Steven H. Grossman

In vitro and in vivo studies investigating possible antioxidant actions of nicotine: relevance to Parkinson’s and Alzheimer’s diseases

An inverse relationship appears to exist between cigarette smoking and the risk of Parkinsons and Alzheimers diseases. Since both diseases are characterized by enhanced oxidative stress, we investigated the antioxidant potential of nicotine, a primary component of cigarette smoke. Initial chromatographic studies suggest that nicotine can affect the formation of the neurotoxin 6-hydroxydopamine resulting from the addition of dopamine to Fentons reagent (i.e., Fe2+ and H2O2). Thus, under certain circumstances, nicotine can strongly affect the course of the Fenton reaction. In in vivo studies, adult male rats being treated with nicotine showed greater memory retention than controls in a water maze task. However, neurochemical analysis of neocortex, hippocampus, and neostriatum from these same animals revealed that nicotine treatment had no effect on the formation of reactive oxygen species or on lipid peroxidation for any brain region studied. In an in vitro study, addition of various concentrations of nicotine to rat neocortical homogenates had no effect on lipid peroxidation compared to saline controls. The results of these studies suggest that the beneficial/protective effects of nicotine in both Parkinsons disease and Alzheimers disease may be, at least partly, due to antioxidant mechanisms.

Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13‐fold and 4‐fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the KD for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate‐induced conformational change. Creatine kinase labeled with two molecules of 5′‐iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle‐type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme‐MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 ‐ (4 ‐ maleimidylphenyl) ‐ 7 ‐ diethylamino ‐ 4 ‐ methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.

Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.

Purification and characterization of arginine kinase from the sea cucumber Caudina arenicola

Abstract 1. 1. Arginine kinase from Caudina arenicola, a Pacific sea cucumber, has been purified from muscle to apparent homogeneity and shown to be a dimeric protein of mol. wt 80,000. 2. 2. The isoelectric point of arginine kinase from Caudina arenicola occurs at pH 7.8, unusually high compared to the monomeric arginine kinase from lobster muscle, but similar to muscle type creatine kinase from vertebrate. The enzyme is also unusually unstable to heat, losing half its activity after exposure to 40°C for about 10 min. The pH optimum of arginine kinase is approximately 7.9. 3. 3. Kinetic analysis reveals Michaelis constants of 1.3 mM for MgATP and 0.5 mM for arginine. The dissociation constant for arginine from the binary enzyme—substrate complex is four times greater than for the ternary complex containing MgATP. Dissociation constants for MgATP from the binary and ternary complexes do not differ significantly. Inhibition of arginine kinase by MgADP is potentiated by nitrate anion, presumably by formation of a transition state analog in which nitrate stabilizes the dead-complex between enzyme, MgADP and arginine. 4. 4. Arginine kinase from Caudina arenicola and creatine kinase from rabbit brain will hybridize in vitro to form a bifunction dimer.

SUBUNIT CONFORMATION AND DYNAMICS IN A HETERODIMERIC PROTEIN : STUDIES OF THE HYBRID ISOZYME OF CREATINE KINASE

Several physical properties of creatine kinase (EC 2.7.3.2) isozymes MM (CK-MM, muscle-type) and BB (CK-BB, brain-type), both homodimers, and isozyme MB (CK-MB), a heterodimer, were compared to determine how formation of the hybrid modifies subunit conformation and dynamics. Circular dichroic spectra revealed additional alpha-helical content for the hybrid isozyme. Double-beam absorption difference spectra between CK-MB and a stoichiometric mixture of CK-MM and CK-BB revealed decreased exposure of intrinsic chromophores in the hybrid. The relative intensity of the intrinsic fluorescence of CK-MB was between the two homodimers, but was 16% closer to the less fluorescent CK-MM. Steady state anisotropy spectra and decay of the anisotropy of CK derivatized on a single subunit with the fluorescent sulfhydryl reagent 5-[2-(iodoacetyl)amino-ethyl]aminonaphthalene-1-sulfonate indicated that the derivatized sites are more flexible in the heterodimer. The slow component in the anisotropy decay suggests that hybridization results in a small increase in the packing density or contraction of overall conformation of the B-subunit. The KM for MgATP with singly derivatized CK-MB was the same as the KM for the native enzyme. However, derivatization of a single subunit caused the Vmax to decrease by greater than 50%, which indicates that subunit-subunit interactions may modulate the activity of CK. A model for assembly of CK-MB is proposed which includes subunit characteristics more similar to those found in the muscle-type homodimer than in the brain-type homodimer and increased flexibility of the active site domain of both subunits.

An equilibrium study of the dependence of secondary and tertiary structure of creatine kinase on subunit association

Several physical properties of dimeric creatine kinase in increasing concentrations of guanidine hydrochloride (GDN/HCl) were evaluated and correlated with degree of subunit dissociation, determined by isozyme competitive hybridization. Three distinct stages were observed that correlated with phases before, during and after dissociation. In 0.2 M GDN/HCl, before significant dimer dissociation, creatinine kinase has 75% of its original activity, and exhibits only small decreases in circular dichroism, intrinsic fluorescence and emission maximum. The spectral characteristics of creatine kinase (CK) derivatized with 5-[(((2-iodoacetyl)amino)ethyl)amino]naphthalene-1- sulfonate (AEDANS-CK) at the cysteine near the active site are suggestive of a slightly more non-polar environment. A decrease in steady-state anisotropy (0.140 to 0.132) was characterized by time-resolved methods. The slow component of the time-resolved anisotropy decay law, which reflects global protein rotation, is decreased only from 36.6 to 33.4 ns. The faster component decreases from 1.95 to 0.74 ns which suggests the active-site domain is more sensitive to conformation perturbation than the protein as a whole. Overall these observations suggest the subunits within the dimeric state are rather stable in dilute denaturant, but undergo a minor contraction in conformation. The region of the active site, as reported by the extrinsic fluorophore, is less polar but apparently more flexible in dilute denaturant. Between 0.5 M and 1 M GDN/HCl, most of the dimers dissociate, 63% of helical content is lost and inactivation is complete. The intrinsic fluorescence shifts 8 nm to the red and increases by 35%, indicating exposure of tryptophans to solvent and release of quenching, perhaps between residues on separate subunits. Over the same denaturant range, the spectral characteristics and lifetime of AEDANS-CK suggests less exposure of the active site to solvent. Time-resolved anisotropy measurements show that the sharp decrease in steady-state anisotropy to 0.086 is due to a decrease in macromolecular rotation to 22 ns. This may represent the rotational correlation time of a relatively intact subunit, and suggests limited subunit unfolding accompanying dissociation. Dissociation is complete in 1.5 M GDN/HCl. The subunits still retain 20% helical content in 2 M denaturant and not until 5 M GDN/HCl is all helical structure eliminated. Above 2 M GDN/HCl, AEDANS-CK exhibits sharp decreases in steady-state anisotropy, fluorescence lifetime and the long-lived component in the time-resolved anisotropy decay law. These results reveal a catastrophic loss of tertiary structure by the subunits and may define the physical properties of the random coil.

Conformational heterogeneity of creatine kinase determined from phase resolved fluorometry

Fluorescence lifetimes of dimeric rabbit muscle creatine kinase specifically dansylated at both active sites and the homologous monomeric lobster muscle arginine kinase singly dansylated were determined using phase-modulation methods with global analysis of overdetermined data sets. For both proteins, the data is adequately described by three discrete exponential decays or a Lorentzian double distributed decay. Analogue phase resolved spectroscopy also reveals the presence of at least two distinct fluorophore domains for the dansyl moieties of creatine kinase. The model fluorophore, dansyllysine, exhibits a monoexponential decay with a value that is highly solvent dependent. Because the monomeric arginine kinase exhibits essentially the same decay law as doubly derivatized dimeric creatine kinase, it is proposed that the multiple lifetimes of creatine kinase reflect two or more isomeric dimeric states and not subunit asymmetry within a conformationally homogeneous dimeric population. Exposure of arginine kinase to 6 M guanidinium chloride results in a shift to shorter lifetimes and narrowing of the lifetime distributions. Creatine kinase displays a small narrowing of the distribution, but little change in fractional populations or lifetimes. These results suggest the presence of structural elements resistant to denaturation. The longest lifetime component in the triexponential discrete decay law of doubly dansylated creatine kinase is totally unquenched by acrylamide, whereas the two shorter lifetime components exhibit limited dynamic quenching. Steady-state quenching by acrylamide is significant and reveals a sharp distinction between accessible and non accessible dansyl groups. The major mechanism for interaction between the dansyl moieties and acrylamide is, atypically, static quenching. The results are consistent with two dansyl domains, one accessible and hydrophilic according to lifetime values and the other inaccessible and hydrophobic in solvent characteristics.Energy transfer between the dansyl group and the eight tryptophan residues of dimeric creatine kinase give similar results(~ 35%) from measurements of lifetimes, steady-state donor quenching and sensitized acceptor emission. The similarity suggests that the overall flexibility of the dimeric protein is limited. The occurrence of multiple conformers of muscle creatine kinase provides an explanation for several previous observations, most notably the structural origins for compartmentation of the muscle isozyme observed in the myofibril.

An analysis of the reassembly of denatured creatine kinase from monkey brain

Creatine kinase isolated from monkey brain was characterized with respect to denaturation/inactivation and renaturation/reactivation/reassociation in order to determine the mechanism of reassembly. Enzyme unfolded in 8 M urea exhibits several characteristics of denatured protein: complete loss of enzymatic activity, decrease in intrinsic fluorescence with a red shift in the emission maximum and loss of circular dichroism at 220 nm. The renatured protein reassembles to its apparently native condition as judged by these criteria, but small differences of uncertain origin persist. Dependence of activity and fluorescence on denaturant concentration indicate that inactivation is more sensitive to urea than is unfolding; spectral changes at the intermediate urea concentrations suggest formation of aggregated protein. Upon dilution, enzyme previously exposed to 8 M urea for 40 min regains 70-80% native activity, independent of protein concentration over the range of 0.56-160 nM. Reactivation kinetics, measured using the assay mixture with and without trypsin, are independent of protein concentration, and are adequately described by a single rate constant, 3.2 X 10(-3) s-1 and 4.2 X 10(-3) s-1, respectively. Reactivation is completed 20-30 min after initiation of renaturation. Fluorescence changes during refolding are at least biphasic, exhibiting a rapid increase, then a slow decrease completed at approximately 15-20 min after initiating refolding. Reassociation is measured by competitive hybridization between dimerizing B subunits and M subunits to form MB heterodimer. The time dependent decay in heterodimer formation during competitive dimerization shows that reassociation is completed between 60 and 90 min after initiation of reassembly. These results indicate that the brain isozyme of creatine kinase, like the muscle form, is composed of subunits which do not require association for expression of catalytic activity. Furthermore, a comparison of spectral data and susceptibility to trypsin inactivation between the muscle and brain isozymes supports previous suggestions that in the native state, the brain isozyme is a conformationally looser, more open protein.

Mechanistic role of each metal ion in Streptomyces dinuclear aminopeptidase: Peptide hydrolysis and 7 × 1010-fold rate enhancement of phosphodiester hydrolysis

The dinuclear aminopeptidase from Streptomyces griseus (SgAP) and its metal derivatives catalyze the hydrolysis of the phosphoester bis(p-nitrophenyl) phosphate (BNPP) and the phosphonate ester p-nitrophenyl phenylphosphonate with extraordinary rate enhancements at pH 7.0 and 25 degrees C [A. Ercan, H. I. Park, L.-J. Ming, Biochemistry 45, (2006) 13779-13793.], reaching 6.7 billion-fold in terms of the first-order rate constant of the di-Co(II) derivative with respect to the autohydrolytic rates. Since phosphoesters are transition state-like inhibitors in peptide hydrolysis, their hydrolysis by SgAP is quite novel. Herein, we report the investigation of this proficient alternative catalysis of SgAP and the role of each metal ion in the dinuclear site toward peptide and BNPP hydrolysis. Mn(II) selectively binds to one of the dinuclear metal sites (M1), affording MnE-SgAP with an empty (E) second site for the binding of another metal (M2), including Mn(II), Co(II), Ni(II), Zn(II), and Cd(II). Peptide hydrolysis is controlled by M2, wherein the k(cat) values for the derivatives MnM2-SgAP are different yet similar between MnCo- and CoCo-SgAP and pairs of other metal derivatives. On the other hand, BNPP hydrolysis is affected by metals in both sites. Thus, the two hydrolytic catalyses must follow different mechanisms. Based on crystal structures, docking, and the results presented herein, the M1 site is close to the hydrophobic specific site and the M2 site is next to Tyr246 that is H-bonded to a coordinated nucleophilic water molecule in peptide hydrolysis; whereas a coordinated water molecule on M1 becomes available as the nucleophile in phosphodiester hydrolysis.

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.