Showing papers on "Conformational change published in 1975"
TL;DR: The role of SH groups in opiate-receptor interactions has been further examined and it is suggested that the same conformational change is involved in the two phenomena.
Abstract: The role of SH groups in opiate-receptor interactions has been further examined. In activation by N-ethylmaleimide of sterospecific opiate binding by rat brain membrane fractions follows pseudo-first order kinetics and exhibits strong temperature dependence. The kinetics indicate that alkylation of a single SH group suffices to block opiate binding. Considerable protection from SH group inactivation is observed when treatment with N-ethylmaleimide is carried out in the presence of an opiate or an antagonist, suggesting close proximity of the SH group to the opiate binding site. The rate of inactivation of receptor binding by N-ethylmaleimide is markedly slower in buffers containing 100 mM NaCl (t1/2 equals 30 plus or minus 1.4 min) than in sodium-free buffers (t1/2 equals 10 plus or minus 1.0 min). Since the rate of alkylation of model SH compounds is unaffected by sodium ions, this protection seems best explained by a conformational change in the receptors that renders the SH groups less accessible to alkylation. The rate of inactivation is not affected by K+, Rb+, or Cs+ and only slightly by Li+. This cation specificity as well as the concentration-response to Na+ are remarkably similar to those previously shown to lead to increased antagonist and decreased agonist binding. We suggest that the same conformational change is involved in the two phenomena.
145 citations
TL;DR: It is shown that a bridge is formed across the polyphosphate binding site between the two β chains demonstrating that the cross-link stabilizes the deoxy conformation but does not prevent the conformational change associated with oxygenation.
116 citations
TL;DR: Conformational changes induced in antibody molecules and in their Fab fragments by binding of antigen were investigated by the circular polarization of the fluorescence emitted by the tryptophan residues, suggesting that the disulfide bonds at the hinge region of the antibody are required for the transmission of the conformational change from the Fab to the Fc.
Abstract: Conformational changes induced in antibody molecules and in their Fab fragments by binding of antigen were investigated by the circular polarization of the fluorescence emitted by the tryptophan residues. This property of the fluorescence is related to the asymmetry, and thus to the conformation and environment, of the emitting chromophore. Changes in the circular polarization of the fluorescence of the antibody were observed upon binding of RNase to anti-RNase, of poly(DL-alanyl)-poly(L-lysine) to antipoly(D-alanine), and of the "loop" of lysozyme, a monovalent antigenic determinant, to anti"loop." The spectral changes were observed at different antigen-antibody ratios, including high antigen excess, indicating that they are due to antigen binding and not to aggregation. The circular polarization of fluorescence also detects changes in conformation of the different Fab fragments upon binding of the corresponding antigens. These changes in conformation were, however, markedly different from those observed for the whole antibody molecules, and indicated an interaction between the Fc and Fab fragments in the antibody molecule, and probably a change in the conformation of Fc upon binding of antigen to the antibody. In contrast, the small hapten, phosphorylcholine, did not induce a change in the circular polarization of the fluorescence of its antibody or corresponding Fab fragments. Reduction of the interchain disulfide bonds of the antibodies abolished the antigen-induced spectral changes due to the presence of the Fc portion in the molecule, but not the changes observed in Fab, suggesting that the disulfide bonds at the hinge region of the antibody are required for the transmission of the conformational change from the Fab to the Fc.
100 citations
TL;DR: The spectral changes of cytochrome P-450 caused by the addition of small molecules to liver microsomes were investigated precisely and the following conclusions were reached.
Abstract: The spectral changes of cytochrome P-450 caused by the addition of small molecules to liver microsomes were investigated precisely and the following conclusions were reached. 1. The Type I spectral change was entirely due to the interaction of the cytochrome with a hydrocarbon residue in a ligand. To induce the modified Type II spectral change, the presence of a hydroxyl group in a ligand was required. Compounds which contain a basic amino group induced the Type II spectral change. 2. The Type I spectral change was caused by the interaction of a ligand with the 419-nm form of cytochrome P-450, with its concomitant conversion to the 394-nm form. Whereas, compounds inducing modified Type II spectral change interacted with the 394nm form of the cytochrome. In this case, however, the 394-nm form was not converted back to the 419-nm form but was converted to a new state showing an absorption peak at 416 nm. The Type II spectral change-inducing interaction of a ligand with the cytochrome could occur with all forms of the cytochrome. 3. Both Type II and modified Type II compounds bound to the cytochrome at heme iron, and converted the cytochrome into modified ferrihemochromes. On the other hand, the Type I interaction occurred ina protein moiety of the cytochrome, and probably caused a conformational change of the cytochrome accompanied either by weakening of the internal ligand interaction or by displacement of the ligand with another one having a weaker field at the heme iron. 4. Type I and each of other two types of binding of compounds with cytochrome P-450 could occur simultaneously.
67 citations
TL;DR: A mechanism for cyroprecipitation was proposed wherein precipitation in the cold is a direct consequences of binding rather than a property inherent in a nature of the soluble complexes.
51 citations
TL;DR: The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme, and it is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histazine-48 peak observed in the acid-stable form of the enzyme.
Abstract: The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme in which resolution of the C(2)-H resonance of histidine-48 was simplified. The NMR titration curves at 100 and 250 MHz of histidine-48 of ribonuclease A are discontinuous both for the enzyme alone in 0.3 M chloride and for its complex with cytidine 3'-phosphate. This suggests that titration of histidine-48 occurs only as the result of a slow conformational transition. The sum of the peaks corresponding to histidine-48 in the acid-stable and base-stable forms of the enzyme is less than one proton in the transition region, which indicates that there exists at least one intermediate conformational form of the enzyme. The transition from the acid-stable form to an intermediate form has a pHmid of 5.6, and the transition from an intermediate form to the base-stable form has a pHmid of 6.9. In ribonuclease S and in ribonuclease A in the presence of 0.3 M acetate, the titration curve of histidine-48 is continuous, and the area of the peak is uniform throughout the titration. Proton NMR difference spectra at 100 and 250 MHz reveal a pH-induced conformational change with a pHmid of 5.7 that affects the chemical shift of a single tyrosine residue. This conformational transition is absent in ribonuclease S and is altered in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. It is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histidine-48 peak observed in the acid-stable form of the enzyme. It is proposed that the perturbed tyrosine is tyrosine-25. The transition with pHmid 5.6 is attributed to dissociation of aspartic acid-14, and the transition with pHmid 6.9 is assigned to dissociation of histidine-48. A peak in the aromatic region that moves upfield on addition of the competitive inhibitor cytidine 3'-monophosphate is assigned to a tyrosine, and evidence is presented that this tyrosine is tyrosine-25. Inhibitor binding appears to induce a conformational change in the histidine-48/tyrosine-25 region which is remote from the active site.
51 citations
TL;DR: A conformational change at low pH in bovine β-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate and it seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0.
50 citations
TL;DR: Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.
49 citations
TL;DR: The Raman spectra of human IgG, IgM, and rabbit IgG in lyophilized form and solution are reported and indicate that the predominant structure in these immunoglobulin proteins is the antiparallel β‐sheet.
Abstract: The Raman spectra of human IgG, IgM, and rabbit IgG in lyophilized form and solution are reported. The spectral results indicate that the predominant structure in these immunoglobulin proteins is the antiparallel β-sheet. The Raman spectra have also been obtained of rabbit anti-ovalbumin, and this antibody molecule precipitated with its respective antigen. The spectra reflect a conformational change on binding of antibody with antigen. The conformational change occurs in the direction of disordering.
47 citations
TL;DR: The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18, and a model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.
43 citations
TL;DR: The fact that not only the magnitude but also the sense of the change in the "spin--spin" interaction is a function of increasing saturation with heavy meromyosin subfragment-1 indicates that the monomers of the actin filament are capable of cooperative interaction in the absence of tropomyOSin.
Abstract: The decrease in amplitude of the electron spin resonance spectrum of the cysteine-bound spin-label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinoxyl, brought about by the magnetic interaction with tightly bound manganous ion, was used as a probe of conformational change in actin on binding myosin. The magnitude of this "spin--spin" interaction first decreased then increased on increasing saturation of the actin filament with heavy meromyosin subfragment-1. That the "spin--spin" interaction occurred between spins of adjacent monomers was demonstrated by the observation that the change in magnitude of the "spin--spin" interaction was maintained on binding of heavy meromyosin subfragment-1 to copolymers in which actin monomers containing both manganous ion and spin label were diluted 7-fold with native actin monomers. These data provide evidence for a conformational change in actin on interacting with heavy meromyosin subfragment-1. Further, the fact that not only the magnitude but also the sense of the change in the "spin--spin" interaction is a function of increasing saturation with heavy meromyosin subfragment-1 indicates that the monomers of the actin filament are capable of cooperative interaction in the absence of tropomyosin.
TL;DR: All the information required to construct a ribosome must be present in its components, and it seems that cells use an additional mechanism to facilitate their assembly.
Abstract: RECONSTITUTION of bacterial ribosomes occurs in vitro in a system containing ribosomal components only1, which suggests that ribosomes also form by self-assembly in vivo. This inference is reinforced by the fact that intermediary particles, containing a similar fraction of ribosomal proteins, are observed both in vitro and in vivo1–3. The in vitro reconstitution process, however, requires a very high energy of activation, presumably to force the intermediates into a rare conformational state1,4. This requirement is too high to be compatible with the rate of ribosome formation in vivo5. Furthermore, there is evidence that the rate-limiting step which leads to the accumulation of the precursor particles in vivo does not involve spontaneous conformational change of these particles6. Although all the information, therefore, required to construct a ribosome must be present in its components, it seems that cells use an additional mechanism to facilitate their assembly.
TL;DR: Data are presented which suggest that photosystem II dependent hydrogen ion accumulation and ATP synthesis can occur only after the lamellar membranes have undergone a conformational change.
Abstract: Data are presented which suggest that photosystem II dependent hydrogen ion accumulation and ATP synthesis can occur only after the lamellar membranes have undergone a conformational change. This membrane conformational change is detected by the electron transport dependent incorporation of diazonium benzene[35S]sulfonate into membrane components. Previously it was established that electron flux from the photosystem II primary acceptor to plastoquinone is a necessary event for the occurrence of the diazonium-detected conformational change. These studies indicate that the release of hydrogen ions during photosystem II oxidation of the primary reductant is also a necessary event for the diazonium-detected conformational change. When iodide were substituted for water (or other proton-releasing donors) as the primary reductant of system II the conformational change did not occur even though a substantial rate of electron flow from the primary acceptor to plastoquinone occurred.
TL;DR: The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements and a simple mechanism is proposed to account for the results.
Abstract: The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.
TL;DR: It is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48, and a model assuming mutual interaction of the Active Site Histidine residues does not account for the low pH inflections in these curves.
TL;DR: A new model is presented in which an obligatory conformational change upon binding of substrates results in an alteration in the relative orientation of c versus r that accounts satisfactorily for the observation by Kerbiriou and Herve that homotropic effects can be abolished whereas heterotropic effects are retained in the altered enzyme from Escherichia coli grown in the presence of 2-thiouracil.
TL;DR: Kinetic data for these various transitions suggest that dissociation is a rate-limiting step while conformational changes of the polypeptide chains are relatively much more rapid, according to the conditions of temperature and concentration.
TL;DR: In this article, the conformational difference between polypeptide chain elongation factor Tu (EF-Tu)-GTP and EF-Tu-GDP has been studied using hydrophobic and fluorescent probes.
Abstract: The conformational difference between polypeptide chain elongation factor Tu (EF-Tu)-GTP and EF-Tu-GDP has been studied using hydrophobic and fluorescent probes. The interaction of EF-Tu-GDP with 1-anilino-8-naphthalenesulfonate (ANS) was measured in terms of the enhancement of the fluorescence intensity at the emission maximum of 475 nm. When EF-Tu-GDP-ANS complex was converted to EF-Tu-GTP-ANS complex by incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], there was a roughly 2-fold increase in fluorescence intensity and a blue shift of the emission maximum from 475 to 467 nm, indicating a conformational transition of the protein. The conformational change was found to be reversible and the spectrum promptly returned to that of EF-Tu-GDP-ANS complex upon addition of excess GDP. A similar change in the spectrum was also observed when aminoacyl-tRNA, but not deacylated tRNA, was added to EF-Tu-GDP-ANS complex. Measurement of the number of binding sites by gel filtration indicated that EF-Tu-GTP and EF-Tu-GDP bind 2.9 and 1.7 molecules of ANS, respectively. These results suggest that in EF-Tu-GTP the conformation was altered and one additional binding site for ANS was created at or near the site interacting with aminoacyl-tRNA. Another reagent, N-(1-anilinonaphthyl-4) maleimide (ANM) was covalently bound to the sulfhydryl group in EF-Tu-GDP which is essential for interaction with aminoacyl-tRNA. The binding could be determined spectrofluorometrically, since the reagent, which is nonfluorescent in aqueous solution, emitted a strong fluorescence upon binding with the sulfhydryl group, indicating a marked hydrophobicity of the local environment. Measurements of the kinetics of the binding revealed that ANM reacted rapidly with the sulfhydryl group in EF-Tu-GTP, while the reaction with that in EF-Tu-GDP proceeded more sluggishly. The difference in the reactivity of the sulfhydryl group essential for aminoacyl-tRNA binding between EF-Tu-GTP and EF-Tu-GDP probably reflects a conformational transition of the protein near the active site. These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Onishi (1974) J. Biol. Chem. 249, 3311), demonstrate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.
TL;DR: The conformational parameters of these compounds were determinated and correlation appears to exist between the various conformations.
Abstract: Proton magnetic resonance spectra at 250 MHz were measured as a function of temperature and pH of the three guanosine phosphates. From these data and previously published work the conformational parameters of these compounds were determinated. The phosphate group of Guo-5'-P changes its conformation around the C-O bond and its rotation is relatively slow at 20 degrees. At neutral pD the S conformation is favoured and the N form at acid pD. This conformational change is paralleled by a change in exocyclic rotamer distribution and takes place at the pK of the protonation of the base on N-7. Although correlation appears to exist between the various conformations, notable exceptions exist.
TL;DR: It was concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein.
Abstract: In order to clarify how the electrophoretic behavior reflects the conformational transition of globular proteins, moving boundary electrophoresis was applied to analysis of the acid conformational change of alpha-lactalbumin. The appearance of only a single electrophoretic boundary in the transition region of the protein suggests a very rapid transition with a half-time estimated to be smaller than 7 min on the basis of the theory of isomerizing systems in electrophoresis. The transition is clearly reflected in the dependence of the mobility on the protein net charge, which shows a sigmoidal curve closely similar to that obtained by a Linderstrom-Lang pH-tritration plot for the carboxyl groups of alpha-lactalbumin. It was also concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein. All results obtained by electrophoresis were also supported by the results of pH-jump studies, analytical gel chromatography, and CD measurements.
TL;DR: Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg-2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyoson.
Abstract: TN-C was purified from bovine cardiac muscle. In the absence of Ca2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyosin. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational Change upon binding Ca2+. A similar response is elicited by Sr2+, whereas Mg2+ has a much less pronounced effect. The presence of Mg2+ does not alter the net effects of either Ca2+ or Sr2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.
TL;DR: A conformational change is postulated to occur in the light which exposes the oxygen evolution system to the internal face and internal pH of the thylakoid membrane, and a physiological function of this conformation change is discussed in terms of a flexibility of energy coupling in photosystem II.
TL;DR: The results suggest that myosin-substrate interaction in vitro does not result in a delocalized conformational change.
Abstract: The effect of substrate on the far UV (185-250 nm) and near UV (250-325 nm) circular dichroism (CD) of myosin and heavy meromyosin (HMM) was studied. The following results were obtained with the addition of ATP (during various conditions of hydrolysis), ADP, and pyrophosphate: (1) no changes were observed in the far UV CD, (2) ATP and ADP perturbed the near UV CD only at spectral regions below 280 nm coinciding with the regions of their optical activity, (3) the optically inactive pyrophosphate caused no change in the near UV CD, and (4) myosin and HMM gave exactly the same results. These results suggest that myosin-substrate interaction in vitro does not result in a delocalized conformational change.
TL;DR: It is demonstrated that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that protonRelease is probably coupled to the conformationalchange.
TL;DR: It is suggested, by analogy with haemoglobin, that the molecular basis of the negative cooperativity may be the creation of additional intersubunit bonds during the binding of the first two NAD molecules to the tetramer, and a change from a "relaxed" quaternary structure to a "tense" structure at half-saturation.
TL;DR: In this paper, it was shown that the lysozyme molecule undergoes a conformational transition around 25°C which would account for the existence of the two crystalline forms.
Journal Article•
TL;DR: In reconstituted troponin, the conformation of TN-C was more influenced byTN-I in the presence of calcium and by TN-T in its absence as indicated by the imidazole label, whereas with the maleimide label, TN-I was more influential in the absence of calcium.
Abstract: The three troponin components, TN-C, TN-I, and TN-T, were spin-labeled with two different derivatives of the nitroxide radical, a maleimide and an imidazole reagent. The ESR spectra of various combinations of labeled and unlabeled components were measured both in the presence and absence of calcium. Conformational changes due to the binding of the components and also due to the binding of calcium were sensitively detected in many combinations as large changes in the spectrum. The conformation of TN-C was modified by both TN-T and TN-I. The effects were larger in the presence of calcium than in its absence. In the presence of calcium, TN-T and TN-I both showed large effects with the maleimide label, while TN-I showed a larger effect than TN-T with the imidazole label. In the absence of calcium, the effect of TN-I was larger than that of TN-T. The senstivitiy of TN-C to calcium was magnified by component binding, since the conformation of TN-C itself was not greatly affected by calcium. The conformation of TN-I was greatly altered only in the presence of both TN-C and calcium. This indicates that the calcium-induced conformational change in TN-C is transmitted to the adjacent TN-I. In reconstituted troponin, the conformation of TN-C was more influenced by TN-I in the presence of calcium and by TN-T in its absence as indicated by the imidazole label. With the maleimide label, TN-I was more influential in the absence of calcium. The effect of calcium on the troponin complex was to make the local environment of the label more rigid. The half-maximal effect was observed at 2 X 10(-6)M calcium with TN-I in various complexes, while it was 10(-5)M with TN-C in the complexes. In any case the calcium effects became discernible at 10(-6)M and saturated at 10(-4)M.
TL;DR: The spectroscopic data obeyed a linear relationship with protein concentration at 0·9 m to 2·4 m-MgCl2 despite the increased subunit dissociation with a lowering of protein concentration that has been observed (Hsu # Neet, 1973).
TL;DR: The measurements of ultraviolet linear dichroism on glycerinated muscle fibers of rabbit psoas and crab leg having various sarcomere lengths and the effect of interaction of the thin filament with myosin has been investigated, indicating a conformational change in F-actin in thethin filament.
Abstract: The measurement of ultraviolet linear dichroism has been carried out on glycerinated muscle fibers of rabbit psoas and crab leg having various sarcomere lengths and the effect of interaction of the thin filament with myosin on the dichroism has been investigated under various conditions.
The dichroism spectrum of the muscle fiber has a large positive peak at 285 nm (smaller absorption of light polarized perpendicular to the fiber axis) and a small negative peak at 297.5 nm. The negative peak at 297.5 nm, which comes from the dichroism of F-actin in the thin filament, became smaller in the presence of Ca2+ than in its absence. The positive peak at 285 nm, where the thick filament has main contribution, showed no appreciable change by Ca2+. The dichroism at 292.5 nm, where the thin filament has no contribution, showed no change either. Thus, the decrease of negative dichroism at 297.5 nm by Ca2+ indicates a conformational change in F-actin in the thin filament.
With decreasing sarcomere length or increasing overlapping between thin and thick filaments, the dichroism decrease at 297.5 nm by Ca2+ increased, and attained a maximum at maximum overlapping. That is, the conformational change of F-actin by Ca2+ was amplified by interaction with myosin. With further decrease of the sarcomere length, the dichroism decrease by Ca2+ decreased.
In the absence of Ca2+, the addition of ATP or pyrophosphate induced a large change of transmittance and transmittance anisotropy, and also a large increase of birefringence at long wavelengths.
TL;DR: The enzyme study is presented as a source of strategy in drug design for the study of enzymes for drug design by considering the mechanism whereby less than one bound mole of inhibitor per mole of enzyme can in time bring about complete inactivation of an active site.
Abstract: Publisher Summary This chapter presents the enzyme study as a source of strategy in drug design. The mechanism whereby less than one bound mole of inhibitor per mole of enzyme can in time bring about complete inactivation of an active site has great importance for the study of enzymes for drug design. The process is best explained by considering that, when a ligand is adsorbed at a control site or a receptor site, it produces a small conformational change after desorption. If the relaxation time of the macromolecule is slow, it will not completely return to the original state before a second molecule is adsorbed and an added increment in conformational distortion produced. This gradual change in activity can continue until all activity is destroyed. This means that in instances where this process can occur, the Michaelis constant Km or the inhibition constant Ki are not really constant but time-dependent. If this type of time-dependent deactivation of a receptor occurs in the whole animal in a manner similar to that in vitro, then in vitro data will be applicable to in vivo work. If, on the other hand, the enzyme is more stabilized against such time-dependent deactivation by the constellations of macromolecules surrounding it in living tissue, then results obtained in vitro could be misleading.