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Biomedical subjects

M Gutman

Publications and source records attributed to M Gutman.

At least 145 records · Page 8Linked to original sources
PubMed1983

Fluorescence decay time measurements of Eu3+-ATP-enzyme complexes. Replacement of the metal hydration water by active site ligands.

Measurements of the fluorescent lifetimes of the rare earth metal Eu3+ in varying mole fractions of H2O/D2O were used to determine the hydration of the metal in the presence of ATP and/or hexokinase or chloroplast reversible ATPase. The number of water molecules coordinated to the metal in Eu3+-ATP was estimated to be 2.6; when this complex is bound to hexokinase, 1 water molecule is displaced. Upon binding to chloroplast reversible ATPase, the metal coordinates 1 water molecule while the Eu3+-ATP complex does not retain any associated solvent. These numbers are in contrast to the 9 solvent molecules coordinated to the naked metal ion. These results are discussed in reference to mechanistic and structural considerations of the two enzymes.

Adenosine Triphosphatases↗
PubMed1983

Kinetic analysis of the protonation of a surface group of a macromolecule.

The dynamics of proton transfer between a surface-attached acidic moiety and the bulk of the solution was measured using the laser-induced proton pulse technique. [Gutman, M., Huppert, D. and Pines, E. (1981) J. Am. Chem. Soc. 103, 3709-3713]. The model system for this study consists of pH indicators (either neutral red or bromcresol green) adsorbed on Brij 58 micelles, as defined targets for protonation and a non-adsorbed proton emitter (2-naphthol-3,6-disulfonate) for generation of protons in bulk. The reaction was measured with 50-ns time resolution over a time period of about 200 microseconds. The results were analyzed by a numerical solution of the coupled nonlinear differential equation corresponding with the reaction system. [Gutman, M., et al. (1983) J. Am. Chem. Soc. 105, 2210-2216]. Quantitative analysis reveals two independent reactions which govern the observed dynamics: (a) a diffusion-controlled reaction between the proton and the surface targets; (b) translocation of the protonated target between the hydration layer of the interface and a more hydrophobic one. The contribution of the translocation reaction to the dynamics of surface protonation is more pronounced for compounds like carboxylates or phenolates which increase their hydrophobicity upon protonation. Amines and azoaromatic structures are more hydrophilic in their protonated states, the dynamics of their protonation is less affected by post-protonation distribution within the microenvironments of the interface. The interrelation between the partial rate constants and the macroscopic time constants and equilibrium parameters is analyzed.

Bromcresol Green↗
PubMed1983

Unknown primary melanoma.

Of 230 melanoma patients treated during the past 8 years, 12 (5.2%) were found to have unknown primary lesions. Nine of these "unknown primary melanoma' patients presented with metastases in regional lymph nodes, one inside the parotid gland, and two presented with disseminated melanoma and no detectable primary tumor. The patients with melanoma confined to a regional lymph node underwent block dissection followed by adjuvant chemotherapy and immunotherapy. Four patients with metastasis in only one lymph node are disease free 4-6 years after diagnosis. One patient with multiple metastases in the groin is alive 8 years after lymphadenectomy. The other five patients with metastases in multiple regional lymph node died 16 months to 3 years after surgery. Both patients with disseminated melanoma succumbed to their disease within a month of diagnosis. The prognosis of unknown primary melanoma seems to be no worse than the typical melanoma at the same stage. This justifies the aggressive surgical approach to this unusual entity of melanoma.

Adult↗
PubMed1982

Direct measurement of proton transfer as a probing reaction for the microenvironment of the apomyoglobin heme-binding site.

Aromatic alcohols fluoresce at different wavelengths in their neutral (phiOH*) and anionic (phiO-*) excited states. Consequently, time-resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state. 8-Hydroxypyrene 1,3,6-trisulfonate (pK = 7.7, pK* = 0.5) is bound to apomyoglobin with a stoichiometry of 1:1. In the bound state its neutral form fluorescence increase 20-fold. The binding affinity is pH-dependent. Two protonatable groups, with pK = 6.5, participate in the stabilization of the negatively charged ligand in the binding site. The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin. Time-resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin. The rate of proton dissociation is slowed to 7% of the rate measured for the free ligand. Such a slow dissociation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637-642]. The water activity in the site is equivalent to alpha (H2O) = 0.67.

Apoproteins↗
PubMed1982

Kinetic studies of proton transfer in the microenvironment of a binding site.

Excitation of 8-hydroxypyrene 1,3,6-trisulfonate to its first electronic singlet state converts the compound from weak base (pK degrees = 7.7) into a strong acid (pK* = 0.5). The dissociation of the proton in water or dilute salt solution is a very fast reaction, K12 = 1 X 10(10) S-1. In concentrated salt solutions the dissociation is slowed as an exponential function of the chemical activity of the water in the solution. This kinetic parameter has been used to gauge the properties of the microenvironment of the binding sites of bovine serum albumin at which this compound is bound. Time-resolved fluorometry reveals two distinct steps: a rapid dissociation of the proton with tau = 300 +/- 40 ps which lasts approximately 0.5 ns, followed by a slower reaction with tau = 3.3 ns. The first rapid phase represents proton dissociation taking place in the binding site. From the rate constant K = 3.3 X 10(9) s-1 we estimate that the ability of the water molecules in the site to hydrate the ejected proton is equivalent to a salt solution with water activity of 0.85. The slow phase represents the escape of the proton from the binding site. The rate of the escape, 1.4 X 10(8) s-1, is significantly slower than diffusion-controlled dissociation. It is concluded that the shape of the site or its lowered proton conductivity do not allow a rapid escape of the proton to the bulk. Still it should be remembered that the escape of the proton is 10(5)-10(6)-times faster than a typical turnover of an enzyme.U

Arylsulfonates↗
PubMed1981

Modulation by divalent metal ions of the autocatalytic reactivity of adenosinetriphosphatase from chloroplasts.

A nonlinear, pre-steady-state initial rate of ATP hydrolysis is obtained on the addition of a divalent metal ion--ATP complex to a heat-activated coupling factor 1 isolated from chloroplasts. The acceleration of the initial rate follows first-order kinetics. The observed first-order kinetic constant (kobsd) changes with the concentration of the substrate, reaching half-maximal value at the Km for ATP hydrolysis. Preincubation of the enzyme with divalent metal ions decreases the kobsd from 1 to 0.04 s-1. Saturation of the divalent metal ion effect was obtained at the micromolar range. It is suggested that the autocatalysis is a result of early stages in ATP hydrolysis which induce conformational changes in the enzyme. Binding of divalent metal ions in the absence of ATP slows down this change.

Adenosine Triphosphatases↗
PubMed1981

Probing the micelle/water interface by a rapid laser-induced proton pulse.

The laser-induced pH jump (Gutman, M. and Huppert, D.J. (1979) Biochem. Biophys. Methods 1, 9-19) has a time resolution capable of measuring the diffusion-controlled rate constant of proton binding. In the present study we employed this technique for measuring the kinetics of protonation-deprotonation of surface groups of macromolecules. The heterogeneous surface of proteins excludes them from serving as a simple model, therefore we used micelles of a neutral detergent (Brij 58) as a high molecular weight structure. The charge was varied by the addition of a low concentration of sodium dodecyl sulfate and the surface group with which the protons react was an adsorbed pH indicator (bromocresol green or neutral red). The dissociation of a proton from adsorbed bromocresol green is slower than that from free indicator. This effect is attributed to the enhanced stabilization of the acid form of the indicator in the pallisade region of the micelle. The pK shift of bromocresol green adsorbed on neutral micelles is thus quantitatively accounted for by the decreased rate of proton dissociation. Indicators such as neutral red, which are more lipid soluble in their alkaline form, do not exhibit such decelerated proton dissociation in their adsorbed state nor a pK shift on adsorption to neutral micelles. The protonation of an indicator is a diffusion-controlled reaction, whether it is free in solution or adsorbed on micelles. By varying the electric charge of the micelle this rate can be accelerated or decelerated depending on the total charge of the micelle. The micellar charge calculated from this method was corroborated by other measurements which rely only on equilibrium parameters. The high time resolution of the pH jump is exemplified by the ability to estimate the diffusion coefficient of protons through the hydrated shell of the micelle.

Colloids↗
PubMed1980

Modulation of the flavin redox potential as mode of regulation of succinate dehydrogenase activity.

The redox properties of flavin in active and non-active (oxaloacetate reacted) soluble succinate dehydrogenase were studied. Quantitative analysis of reductive activation titrations of redox titrations of active and non-active enzyme reveal that the redox potential of the histidyl-flavin in the active enzyme (-3 +/- 15 mV) is high enough to allow reduction by succinate, whereas in the non active enzyme it is -196 +/- 19 mV, far to low to be reduced by substrate. The flavin radical in the active enzyme attains 60% of total flavin at a poised redox potential of about +60 mV, upon addition of oxaloacetate the magnitude of the signal is diminished and the potential where it reaches maximal concentration is shifted by about -200 mV. A mechanism is proposed which ascribes the fundamental difference between active and non-active enzyme to the inability of the latter to be reduced by substrate.

Animals↗
PubMed1980

The effect of membrane potential on the redox state of cytochrome b561 in antimycin-inhibited submitochondrial particles.

The oxidation of cytochrome b561 by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochrome b was calculated and expressed as a change in redox potential (Eh) measured in millivolts. The oxidation of cytochrome b561 is an energy-driven reaction affected only by the delta psi component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at delta G'ATP less than - 12 kcal/mole. The maximal measured value of ATP-dependent delta psi was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochrome b561. In contrast to the ATP-induced oxidation of cytochrome b561, cytochrome b566 is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation of b561 is explained within the term of the Q cycle as a reflection of delta psi on the electron electrochemical potential. The positive electric potential of the C phase causes cytochrome b566 to act as oxidant with respect to cytochrome b561. In the presence of antimycin cytochrome b561 cannot equilibrate with the quinone and undergoes oxidation, while cytochrome b566 reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.

Adenosine Triphosphate↗
PubMed1979

Rapid pH and deltamuH+ jump by short laser pulse.

The excited state (S1) of sulfononaphthols has a pK value well below that of the ground state, consequently intensive illumination of their aqueous solution should lead to acidification. In this study the second harmonies of a ruby laser pulse (10 MW, 30 ns at 347.2 nm) were used for excitation of a sulfononaphthol solution, resulting in a lowering of the pH from 8 to 4. The change in pH was demonstrated by spectral changes of the pH indicators Bromocresol Green, Bromothymol Blue, Bromocresol Purple and Phenol Red. The magnitude of the pH change was calculated from the kinetics of the changes in the indicators' absorbance and from fluorescence intensity of naphtholate. Sulfononaphthols, due to their hydrophylic nature, cannot permeate across phospholipid membranes. Taking advantage of this property, liposomes containing sulfononaphthol were prepared and irradiated by the laser pulse. Evidence is given that under such conditions the change in pH was limited to the space enclosed in the liposomes. The resulting proton-motive force (deltamuH+ = 180 -- 240 mV) is adequate for perturbing the energy-coupled reactions of oxidative phosphorylation. Possible applications of this technique in chemical physics, chemistry, biochemistry and bioenergetics are discussed.

Hydrogen-Ion Concentration↗