Glucosyl transferase activity of bovine galactosyl transferase.

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.

Immobilized bovine lactose synthase. A method of topographical analysis of the active site.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.

Mapping fatty acid binding to beta-lactoglobulin: Ligand binding is restricted by modification of Cys 121.

Native beta-lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 microM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4'-bis 1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) binding to the protein, indicating that the incorporated appendages do not perturb the internal hydrophobic site within the beta-barrel of Blg (i.e., the retinoid site is unaffected). On the other hand, methylation of Cys 121, reduces the affinity of Blg for palmitic acid by 10-fold as monitored by intrinsic fluorescence. Modification of the Cys 121 with methylmethanethiosulfonate or a thiol-specific spin label appears to either further weaken or totally eliminate fatty acid binding, respectively, due to steric hindrance. Furthermore, this binding pattern has been independently verified using a spin labeled fatty acid analog and monitoring ESR as well as by bis-ANS fluorescence when bound to the protein. These results suggest that fatty acids bind at the "external site" of beta-lactoglobulin, between the sole alpha-helix and the beta-barrel. In addition, structural stability studies of native and chemically modified Blg appear to confirm this observation as well.

Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.

alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 degrees C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.

alpha-Lactalbumin: structure and function.

Small milk protein alpha-lactalbumin (alpha-LA), a component of lactose synthase, is a simple model Ca(2+) binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca(2+) binding site, which binds Mg(2+), Mn(2+), Na(+), and K(+), and several distinct Zn(2+) binding sites. The binding of cations to the Ca(2+) site increases protein stability against action of heat and various denaturing agents, while the binding of Zn(2+) to the Ca(2+)-loaded protein decreases its stability. Functioning of alpha-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of alpha-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.

Unique in vivo applications of spin traps.

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.

Detection of free radicals in blood by electron spin resonance in a model of respiratory failure in the rat.

Previous work has suggested that a free radical mechanism is involved in some types of muscle fatigue and that there can be free radicals released extracellularly. Because muscle fatigue may be an important factor in respiratory failure, the authors tested the hypothesis that increased concentrations of free radicals could be detected in the blood of animals undergoing severe resistive loading to respiratory failure. An ex vivo spin trapping technique with alpha-phenyl-N-tert-butylnitrone (PBN) was used to investigate the possible formation of free radicals in systemic blood samples by electron spin resonance (ESR) spectrometry. After 2.5-3 h of severe inspiratory resistive loading with 70% supplemental inspired oxygen, free radical levels in the form of PBN-adducts were found to rise significantly over the control group breathing room air and the control group breathing 70% oxygen (p < 0.05, N = 8). There were no significant differences between control groups breathing room air and control groups breathing 70% oxygen. This study presents direct evidence that free radicals are produced ex vivo and that they can be detected in the systemic circulation due to excessive resistive loading of the respiratory muscles.

Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state.

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.

NMR spin trapping: detection of free radical reactions with a new fluorinated DMPO analog.

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.

Binding of fluorescent and spin-labeled C-terminal hirudin analogs to thrombin.

Synthetic peptides based on the sequence of the negatively charged carboxyl tail of hirudin exhibit anticoagulant activity. Several antithrombin agents are being developed by chemical and structural optimization of these "hirupeptides". The present work demonstrates the design and use of novel spin-labeled and fluorescent-labeled C-terminal hirudin analogs to study the interactions of these antithrombin agents with thrombin in solution. Three labeled hirulabels were synthesized based upon the amino acid sequence of the antithrombin agent MDL 28050, X-NH-(CH2)7-CO-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH, where X = anthraniloyl, 1,5-dansyl, or 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl. The modifications did not significantly alter the potency of these inhibitors which showed Ki values of 100 nM. Their interactions with human and bovine thrombin were studied by ESR and fluorescence techniques. The spin-labeled hirupeptide was able to discern subtle differences in binding to human versus bovine thrombin. The 8-aminooctanoic acid spacer arm placed the nitroxide moieties near the active site, near regions of the autolysis loops which differentiates between human alpha- and gamma-thrombin. It was also able to discern paramagnetic quenching and fluorescence energy transfer interactions, respectively, between covalently attached spin labels and fluorescent probes at the active site Ser 195 and the fluorophore on the hirupeptide.

Magnetic resonance and biochemical studies during pentylenetetrazole-kindling development: the relationship between nitric oxide, neuronal nitric oxide synthase and seizures.

The major aim of this study was to elucidate the role of nitric oxide (NO) in the development of pentylenetetrazole (PTZ)-kindling as an animal model of primary generalized epilepsy. The daily administration of PTZ is associated with an increase in the amount of neuronal nitric oxide synthase (nNOS). NO generation was measured directly by in vivo and ex vivo electron paramagnetic resonance on rodents undergoing progressive convulsions. We found that primary generalized epilepsy is caused by NO induction during the persistent up-regulation of nNOS expression, but that NO induction is not associated with severe generalized seizures following long-term kindling phenomena after PTZ withdrawal. Morphological changes in the brain structure of rats were measured by magnetic resonance imaging during epileptic convulsions induced by repetitive administration of PTZ. Cerebellum volume for kindled rats decreased 20% but not in rats treated with the nNOS inhibitor, 3Br-7NI, suggesting that generation of NO in the cerebellum is related to decrease in cerebellum volume following PTZ-kindling.

Subtle differences in active site structure between bovine and human thrombins: ESR and fluorescence studies.

The primary structures of bovine and human alpha-thrombins are highly homologous yet their x-ray structures are not yet complete enough to distinguish differences. In order to probe and compare their dynamic conformations in solution, we examined bovine and human alpha-thrombins with a series of active site directed fluorosulfonylphenyl spin labeled inhibitors and fluorophores which probe a region within 10-15 A of the catalytic serine residue. Overall, the nitroxide moieties were more immobilized in the bovine vs human derivatives reflecting either more apolar binding regions or steric obstructions to the motion of the nitroxide in bovine thrombin. Most of the labels which distinguish indole (apolar ligand) binding in human thrombin were found to display similar interactions in bovine thrombin, although slight differences in the general topography of this region were suggested. The two active site directed fluorophores, dansyl fluoride and p-nitrophenyl anthranilate showed differences in both lambda emmax and lambda exmax of the complexes with bovine and human-alpha-thrombin, respectively, Several of the effects observed i.e., ligand binding (indole or benzamidine) and the subtle hydrophobic interactions between the nitroxide moiety and the protein active site would be difficult to assess from an x-ray structure determination alone.

Hirudin C-terminal fragments inhibit thrombin induced neutrophil chemotaxis.

Since native hirudin blocks the thrombin induced chemotaxis response of neutrophils, we examined whether hirudin C-terminal peptides were also capable of this inhibition. The studies showed that thrombin induced human neutrophil chemotaxis was effectively blocked by the C-terminal hirudin peptide analogs, Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln (12-mer[54-65]) and Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- Tyr- Leu-Gln (21-mer[45-65]). Furthermore, neither peptide had an effect on formyl-L-methionyl-L-leucyl-L-phenylalanine induced chemotaxis. The results suggest that binding of the hirudin C-terminal peptides block the thrombin chemotactic domain.

Synthesis and evaluation of 19F labeled sulfonyl fluorides as probes of protease structure: alpha-chymotrypsin.

Active site Ser-195-fluorine-labeled derivatives of alpha-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new 19F spin labels varied in the position of the -CF3 substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of "tosyl-chymotrypsin" were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.

The spin-label approach to labeling membrane protein sulfhydryl groups.

Labeling experiments with viable membrane preparations are optimally undertaken where conditions are extremely mild (minimal excess of modification reagent, very short reaction times) in order to reduce the possibilities of introducing artifacts (cell aging and death, denaturation, etc.). Furthermore, specificity of labeling is a critical requirement where the system is complex and contains many proteins. The spin label IV described above offers several advantages over previously employed reagents. By virtue of its reversibility and ease of quantitation, one may repetitively label and remove label from the same membrane preparation in order to check reproducibility while using the same sample as a control.

Biological reduction of aromatic nitroso compounds: evidence for the involvement of superoxide anions.

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O2-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.

Biological reactions of peroxynitrite: evidence for an alternative pathway of salicylate hydroxylation.

Salicylate hydroxylation has often been used as an assay of hydroxyl radical production in vivo. We have examined here if hydroxylation of salicylate might also occur by its reaction with peroxynitrite. To test this hypothesis, we exposed salicylate to various concentrations of peroxynitrite, in vitro. We observed the hydroxylation of salicylate at 37 degrees C by peroxynitrite at pH 6, 7 and 7.5, where the primary products had similar retention times on HPLC to 2,3- and 2,5-dihydroxybenzoic acid. The product yields were pH dependent with maximal amounts formed at pH 6. Furthermore, the relative concentration of 2,3- to 2,5-dihydroxybenzoic acid increased with decreasing pH. Nitration of salicylate was also observed and both nitration and hydroxylation reaction products were confirmed independently by mass spectrometry. The spin trap N-t-butyl-alpha-phenylnitrone (PBN), with or without dimethyl sulfoxide (DMSO), was incapable of trapping the peroxynitrite decomposition intermediates. Moreover, free radical adducts of the type PBN/.CH3 and PBN/.OH were susceptible to destruction by peroxynitrite (pH 7, 0.1 M phosphate buffer). These results suggest direct peroxynitrite hydroxylation of salicylate and that the presence of hydroxyl radicals is not a prerequisite for hydroxylation reactions.

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.

An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes.

alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide: prospects and perspectives of in vivo detection by L-band EPR spectroscopy.

This paper discusses, compares and evaluates various in vivo EPR methods of detection of nitric oxide (NO). In particular the various classes of agents are: Fe(II)-dithiocarbamate derivative complexes of MGD (N-methyl-D-glucamine dithiocarbamate) and DTCS [N-(dithiocarboxy)sarcosine], stable imidazolineoxy N-oxides and nitronyl N-oxides, and NO-sensitive chars. As a specific example direct, real-time, in vivo measurements of nitric oxide (NO) in mice are described with the water soluble metal chelator complex (MGD)2-Fe(II), as monitored at L-band EPR. The three-line EPR spectrum of [(MGD)2-Fe(II)-NO] was observed non-invasively in both control animals injected with the preformed product [(MGD)2-Fe(II)-NO] and from lipopolysaccharide (LPS) treated mice subsequently injected with (MGD)2-Fe(II) complex. The [(MGD)2-Fe(II)-NO] spectrum was markedly suppressed after administration of phenyl N-tert-butyl nitrone (PBN) prior to LPS injection as PBN inhibits the expression of inducible nitric oxide synthase (iNOS). When 15N-arginine was administered to LPS-treated mice, an EPR spectrum consisting of both three- and two-line EPR signals (due to (MGD)2-Fe(II)-14NO and (MGD)2-Fe(II)-15NO respectively) was observed, confirming that the trapped NO was generated through the NOS enzyme and not other chemical routes.