Finally, data obtained from oxygen consumption, the release of acetate and the accumulation of sn-glycerol 3-phosphate do not support the involvement of the microsomal ethanol-oxidizing system in the catalysis of ethanol oxidation, even at high doses of alcohol.

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.

Real-time monitoring of spin-trapped oxygen-derived free radicals released by the isolated ischemic and reperfused rat heart has been achieved by ESR analysis of the coronary effluents using continuous flow detection and high-speed acquisition techniques. Two nitrone spin traps 5,5-dimethyl pyrroline 1-oxide (Me2PnO) and 3,3,5,5-tetramethyl pyrroline 1-oxide (MePnO) have been separately perfused at a concentration of 40 mM during a sequence of 50 min of low-flow ischemia (1 ml/min) followed by 30 min of global ischemia and subsequent reperfusion at the control flow rate (14 ml/min). ESR spectra were sequentially obtained in 5-min or 30-s blocks during low-flow ischemia and reperfusion, respectively. 1. The results show the formation of OH. free radicals in the ischemic and reperfused heart, as demonstrated by the observation of Me2PnO-OH (aN = aH = 14.9 G; g = 2.0053) and Me4PnO-OH (aN = 15.2 G, aH = 16.8 G; g = 2.0055) spin adducts. There is no evidence of significant biological carbon-centered or peroxyl free radicals spin-adduct formation in the coronary effluents or in lipid extracts analyzed after reflow. 2. The OH. generation began 15-20 min after the onset of ischemia and was moderate, peaking at 30-40 min. During reperfusion, an intense formation of OH. spin adducts was observed, with a maximum at 30-60 s and a further gradual decrease over the following 2 min. 3. Cumulative integrated values of the amount of spin adducts released during the ischemic period show a Me2PnO-OH level fourfold greater than that of Me4PnO-OH. It was 2.5 times greater during reflow, reflecting slower kinetics with the more stable Me4PnO. 4. The original ESR detection technique developed in this study allows accurate real-time quantitative monitoring of the oxygen-derived free radicals generated during myocardial injury. It might provide a quick and reliable new means for assessing the efficacy of free-radical inhibitors.