Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

ThepH dependence of the equilibrium constantK Hyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond in bovine pancreatic trypsin inhibitor (aprotinin)

ThepH dependence of the equilibrium constant K_Hyd for the hydrolysis of the Lys¹⁵-Ala¹⁶ reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over thepH range 2.3–6.5. Solutions of aprotinin, modified aprotinin with the Lys¹⁵-Ala¹⁶ peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine β-trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The K_Hyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. ThepH-independent equilibrium constant is 0.90 and thepK values of the Lys¹⁵ carboxyl group and of the Ala¹⁶ amino group are 3.10 and 8.22, respectively. ThepK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.     

The pH dependence of the equilibrium constant KHyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond in bovine pancreatic trypsin inhibitor (aprotinin)

ThepH dependence of the equilibrium constant K_Hyd for the hydrolysis of the Lys¹⁵-Ala¹⁶ reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over thepH range 2.3–6.5. Solutions of aprotinin, modified aprotinin with the Lys¹⁵-Ala¹⁶ peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine -trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The K_Hyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. ThepH-independent equilibrium constant is 0.90 and thepK values of the Lys¹⁵ carboxyl group and of the Ala¹⁶ amino group are 3.10 and 8.22, respectively. ThepK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.     

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.     

The effect of guanidine hydrochloride on the conformation of bovine pancreatic DNAase I as measured with circular dichroism

The denaturation of bovine pancreatic DNAase I (EC 3.1.21.1) by guanidine hydrochloride (GdnHCl) has been investigated with circular dichroism in the presence and absence of 1 mM Ca2+ at the wavelength region of 210-240 nm at 12.25 and 36 degree C. The change of the molar ellipticity at 220 nm by GdnHCl titration showed cooperative transition at each temperature and the midpoints of the titrations occurred near 2 M GdnHCl. At each temperature, the denaturation of DNAase I in the presence of 1 mM Ca2+ occurred a little slowly as compared with that in the absence of Ca2+. This suggests that 1 mM Ca2+ can to some extent stabilize the secondary structure of DNAase I against GdnHCl denaturation. The apparent free energy for the denaturation of DNAase I obtained by GdnHCl titration was calculated as 9.3 +/- 0.3 kcal/mol and 8.9 +/- 0.2 kcal/mol at 25 degree C in the presence and absence of 1 mM Ca2+, respectively. The possible regions for the alpha -helix and beta -structure of DNAase I were predicted from the amino acid sequence by probability calculation of Chou, P.Y. and Fasman, G.D., Adv. Enzymol. 47, 45-148. The characteristic feature is that the NH2-terminal half of DNAase I is rich in beta -structure and the COOH-terminal half contains mainly alpha -helix.     

Effects of detergents on the conformation of Escherichia coli tRNA as measured by circular dichroism.

The effect of a cationic detergent, lauryl pyridiniumchloride (LPC), and an anionic one, sodium n-octylbenzenesulfonate (SOBS), on the conformation of unfractionated Escherichia coli tRNA was investigated at various molar ratios of detergent to tRNA (D/R) in the presence and absence of Mg2+ and Na+ ions by measuring the circular dichroism (CD) at 265 nm and 340 nm, which reflects conformational change involving base pairs and/or base stacking, and the disymmetry in the vicinity of 4-thiouridylate (4-TU), respectively. In the presence of Mg2+ and Na+ ions, the tRNA retains its native structure even in the presence of high molar ratios of detergent to tRNA (D/R congruent to 40 at 265 nm and D/R congruent to 20 at 340 nm). However, in the absence of these metal ions, the ellipticity at 340 nm was very sensitive to LPC concentration and decreased from 5,600 to nearly--1,600 at 25 degrees C with the increase of D/R ratios up to 20, and a similar decrease in the ellipticity at 340 nm was observed on thermal denaturation. This result suggests that the local environment involving the 4-TU region might be readily influenced by LPC, reflecting a large conformational change. However, no effect was observed in the case of the SOBS: tRNA system. On the other hand, secondary base pairing and/or base stacking structure was virtually invariant on adding both LPC and SOBS even at high D/R ratios in the absence of Mg2+ and Na+ ions.     

Organization of intracytoplasmic membranes in a novel thermophilic purple photosynthetic bacterium as revealed by absorption, circular dichroism and emission spectra

The chromatophore of a novel thermophilic purple photosynthetic bacterium, Chromatium tepidum, had light-harvesting BChl proteins which gave absorption maxima at 917, 855 and 800 nm at 20°C. These antenna complexes were found to have BChl of the a type [4]. This is, therefore, the first example of a BChl a antenna complex which shows a long-wavelength absorption up to 917 nm. Treatment by Triton X-100 and successive sodium dodecyl sulfate polyacrylamide gel electrophoresis separated these antenna complexes into two groups. One of them has one antenna component which absorbs around 917 nm (B917). The other contains at least an antennae which absorb maximally at 800 and 855 nm (B800–855). The temperature-dependent changes of absorption, circular dichroism and emission spectra were reversible up to 70°C in the intact chromatophore and in the isolated B800–855 complex. On the contrary, the isolated complex B917 lost its absorption irreversibly over the temperature of 50°C. These results suggest a membrane structure which is essential for the thermostability of chromatophores from C. tepidum.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Effect of bovine growth hormone on rat liver plasma membranes as studied by circular dichroism and fluorescence using the extrinsic probe 7,12-dimethylbenzanthracene

Conformational changes produced by in vitro bovine growth hormone addition to plasma membranes of hypophysectomized rat liver proteins and lipids have been studied by circular dichroism as well as intrinsic and extrinsic fluorescence. 7,12-Dimethylbenzanthracene has been used as a fluorescent probe of changes in membrane structure. The exposure of membranes to bovine growth hormone produced a change in membrane negative ellipticity. Dimethylbenzanthracene at concentrations similar to those employed in fluorescence studies had no effect on the membrane circular dichroism spectrum. Its presence did, however, prevent a response to growth hormone. There was a decrease in peak fluorescence intensity and a peak shift when bovine growth hormone (0.5 · 10^?12 M) was added to liver membranes. The addition of dimethylbenzanthracene (1.6 · 10^?6 M) to membranes resulted in a decrease in the intensity of the protein fluorescence peak at 335 nm and the appearance of two peaks at 430 and 407 nm, assignable to the probe. The addition of bovine growth hormone (0.5 · 10^?12 M) produced a decrease in fluorescence at 335 nm and also in the peaks at 407 and 430 nm. These data are consistent with the conclusion that bovine growth hormone produces a conformational change in rat liver plasma membrane proteins and lipids.     

Carbohydrate-containing derivatives of the trypsin-kallikrein inhibitor aprotinin from bovine organs. II. Inhibitor coupled to the (carboxymethyl)dextran derivatives of D-galactose

The trypsin-kallikrein inhibitor aprotinin was coupled to (carboxymethyl)dextran derivatives of D-galactose. The conjugates contained 14 and 38 D-galactose residues/mol of protein, respectively. The apparent dissociation constants Ki of the complexes between trypsin and modified aprotinins proved to be one order of magnitude higher than the respective values for native aprotinin. The distribution of the modified aprotinins in rat organs after endocardial injection has been studied. The conjugates of aprotinin with (carboxymethyl)dextran derivatives of D-galactose were characterized by decreased clearance rates; they accumulated in the active form in liver. The accumulation was 2.5-10 times higher than native aprotinin for the time of observation (5 min-2 h).     

Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.     

Conformational differences between the E1 and E2 states of the calcium adenosinetriphosphatase of the erythrocyte plasma membrane as revealed by circular dichroism and fluorescence spectroscopy

Different conformational states of the purified plasma membrane Ca2+-ATPase from pig erythrocytes have been detected by circular dichroism (CD) and fluorescence spectroscopy. The helical content of the enzyme decreased by about 10% in the transition from the Ca2+ high-affinity form (10 microM free Ca2+ = E1 state) to the VO4(3-)-inhibited state (20 microM VO4(3-) = E2 state). The changes in the CD spectra did not show full reversibility upon reversing the E1-E2 transition, whereas those in the fluorescence spectra did. A temperature-dependent loss of alpha-helical content in the presence of Ca2+ was also observed. Intrinsic fluorescence measurements revealed an increase in fluorescence intensity upon addition of Ca2+. The change was fully reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The increase in fluorescence intensity was partly reversed by adding ATP, an effect which is suggested to correspond to the "Ca2+-occluded" form of the ATPase. The steady-state level of the fluorescence intensity was stable for several minutes in the presence of 100 microM ATP. By contrast, the decrease of fluorescence intensity induced by limiting concentrations of ATP (= 1 microM) was only transient, indicating the decomposition of the phosphorylated intermediate of the ATPase and the reestablishment of the Ca2+ high-affinity form of the enzyme.     

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.