Dissociation kinetics of hemocyanin from Octopus vulgaris.

The native form of hemocyanin (Hc) from Octopus vulgaris can be completely dissociated, at alkaline pH and in the presence of EDTA, from 49S decamers to 11S monomers. The kinetics of this process was studied, using a Bio-Logic stopped flow system, by following the time dependence of the 450-nm light intensity, scattered at 90 degrees, in the 7.9-8.8 pH range. All experimental traces were best fitted by a sum of three exponential decay functions. We then tried to best fit these decay functions with a series of kinetic models, the best of them resulting in one whose dissociation of decamers to monomers takes place in three consecutive and irreversible steps, with a highly cooperative step concerning dissociation of octamers to dimers, which appears to be the only intermediate species. This model was preferred over several others, not only for the best norm value but also for the best accordance between each calculated and experimental kinetic parameter (rate constants and amplitudes). Although other more complex models may be considered, our best fit model represents the simplest one, which is able to describe the observed dissociation kinetics.

Alkaline denaturation and partial refolding of pepsin investigated with DAPI as an extrinsic probe.

The binding parameters of DAPI to porcine stomach pepsin have been described in the previous article in this issue (A. Mazzini et al.). Here we exploit the differences in the spectroscopic (fluorescence and circular dichroism) properties of DAPI bound to either native or alkali denatured pepsin. We follow the kinetics of pepsin denaturation around neutrality (pH range 6.8-7.4), at several phosphate buffer ionic strengths (range 0.02-0.25). The dependence of the apparent dissociation rate constant on pH clearly shows that the rate limiting step follows the dissociation of about three acidic protein residues. The accelerating effect by ionic strength we observed can be accounted for by a simple treatment based on both transition state theory and Debye-Hueckel's limiting law. Furthermore, when a solution of pepsin, rapidly denatured at pH 7, is reacidified to a pH between 4.5 and 5.5, a substantial recovery of protein secondary structure, with no enzymatic activity, is observed, judging by the far UV circular dichroism of the protein. This process of partial refolding can easily be followed using DAPI as an extrinsic reporter group, able to monitor the kinetics of formation and decay of a highly fluorescent intermediate. This process becomes faster at a lower pH, at least in the limited range investigated (pH 4.5-5.5), in which the refolded protein does not aggregate, but, in contrast to unfolding, is almost independent in ionic strength.

Interaction of DAPI with pepsin as a function of pH and ionic strength.

The fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), extensively used with nucleic acids, has also recently been used with membranes and proteins (Favilla et al., Biophys. Chem., 46 (1993) 217-226 and Mazzini et al., Biophys. Chem. 52 (1994) 145-156, respectively). The spectroscopic changes of DAPI observed, namely a considerable enhancement of fluorescence, induced circular dichroism (CD) and absorbance spectral shifts, have been exploited to study the binding of this dye to both native and alkali denatured pepsin. Fluorescence and CD titrations show that nearly two molecules of DAPI bind to either native or alkali denatured pepsin with pH and ionic strength dependent Kd values, whereas absorbance titrations evidentiate an interaction characterized by a lower affinity and a larger number of binding sites. Therefore two kinds of interaction are proposed: a specific one, involving both hydrophobic and electrostatic forces; and a non-specific one, involving surface protein negative charges only. Finally, the behaviour of DAPI as a competitive inhibitor and the remarkable effect of pepstatin A, a specific inhibitor of pepsin, on both the CD and fluorescence spectra of DAPI in the presence of pepsin, show the involvement of the protein active site in the interaction.

Specificity of zinc binding to myelin basic protein.

Zn2+ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of 65Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

The interaction of DAPI with phospholipid vesicles and micelles.

The interactions of the dye 4',6-diamidino-2-phenylindole (DAPI) with phospholipids ordered in single bilayer vesicles of dioleylphosphatidylserine (DOPS) or dimyristoylphosphatidylcholine (DMPC) or micelles of monomyristoylphosphatidylcholine (MPC) have been investigated. Somewhat unexpectedly, the binding of this dye to such ordered structures is not affected by the ionic strength of the external medium, which suggests an embedding of DAPI into the hydrocarbon phase. The fluorescence enhancement of DAPI bound can be accomodated within a model previously proposed for the behaviour of DAPI bound to proteins (Mazzini et al., Biophys. Chem. 42 (1992) 101). From both static and dynamic anisotropy measurements, bound DAPI results severely restricted in its rotational freedom but insensitive to the temperature dependent phase transition of the saturated DMPC vesicles. The considerable tightness and specificity of the interactions between DAPI and ordered phospholipids are also deduced from preliminary fluorescence quenching studies (reduced accessibility of iodide ions towards DAPI bound and quenching effects by the chaotrop Nonidet P-40).

Fluorescence studies on the conformation of litorin in solution and in the presence of model membranes.

The conformation of the nonapeptide hormone litorin has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence. The results obtained show that, in buffer, the hormone probably exists in a collection of flexible conformers, slowly interconverting between them. The marked changes observed in fluorescence spectra and lifetimes upon addition of dimyristoylphosphatidylserine vesicles clearly show that the peptide interacts with lipids assuming lipid specific conformations. Interestingly, no significative spectroscopic changes are produced by exposure to dimirystoylphosphatidylcholine vesicles both in the gel and liquid-christalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.

The interaction of 4', 6-diamidino-2-phenylindole (DAPI) with pepsin.

We recently found that the fluorescent dye DAPI, well-known for its use with nucleic acids, is also able to interact with proteins as well as ordered phospholipids assemblies. The interaction of DAPI with pepsin under different conditions of pH and ionic strength was studied with fluorescence and circular dichroism techniques. From a comparison of the results obtained, the interaction appears to be rather tight and specific, dependent on both electrostatic and hydrophobic forces, and able to probe the tridimensional conformation of the protein.

The binding of 4',6-diamidino-2-phenylindole to bovine serum albumin.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to bovine serum albumin (BSA) has been investigated between pH 6 and 8, in 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations and the results analyzed according to a procedure previously reported (R. Favilla and A. Mazzini, Biochim. Biophys. Acta 788 (1984) 48). The dye binds to the protein with a blue shift of about 4 nm in its fluorescence emission maximum, but with an enhancement factor of 10 of its fluorescence quantum yield. The dissociation constant decreases from 100 microM to 54 microM as the pH is increased from 6 to 8, with a constant number of nearly three equivalent binding sites. The complete displacement of DAPI bound to BSA by Ca2+ suggests a possible specificity of this substantially electrostatic interaction. The fluorescence decay of DAPI bound to the protein shows a double exponential kinetics, with a tau 1 = 0.97 ns and tau 2 = 2.78 ns. These results, compared with those obtained for DAPI alone, tau 1 = 0.16 ns and tau 2 = 2.8 ns, are rationalized in terms of two different rotamers of DAPI. Both rotamers are able to bind to the protein, but only one of them undergoes an intramolecular proton transfer, from the 6-amidinium group to the indole aromatic ring, in the excited singlet state of DAPI alone. When DAPI interacts with BSA this transfer does not occur and consequently a large increase of fluorescence is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cell growth and host protein synthesis during HIV infection in vitro.

During HIV infection of CEM cells cultured in vitro, significant differences in growth rate and protein turnover were observed with different viral preparations. There was a significant inhibition of proliferation after infection with crude HIV supernatants. On the other hand, infection with purified HIV particles obtained by filtration, differential centrifugation, and isopycnic sedimentation led to a progressively increasing stimulation of cell growth. This early stimulation was prevented by neutralizing the virus with soluble CD4 molecules. Study of cell growth in the presence of a purified membrane preparation indicated that membrane fragments contaminating the crude HIV supernatant were responsible for the observed growth inhibition. Interestingly, the stimulation of proliferation was also observed with heat-inactivated virus or after inhibition of viral replication with ZDV. In the presence of purified HIV virions, the rate of general protein synthesis was not inhibited, as is usually observed with crude viral supernatants. However, a marked reduction in protein content and increased protein degradation was found in cultures infected with either crude or purified HIV preparations.

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells.

Commercial serum albumins have been found to be able to stimulate the proliferation of Balb/c 3T3 cells transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with crystalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.

Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase.

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.

Fluorescence stopped-flow studies on the binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

Fluorescence stopped-flow techniques have been used to investigate the binding of the oxidised coenzyme eNAD to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate, a substrate analogue, by following the transient kinetics of fluorescence intensity changes associated with changes in the binding of 1,N6-etheno-NAD (eNAD) to the enzyme, using displacement by NAD, NADP, ADP or GTP. Computer simulations of the various kinetic models provide a detailed picture of the molecular interactions between the active site (site I) and regulatory sites (sites II and III), specific for adenine and guanine nucleotides, respectively. The observed enhancement of the eNAD dissociation rate constant from site I can satisfactorily be accounted for as being due to the effect of ADP or NAD (and to a lesser extent NADP) binding to site II. This provides a mechanism for the allosteric activation of this enzyme via a predominantly intrasubunit interaction. By contrast the isomerisation of the enzyme induced by ADP alone is markedly slowed down by the occupancy of site I by eNAD in the presence of glutarate. The inhibitory effect of the allosteric effector GTP correlates with a tightening of eNAD binding, causing a decrease of the coenzyme dissociation rate constant followed by a slow isomerisation of the enzyme complexed with eNAD and glutarate.

The effect of guanidinium chloride on the self-association of bovine liver glutamate dehydrogenase: a gel filtration study.

The associative behaviour of bovine liver glutamate dehydrogenase has been studied by gel chromatography at neutral pH in 1 M guanidinium chloride and 1 M sodium chloride. In guanidinium chloride both the elution volume and the elution profile of the enzyme are independent of protein concentration, whereas in sodium chloride they are strongly dependent on it. In NaCl the enzyme behaves as expected according to the well established random association model, whereas in guanidinium chloride it appears to have completely lost the self-associative property. Furthermore, since the elution volume of the enzyme in guanidinium chloride corresponds to that of an hexamer, trimer formation reported to occur in these conditions is not confirmed by this technique.

DNA-4'-6-diamidine-2-phenylindole interactions: a comparative study employing fluorescence and ultraviolet spectroscopy.

DAPI is a drug that interacts with double-stranded nucleic acids, binding preferentially to A + T base pairs. The interaction is not intercalative, therefore providing a useful model for mimicking the effect of functional molecules in modifying specific sites, namely, A + T segments, of significance in gene expression. Knowledge of the nature of such interaction has been enriched by additional information obtained from comparative analysis of the data acquired by uv spectroscopy and fluorescence. Two classes of binding sites, defined by different apparent affinity constants and numbers of binding sites, are evident. All types of interaction are dependent on the nucleic acid/dye ratio and on the ionic strength of the medium.

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique.

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

The binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

The binding of 1,N6-etheno-NAD (epsilon NAD) to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations. epsilon NAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound epsilon NAD relative to free epsilon NAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from Fmax/F0 ratio values obtained after extrapolation from double reciprocal plots of 1/delta F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 mumol polypeptide chains/liter), the dissociation constant of epsilon NAD increases progressively from a plateau value of about 50 microM to about 100 microM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards epsilon NAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards epsilon NAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it (Kappd around 6 microM with saturating GTP and around 300 microM with saturating ADP). Furthermore, in the case of GTP, both GTP and epsilon NAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a Kappd approximately equal to 30 microM for the binding of GTP to glutamate dehydrogenase saturated with epsilon NAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of epsilon NAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.

The reaction of bovine glutamate dehydrogenase with periodate-oxidised ADP.

1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site.

The peroxidatic reaction catalyzed by horse liver alcohol dehydrogenase. 2. Steady-state kinetics and inactivation.

The results of steady-state kinetic measurements on the initial rate of the peroxidatic reaction between beta-NAD+ and hydrogen peroxide, catalyzed by horse liver alcohol dehydrogenase, at pH 7 are described. A simple sequential mechanism is deduced from graphical analysis of the data plotted according to Eadie-Augustinsson-Hofstee primary plots and the values of the true kinetic parameters KmNAD, KmH2O2 and V are estimated from the corresponding secondary plots. Ethanol has been found to compete with hydrogen peroxide for the same enzyme active site. During the catalytic process a progressive inactivation of the enzyme occurs caused by H2O2. The rate law of this process is quantitatively described at pH 7 both in the absence and in the presence of NAD+. The coenzyme has been found to protect the enzyme against inactivation by H2O2, which oxidized essential cysteine residues. The results obtained from the study of both catalytic and inactivating processes are finally rationalized on the basis of a general mechanistic scheme.

The peroxidatic reaction catalyed by horse liver alcohol dehydrogenase. 3. Nuclear magnetic resonance spectroscopic study of NADX.

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324-329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide. From the 1H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5')diphospho(5)-beta-D-ribose-NH-CH = C(CHO)-CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX. A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed.