Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

Binding of placental ribonuclease inhibitor to the active site of angiogenin

The importance of specific residues in angiogenin for binding to placental ribonuclease inhibitor (PRI) has been assessed by examining the interaction of angiogenin derivatives with PRI. PRI binds native angiogenin with a Ki value of 7.1 X 10(-16) M [Lee, F. S., Shapiro, R., & Vallee, B. L. (1989) Biochemistry 28, 225-230]. Substitution of a Gln for Lys-40 in angiogenin by site-specific mutagenesis decreases the association rate constant 3-fold and increases the dissociation rate constant 440-fold, resulting in a 1300-fold weaker Ki value. The half-life of the mutant.PRI complex is 3.4 h compared to approximately 60 days for the native angiogenin.PRI complex. The magnitude of the change in Ki value suggests that in the complex, Lys-40 forms a salt bridge or hydrogen bond with an anionic moiety in PRI. Carboxymethylation of His-13 or His-114 with bromoacetate increases the Ki value 15-fold, and oxidation of Trp-89 by means of dimethyl sulfoxide and hydrochloric acid increases it 2.4-fold, suggesting that these residues also form part of the contact region with PRI. The changes in Ki value reflect an increase in the dissociation rate constant. On the other hand, dinitrophenylation of either Lys-50 or Lys-60 with 1-fluoro-2,4-dinitrobenzene does not significantly alter the Ki value, suggesting that these residues are not part of the contact region. These results indicate that PRI inhibition minimally involves the three residues critical for the activity of angiogenin--Lys-40, His-13, and His-114--and to a lesser extent its single tryptophan, Trp-89.     

Slow tight binding inhibition of proteinase K by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.     

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.     

Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer

The interaction of Tet repressor protein with the inducer tetracycline was studied by fluorescence measurements, equilibrium dialysis and nitrocellulose filter binding. The repressor-tetracycline complex was formed from two molecules of tetracycline and one Tet repressor dimer. Formation of the complex requires divalent cations, and results in drastic effects upon the fluorescence spectra of both compounds. The fluorescence of Tet repressor was quenched about 70%, while that of tetracycline was increased between three- and eightfold, depending upon pH. In addition, the emission maximum of the protein was shifted from 330 to 340 nm, and the excitation maximum of tetracycline dropped from 380 to 370 nm. The latter shift is accompanied by a similar change in the absorption spectra. An analogous effect was observed upon changing the environment of the drug by the addition of sodium dodecyl sulphate. These results suggest that tetracycline binds to a hydrophobic region of the protein. A new excitation band in the fluorescence spectrum of the complex is observed. This presumably arises from energy transfer from a tryptophan to the drug. The association rate constant for formation of the complex is 3.3(+/- 0.3) X 10(5) M-1 s-1, and the equilibrium association constant is 2.8(+/- 0.5) X 10(9) M-1. These results are discussed with respect to the biological function of the Tet repressor.     

Structural microheterogeneity of a tryptophan residue required for efficient biological electron transfer between putidaredoxin and cytochrome P-450cam

The carboxy-terminal tryptophan of putidaredoxin, the Fe2S2.Cys4 iron-sulfur physiological redox partner of cytochrome P-450cam, is essential for maximal biological activity [Davies, M. D., Qin, L., Beck, J. L., Suslick, K. S., Koga, H., Horiuchi, T., & Sligar, S. G. (1990) J. Am. Chem. Soc. 112, 7396-7398]. This single tryptophan-containing protein thus represents an excellent system for studying the solution dynamics of a residue directly implicated in an electron-transfer pathway. Steady-state and time-resolved measurements of the tryptophan fluorescence have been conducted across the emission spectrum as a function of redox state to probe potential structural changes which might be candidates for structural gating phenomena. The steady-state emission spectrum (lambda max = 358 nm) and anisotropy (alpha = 0.04) suggest that Trp-106 is very solvent-exposed and rotating partially free of global protein constraints. The time-resolved fluorescence kinetics for both oxidized and reduced putidaredoxin are fit best with three discrete components of approximately 5, 2, and 0.3 ns. The lifetime components were assigned to physical species with iodide ion quenching experiments, where differential quenching of the longer components was observed (k tau = 2 = 5.9 X 10(8) M-1 s-1, k tau = 5 = 1.3 X 10(8) M-1 s-1). These findings suggest that the multiexponential fluorescence decay results from ground-state conformational microheterogeneity and thus demonstrate that the essential tryptophan exists in at least two distinguishable conformations. Small differences in the relative proportions of the components between redox states were observed but not cleanly resolved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats

Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of human placental ribonuclease inhibitor in Escherichia coli

Human placental ribonuclease inhibitor (PRI) has been expressed in and isolated from Escherichia coli. Its apparent molecular weight, immunoreactivity and amino acid composition are virtually identical with those of native PRI. It inhibits the enzymatic activities of either angiogenin, a blood vessel inducing protein homologous to pancreatic RNase (RNase A), or RNase A in a stoichiometry of 1:1. Recombinant PRI binds to angiogenin and RNase A with Ki values of 2.9 x 10(-16) M and 6.8 x 10(-14) M, respectively, comparable to the affinities of native PRI for these enzymes. Thus, these results confirm that PRI inhibits angiogenin more effectively than RNase A.     

Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin

The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse effects of D-galactosamine and inorganic phosphate on glycogenolysis in isolated rat hepatocytes.

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486--493]. At low quencher concentrations, the quenching follows the classical Stern-Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 X 10(9) M-1s-1 and 1.2 X 10(9) M-1s-1 for N-acetyltrypotophanamide and wheat germ agglutinin, respectively. Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent tryptophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.     

An in vitro binding assay for angiogenin using placental ribonuclease inhibitor

A convenient in vitro assay for angiogenin has been developed which greatly facilitates its routine detection and quantitation. The assay is based on the capacity of angiogenin to bind placental ribonuclease inhibitor (PRI); it is less tedious and more versatile than existing procedures that measure blood vessel growth or cleavage of rRNA. The test sample is added to a reaction mixture containing a known quantity of PRI, which complexes any angiogenin present in the sample. A slight excess of RNase A, relative to PRI, is then added, and the amount of RNase A which remains unbound is determined by measuring the generation of acid-soluble fragments from yeast RNA. The assay is sensitive to 30 fmol of angiogenin and is linear over a 17-fold concentration range. Use of the binding assay in parallel with a conventional RNase A assay provides a means of detecting angiogenin in chromatographic fractions and differentiating it from RNases. This procedure makes possible the isolation of angiogenin from new sources, such as nonhuman sera. It may also be applicable to other biologically active proteins with sequence homology to RNase A, e.g., eosinophil cationic protein or eosinophil derived neurotoxin.     

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence.

beta 1-Bungarotoxin has only one tryptophan residue, namely Trp-19 in the phospholipase A2 subunit. The environment of Trp-19 was studied by intrinsic fluorescence and solute quenching. The native protein showed an emission peak at 330 nm. About 90% of the fluorescent tryptophan was accessible to quenching by either acrylamide or KI but not to CsCl. A red-shift in the emission peak occurred between 2.0 M- and 4.0 M-guanidinium chloride, and the helix-coil transition of the polypeptide backbone occurred between 4.0 M- and 6.0 M-guanidinium chloride. These results suggested that Trp-19 was in a less polar medium but near a positive charge. The local conformation around Trp-19 could be disturbed by binding of Tb3+ or Ca2+ or Sr2+ to the toxin molecule. Tb3+ a tervalent lanthanide ion, effectively substituted for Ca2+ in stimulating the phospholipase A2 activity of beta 1-bungarotoxin. Upon the binding of Tb3+ to the toxin, the Tb3+ fluorescence in the 450-650 nm region was enhanced. This resulted from the energy transfer from Trp-19 to Tb3+. The distance between the energy-transfer pair was estimated to be 0.376-0.473 nm at pH 7.6 and 0.486-0.609 nm at pH 6.3. Assuming that there were two Tb3+-binding sites on the toxin molecule, at pH 7.6 the association constants of the high-affinity and the low-affinity sites were determined to be 3.82 x 10(3) M-1 and 2.85 x 10(2) M-1 respectively. At between pH 6.0 and 7.0 Tb3+ bound to the high-affinity site decreased greatly but did not disappear entirely. Both Ca2+ and Sr2+ competed with Tb3+ at the high-affinity sites, but Sr2+ could not substitute for Ca2+ in stimulating the phospholipase A2 activity.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein"

p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.