Oxidation of exogenous [13C]galactose and [13C]glucose during exercise

The present study examined the oxidation of exogenous galactose or glucose during prolonged submaximal cycling exercise. Eight highly trained volunteers exercised on two occasions on a cycle ergometer at 65% of maximal workload for 120 min, followed by a 60-min rest period and a second exercise bout of 30 min at 60% maximal workload. At random, subjects ingested a 8% galactose solution to which an [1-13C]galactose tracer was added or a 8% glucose solution to which an [U-13C]glucose tracer was added. Drinks were provided at the end of the warm-up period (8 ml/kg) and every 15 min (2 ml/kg) during the first 120 min of the test. Blood and breath samples were collected every 30 and 15 min, respectively, during the test. The exogenous carbohydrate (CHO) oxidation was calculated from the 13CO2/12CO2 ratio and CO2 production of the expired air. Peak exogenous CHO oxidation during exercise for galactose and glucose was 0.41 +/- 0.03 and 0.85 +/- 0.04 g/min, respectively. Total CHO and fat oxidation were not significantly different between the treatments. Forty-six percent of the ingested glucose was oxidized, whereas only 21% of the ingested galactose was oxidized. As a consequence, more endogenous CHO was utilized with galactose than with glucose (124.4 +/- 6.7 and 100.1 +/- 3.6 g, respectively). These results indicate that the oxidation rate of orally ingested galactose is maximally approximately 50% of the oxidation rate of a comparable amount of orally ingested glucose during 120 min of exercise.     

Continuous-wave quantum yields of various cobalamins are influenced by competition between geminate recombination and cage escape

Quantum yields of photolysis of the cobalt-carbon bond for three cobalamin compounds were measured with a continuous-wave laser at 442 nm under both aerobic and anaerobic conditions. Aerobically, the initial homolysis product, Co(II) cobalamin, is trapped by oxygen to form aquocobalamin. Use of an excess of the radical trapping reagent 2,2,6,6-tetramethyl-1-piperidinyloxyl, under anaerobic conditions, scavenges the carbon radical and allows detection of the cobalt(II) photoproduct. Quantum yields measured under anaerobic conditions for 5'-deoxyadenosylcobalamin (phi (Co-C alpha),442 = 0.20 +/- 0.03) and methylcobalamin (phi (Co-C alpha),442 = 0.35 +/- 0.03) are in agreement with the values obtained under aerobic conditions (phi (Co-C alpha),442 = 0.19 +/- 0.04 and phi (Co-C alpha),442 = 0.36 +/- 0.04, respectively). Additionally, the quantum yield values for 5'-deoxyadenosylcobalamin and its base-off derivative (phi (Co-C alpha),442 = 0.045 +/- 0.015) match those obtained on a nanosecond time scale [Chen, E., & Chance, M. R. (1990) J. Biol. Chem. 256, 12987-12994]. A comparison of quantum yields obtained anaerobically for 5'-deoxyadenosylcobalamin and methylcobalamin in H2O versus ethylene glycol shows a 4-fold decrease for the former cobalamin and no change for the latter. These quantum yields are evaluated in terms of time-independent radical separation distances.     

Continuous 24-h L-[1-13C]phenylalanine and L-[3,3-2H2]tyrosine oral-tracer studies at an intermediate phenylalanine intake to estimate requirements in adults

The daily rates of whole-body phenylalanine oxidation (phe-ox) and hydroxylation (phe-OH) were determined in young men (n = 10) receiving [13C]phenylalanine and [2H2]tyrosine via primed constant oral infusion (four also received simultaneously [2H4]tyrosine and [2H3]leucine via primed constant intravenous infusions) continuously for 24 h (first 12 h fast and then 12 h fed). The subjects were given a diet supplying a proposed requirement phenylalanine intake (six subjects: 39 mg phenylalanine.kg-1.d-1 without tyrosine; four subjects: 36 mg phenylalanine plus 6.8 mg tyrosine), based on an otherwise adequate L-amino acid mixture for 6 d before the tracer study. Our hypothesis was that the subjects would be in approximate body phenylalanine equilibrium at these intakes. Estimates of the daily rate of phe-ox were 26.9 +/- 7.5 mg.kg-1.d-1 (17.2 +/- 5.2 and 9.7 +/- 3.2 mg.kg-1.d-1 during the 12-h fast and fed periods, respectively), and for phe-OH they were 32.1 +/- 11.9 mg.kg-1.d-1 (21.7 +/- 10.5 and 10.4 +/- 2.5 mg.kg-1.d-1 during the 12-h fast and fed periods, respectively). The daily phenylalanine balance was approximately neutral (P > 0.05) when based on phe-ox or phe-OH (+4.73 +/- 7.34 and -0.41 +/- 12.6 mg.kg-1.d-1, respectively). In comparison with recent, comparable 24-h tracer studies at deficient (22 mg.kg-1.d-1) and generous (100 mg.kg-1.d-1) phenylalanine intakes, these results support the hypothesis that a phenylalanine intake of 39 mg.kg-1.d-1 (without significant tyrosine) approximates the mean requirement in healthy adults. This contrasts with the upper requirement value of 14 mg.kg-1.d-1 for the total of the aromatic amino acids proposed in 1985 by FAO/WHO/UNU.     

A Mn(II)-Mn(III) EPR signal arises from the interaction of NO with the S1 state of the water-oxidizing complex of photosystem II

It was shown recently [Goussias, C., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261-9266] that incubation of photosystem II preparations with NO at -30 degrees C in the dark results in the formation of a new intermediate of the water-oxidizing complex. This is characterized by an EPR signal centered at g = 2 with prominent manganese hyperfine structure. We have examined the detailed structure of the signal using difference EPR spectroscopy. This is facilitated by the observations that NO can be completely removed without decrease or modification of the signal, and illumination at 0 degree C eliminates the signal. The signal spans 1600 G and is characterized by sharp hyperfine structure. 14NO and 15NO cw EPR combined with pulsed ENDOR and ESEEM studies show no detectable contributions of the nitrogen nucleus to the spectrum. The spectrum bears similarities to the experimental spectrum of the Mn(II)-Mn(III) catalase [Zheng, M., Khangulov, S. V., Dismukes, G. C., and Barynin, V. V. (1994) Inorg. Chem. 33, 382-387]. Simulations allowing small variations in the catalase-tensor values result in an almost accurate reproduction of the NO-induced signal. This presents strong evidence for the assignment of the latter to a magnetically isolated Mn(II)-Mn(III) dimer. Since the starting oxidation states of Mn are higher than II, we deduce that NO acts effectively as a reductant, e.g., Mn(III)-Mn(III) + NO--> Mn(II)-Mn(III) + NO+. The temperature dependence of the nonsaturated EPR-signal intensity in the range 2-20 K indicates that the signal results from a ground state. The cw microwave power saturation data in the range 4-8 K can be interpreted assuming an Orbach relaxation mechanism with an excited state at delta = 42 K. Assuming antiferromagnetic coupling, -2JS1.S2, between the two manganese ions, J is estimated to be 10 cm-1. The finding that an EPR signal from the Mn cluster of PSII can be clearly assigned to a magnetically isolated Mn(II)-Mn(III) dimer bears important consequences in interpreting the structure of the Mn cluster. Although the signal is not currently assigned to a particular S state, it arises from a state lower than S1, possibly lower than S0, too.     

Twenty-four-hour intravenous and oral tracer studies with L-[1-13C]-2-aminoadipic acid and L-[1-13C]lysine as tracers at generous nitrogen and lysine intakes in healthy adults

BACKGROUND: This is a continuation of investigations of the relations between amino acid kinetics and amino acid dietary requirements in healthy adults. OBJECTIVE: The aim was to investigate the 24-h pattern and rate of the metabolism of an L-[1-13C]-2-aminoadipic acid ([13C]AAA) tracer and of whole-body L-[1-13C]lysine ([13C]lysine) oxidation and balance in healthy, young adults receiving a generous intake of lysine. DESIGN: Thirteen healthy adults were given an adequate, L-amino acid-based diet supplying 77 mg lysine x kg(-1) x d(-1) for 6 d before the tracer studies. Two subjects received [13C]AAA intravenously and 2 received it orally; 3 subjects received [13C]lysine intravenously and 6 received it orally. We measured 13CO2 output, plasma [13C]AAA and [13C]lysine enrichment, and urinary [13C]AAA. RESULTS: [13C]AAA oxidation was estimated to be higher after the orally administered than after the intravenously administer tracer; plasma [13C]AAA was similar to urinary [13C]AAA. Whole-body lysine oxidation showed a rhythm that was induced by meal feeding. The intravenous [13C]lysine tracer gave mean estimates of lysine balances (lysine intake minus oxidation) that apparently were too low (-15.7 mg x kg(-1) x d(-1)) or too high (16.6 mg x kg(-1) x d(-1), P < 0.05 from zero balance) on the basis of urinary [13C]AAA or plasma [13C]lysine estimates of oxidation, respectively. For the orally administered tracer and plasma [13C]lysine enrichment, the mean balance was slightly positive (8.7 mg x kg(-1) x d(-1), P < 0.05 from zero). CONCLUSIONS: Use of urinary [13C]AAA as an index of the enrichment of the precursor pool did not appear to significantly improve the estimate of the fasting and feeding components of daily lysine balance. For estimates of daily, whole-body lysine oxidation, we propose use of plasma [13C]lysine with a 24-h, orally administered tracer protocol.     

NO reversibly reduces the water-oxidizing complex of photosystem II through S0 and S-1 to the state characterized by the Mn(II)-Mn(III) multiline EPR signal

Incubation of photosystem II preparations with NO at -30 degreesC results in the slow formation of a unique state of the water-oxidizing complex (WOC), which was recently identified as a Mn(II)-Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. Evolution of the Mn(II)-Mn(III) EPR signal proceeds through one or more intermediates [Goussias, C., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261-9266]. In an effort to identify these intermediates, we have examined the time course of the signal evolution in the presence and absence of methanol. An early step of the interaction of NO with the WOC is the reduction of S1 to the S0 state, characterized by the weak Mn-hyperfine structure recently reported for that state. At longer times S0 is further reduced to a state which has the properties of the S-1 state, in that single-turnover illumination restores the S0 signal. The Mn(II)-Mn(III) state forms after the S-1 state and is tentatively assigned to an (iso)S-2 state, although lower states or a modified S-1 state cannot be excluded at present. Following removal of NO 60-65% of the initial S2 multiline signal size or the O2-evolving activity can be restored. The data provide for the first time EPR information on a state lower than S0. Furthermore, the low-temperature NO treatment provides a simple means for the selective population of the S0, S-1 and the Mn(II)-Mn(III) states.     

Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. NiFe(CN)2CO, Biology's way to activate H2

Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with M?ssbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M. , Duin, E. C., Albracht, S. P. J., and Münck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.     

13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase

Side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta 5-3-ketosteroid isomerase (EC 5.3.3.1, homodimer, M(r) = 26.8 kDa) have been examined by measurements of longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz (proton frequencies). The data, analyzed using the model-free formalism, yielded an optimized correlation time for overall molecular rotation (tau m) of 17.9 ns, in agreement with the result (18 ns) of fluorescence anisotropy decay measurements [Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415-7422] and Stokes' law calculation (20 ns). The order parameter of the side chain C epsilon of Tyr-14 (S2 = 0.74), which is a measure of the restriction of its high-frequency (nanosecond to picosecond) motion, was significantly lower than that of the backbone C alpha (S2 = 0.82), indicating greater restriction of backbone motion. Upon binding of the steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S2 of the side chain C epsilon increased from 0.74 to 0.86, while that of the backbone C alpha did not change significantly. Thus, in the steroid complex, the amplitude of high-frequency side chain motion of Tyr-14 became more restricted than that of its backbone which could lower the entropy barrier to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to kcat were detected by exchange contributions to transverse relaxation of both C epsilon and C alpha. Steroid binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and product release, but decreased the exchange contribution to transverse relaxation of the backbone.     

Mutagenic potential of stereoisomeric bay region (+)- and (-)-cis-anti-benzo[a]pyrene diol epoxide-N2-2'-deoxyguanosine adducts in Escherichia coli and simian kidney cells

We have investigated the mutagenic potential of site-specifically positioned DNA adducts with (+)- and (-)-cis-anti stereochemistry derived from the binding of r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) to N2-2'-deoxyguanosine (G1 or G2) in the sequence context 5'TCCTCCTG1 G2CCTCTC. BPDE-modified oligodeoxynucleotides were ligated to a single-stranded DNA vector and replicated in Escherichia coli or simian kidney (COS7) cells. The presence of (+)- or (-)-cis adduct strongly reduced the yield of transformants in E. coli, and the yield was improved by the induction of SOS functions. Both adducts were mutagenic in E. coli and COS cells, generating primarily G --> T transversions. In E. coli, the (-)-cis adduct was more mutagenic than the (+)-cis adduct, while in COS cells, both adducts were equally mutagenic. These results were compared with those obtained with stereoisomeric (+)- and (-)-trans adducts [Moriya, M., et al. (1996) Biochemistry 35, 16646-16651). In E. coli, cis adducts, especially (-)-cis adducts, are consistently more mutagenic than the comparable trans adduct. In COS cells, trans adducts yield higher frequencies of mutations than the two cis adducts and, with the exception of the high-mutation frequency associated with the (+)-trans adduct at G2, relatively small differences in mutation frequencies are observed for the three other adducts. In E. coli, mutation frequency is a pronounced function of adduct stereochemistry and adduct position. These findings suggest that the fidelity of translesional synthesis across BPDE-dG adducts is strongly influenced by adduct stereochemistry, nucleotide sequence context, and the DNA replication complex.     

Measuring DNA synthesis rates with [1-13C]glycine

We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.     

Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.     

The C2 catalytic domain of adenylyl cyclase contains the second metal ion (Mn2+) binding site

Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.     

Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function

Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.     

NMR studies of Ca2+ binding to the regulatory domains of cardiac and E41A skeletal muscle troponin C reveal the importance of site I to energetics of the induced structural changes

Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" of the structure [Gagné, S. M., et al. (1995) Nat. Struct. Biol. 2, 784-789], which is typical of Ca2+-regulatory proteins. However, the recent structures of the E41A mutant of skeletal troponin C (E41A sNTnC) [Gagné, S. M., et al. (1997) Biochemistry 36, 4386-4392] and of cardiac muscle troponin C (cNTnC) [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221] reveal that both of these proteins remain essentially in the "closed" conformation in their Ca2+-saturated states. Both of these proteins are modified in Ca2+-binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca2+-induced structural transition, Ca2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1H and two-dimensional {1H,15N}-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca2+ titration of E41A sNTnC permits us to assign the order of stepwise binding as site II followed by site I and reveals that the mutation reduced the Ca2+ binding affinity of the site I by approximately 100-fold [from KD2 = 16 microM [sNTnC; Li, M. X., et al. (1995) Biochemistry 34, 8330-8340] to 1.3 mM (E41A sNTnC)] and of the site II by approximately 10-fold [from KD1 = 1.7 microM (sNTnC) to 15 microM (E41A sNTnC)]. Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2.6 microM. The Ca2+-induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca2+. These measurements allow us to dissect the mechanism and energetics of the Ca2+-induced structural changes.     

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes ( S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.     

Plasma kinetics of vitamin A in humans after a single oral dose of [8,9,19-13C]retinyl palmitate

The kinetics of vitamin A and its major metabolites were investigated in humans. Eleven healthy male subjects ingested 105 mumol (100,000 IU) of [8,9,19-13C]retinyl palmitate in an oily solution. Twenty-seven blood samples were collected during the 1-week study. Plasma samples were analyzed for retinyl esters and for [12C]- and [8,9,19-13C]retinol. Retinol isotopes were quantified using a newly developed GC-MS method. Total retinyl esters peaked at about 4.45 mumol/L from 3.5 to 12 h after dosing. As a result of the perturbation of the tracee system, the plasma concentration of [12C]retinol increased and then decreased as the concentration of [8,9,19-13C]retinol increased, indicating rapid distribution kinetics. A broad single peak (1.16 +/- 0.32 mumol/L) was observed for [8,9,19-13C]retinol at about 10 to 24 h postdose; this likely reflects hepatic secretion of [8,9,19-13C]retinol associated with retinol-binding protein. Then, declining levels of the tracer and increasing levels of the tracee were observed. At its peak, the ingested [8,9,19-13C]retinol reached about 51% of the observed total plasma retinol concentration. This percentage dropped to 13.4% on day 7 indicating slow final elimination from plasma. Our data support the concept that the liver follows the principle "last in/first out' in maintaining vitamin A homeostasis.     

13C relaxation and dynamics of the purine bases in the iron responsive element RNA hairpin

The iron responsive element (IRE) RNA hairpin contains a conserved six-nucleotide loop. The NMR structure of this loop showed that the positions of four of its bases are not tightly constrained, while the remaining two are hydrogen-bonded [Laing, L. G., and Hall, K. B. (1996) Biochemistry 35, 13586]. To investigate the flexibility of the RNA in the loop and in the stem, 13C NMR relaxation methods have been used to describe the dynamics of the purine bases. IRE hairpins containing [13C]guanosine and [13C]adenosine are used in NMR experiments to measure T1, T1rho, and NOE values of the bases as a function of temperature (20-37 degreesC). Data are analyzed using the Lipari-Szabo model-free formalism [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546] to determine order parameters and time scales of the motion. Results indicate that the purine bases in the stem have order parameters that are independent of temperature, although they show evidence of both fast (6-40 ps) motions and slower motions at 37 degreesC. The three purines in the loop exhibit increasingly complex motions with long (nanoseconds) correlation times as the temperature increases, suggesting that the loop structure has become disordered.     

Determination of 14CO2 in breath and 14C in stool after oral administration of cholyl-1-[14C]glycine: clinical application

Twenty patients with intestinal bacterial overgrowth and 20 control subjects were investigated for bile acid deconjugation, by measuring 14CO2 in the breath after cholyl-1-[14C]glycine administration. 14CO2 output/24h was 11.0 +/- 5.2% (mean +/- SD) in controls and 54.2 +/- 14.0% (mean +/- SD) in bacterial-overgrowth patients (P less than .001). 14CO2 excretion rate in 12h, when normalized to 100% of the dose at the 12th hour, gave an even finer discrimination between the two groups (no false responses). 14C in stool, analyzed in 20 malabsorption patients and 20 controls by two different techniques, was 6.6 +/- 4% and 31.38 +/- 21.7% (mean +/- SD), respectively. Results by the two different techniques described here correlated well (r = .99). Bile acid malabsorption was in reasonable agreement (r = .67) with percentage of "chenoid" (chenodeoxycholic acid plus ursodeoxycholic acid) in the stool by gas-liquid chromatography; a poorer correlation was observed when "chenoid" plus "choloid" (cholic acid plus its epimers) were plotted vs. -4C in stool (r = .57, n = 15).     

L-[1-(13)C] Phenylalanine oxidation as a measure of hepatocyte functional capacity in end-stage liver disease

BACKGROUND: Liver disease is associated with impaired metabolism of these amino acids phenylalanine and tyrosine. Decreased metabolism of these amino acids leads to abnormal plasma elevations and impaired clearance rates. We have developed a noninvasive breath test that measures hepatic cytosolic enzyme activity. METHODS: The rate of hepatic phenylalanine metabolism was quantitatively calculated from the appearance of 13CO2 in the breath using the nonradioactive tracer L-[1-(13)C]phenylalanine. RESULTS: Normal controls (n = 47) oxidized phenylalanine more than twice that of end-stage liver disease patients (n = 117). Significant differences in the percent of phenylalanine oxidized per hour (mean +/- SEM) were found between controls (7.08% +/- 0.33%, 95% CI: 6.42%-7.74%) and Child Pugh classification patients, class A (4.96% +/- 0.69%, 95% CI: 3.50%-6.42%), class B (2.88% +/- 0.13, 95% CI: 2.39%-3.38%) and class C (1.75% +/- 0.13, 95% CI: 1.50%-2.01%). The phenylalanine breath test score significantly correlated with albumin levels, prothrombin time and total bilirubin. CONCLUSION: We have demonstrated that phenylalanine oxidation is significantly decreased with end-stage liver disease and is correlated with the best clinical measures of liver disease.