Determination of kinetic constants in the general case of cooperative type or Michaelis enzymes inhibited by their substrate: theoretic analysis

For an enzyme (E) susceptible to substrate (S) inhibition, (S) can bind on one hand to (E) and on the other hand to (ES), leading to the dead-end complexes (SE) and (SES). In the general case where the (E)/(S) interaction obeys the Hill equation, the theoretical maximum velocity VM can be estimated when n not equal to 1, from the determination of velocities v beta at substrate concentrations S beta = Sm beta where Sm is the value corresponding to the actual maximum velocity vm. The Hill coefficient (n) as well as the constants KS, KSE and KSES corresponding to the respective dissociations of the complexes (ES), (SE) and (SES) are then determined from the equation: Ln (v/(VM-v] = nLnS-LnKS(1 + Sn/KSE + S2n/KS KSES) and its two asymptotes.     

Thermodynamics of interaction of water vapour with 20 different poly-L-amino acids

The uptake of water vapour by 20 different polyaminoacids have been evaluated by an isopiestic vapour pressure technique in absence of solute at three different temperatures (22 degrees C, 30 degrees C and 37 degrees C). The water vapour adsorption isotherm for different polyaminoacids in the range of water activity varying between zero and unity apparently agreed with that expected from a type III BET isotherm. From the linear BET plots, obeyed in the lower range of water activity, the BET constants n(m) and Qm for different polyamines have been evaluated. The amount of water vapour adsorbed (n1) was calculated in moles/kg of polyaminoacids as well as in moles/mole of amino acid residue. Its value at unit water activity (deltan(o)1) has been evaluated by an extrapolation method and the results support that the multilayer adsorption of water vapour at the interface of polyaminoacids takes place. Values of deltan(o)1 are strictly comparable in terms of moles per kg rather than mole per mole unit. In case of beta lactoglobulin (betalg), lysozyme and BSA, theoretically obtained deltan(o)1 values were observed to be considerably larger than experimental values of deltan(o)1. This indicated that amino acid residues in the polypeptide release a large amount of water due to the formation of a globular structure. Using the Bull equation in the integrated form, standard free energies, deltaGo(w) for water-polyamino acid binding interaction at two different temperatures have been evaluated. Based on the Clausius-Clapeyron equation in an integrated form, the integral enthalpy for water-polyamino acid interaction has also been evaluated.     

The effect of the aflatoxins B1, G1 and G2 on protein and nucleic acid synthesis in rat liver

A comparison has been made of the difference spectra obtained by causing various aflatoxins (B(1), G(1) and G(2)) to interact with calf-thymus DNA. The effect of these toxins on RNA and protein synthesis by rat-liver slices has been measured. The extent of their inhibitory action on the synthetic reactions was proportional to the degree of spectral shift obtained with their interaction with DNA. It is proposed that their toxicity depends on this interaction. It was demonstrated that the RNA polymerase of nucleoli isolated from the livers of aflatoxin B(1)-poisoned rats was inhibited. This finding is in agreement with the proposed mechanism for the hepatotoxic action of aflatoxin.     

Resonance Raman studies of CO and O2 binding to elephant myoglobin (distal His(E7)----Gln)

Carbon monoxide and dioxygen were employed as resonance Raman-visible ligands for probing the nature of the heme-binding site in elephant myoglobin, which has glutamine in the distal position (E7) instead of the usual histidine. The distal histidine (E7) residue has been thought to be responsible for weakening carbon monoxide binding to hemoproteins. It is of interest to see how the His(E7)----Gln replacement affects such parameters as nu(Fe-N epsilon), nu(Fe-CO), delta(Fe-C-O), nu(C-O), delta(Fe-O-O), and nu(O-O) vibrational frequencies and relative intensities. Elephant myoglobin has a CO affinity approximately 6 times higher than that for human/sperm whale myoglobin (Mb). If this enhanced affinity were solely due to the removal of some of the steric hindrance that normally tilts the CO off the heme axis, one would expect the nu(Fe-CO) frequency to decrease and the nu(C-O) frequency to increase relative to the corresponding values in sperm whale Mb. However, the opposite was found. In addition, strong enhancement of the Fe-C-O bending mode was observed. These results suggest that the Fe-C-O linkage remains distorted. In elephant Mb, new interactions resulting from the conformational change accompanying ligand binding may be responsible for the increased CO binding. Similar spectra were obtained for elephant and sperm whale oxymyoglobin. This suggests that the interactions of bound O2 are not markedly affected by the glutamine replacement.     

Kinetics of heat- and acidification-induced aggregation of firefly luciferase

The general approach to analysis of the kinetics of protein aggregation registered by the turbidimetric method has been elaborated. The terminal part of the kinetic curves is analyzed using a theoretical equation connecting the derivative of the apparent absorbance (A) with respect to time (dA/dt) and A (t is time). This analysis allows the limiting value of A at t--> infinity (A(lim)) and the order of aggregation with respect to protein (n) to be calculated. Approach proposed was applied to analysis of thermal and acidification-induced aggregation of firefly luciferase. In both cases the A(lim) value is a linear function of the protein concentration. The terminal part of the kinetic curves of thermal aggregation follows the first-order kinetics (n=1), whereas the kinetics of acidification-induced aggregation are characterized by the value of n higher than unity (n=1.29). The mechanism of nucleation-dependent aggregation has been discussed.     

Production of IgG antibodies and enhanced response of nude mice to DNP-AE-dextran.

DNP-AE-dextran, prepared by the binding of DNP-epsilon-aminocaproyl residues to animoethylated dextran (predominantly alpha-1,6-linked), induced a T-independent anti-DNP antibody response in mice. However, certain differences were observed between the response to this antigen in normal andnude mice. Thus, the antibody titers of nu/nu mice from day 10 to 38 after immunization were significantly higher than those of nu/+ controls. Furthermore, DNP-AE-dextran induced a weak secondary response in nu/+ but not in nu/nu mice. For both thymusless and normal mice the production of IgG in addition to IgM antibodies to DNP-AE-dextran could be established. The former included antibodies of the IgG1 subclass which were considered to be particularly thymus dependent (1). The higher response of nu/nu mice was reflected mainly in the increased production of IgG antibodies. Under the influence of a graft-vs-host reaction, a 10-fold increase in antibody titers to DNP-AE-dextran was observed, due entirely to an enhanced IgG response.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.     

Isotope Flows and Flux Ratios in Biological Membranes

Precise evaluation of permeability of biological tissues is often prevented by imprecise knowledge of operative forces. This problem has been approached by analysis of fluxes of isotopic species applied to opposite surfaces of a membrane. A simple and rather general flux ratio equation has been derived which may permit evaluation of membrane permeability, even without knowledge of forces, or of the nature of active transport processes. Permeability as thus defined should be insensitive to coupled flows, either of other species or of metabolism. In appropriate circumstances application of the equation may permit evaluation of the contributions of the various processes to the transport of the examined species. Composite series membranes would be expected to obey the unmodified general equation. Heterogeneous parallel pathways would alter the relation in a predictable manner. The effect of isotope interaction is specifically incorporated. The formulation is applied to consideration of energetics of active transport.     

Kinetics of oxygen consumption after a single isometric tetanus of frog sartorius muscle at 20 degrees C

The time-course of the rate of oxygen consumption (QO2) has been measured in the excised frog sartorius muscle after single isometric tetani of 0.1-1.0 s at 20 degrees C. To measure deltaQO2(t), the change in QO2 from its basal level, a novel method was devised, based on the validity in this tissue of the one-dimensional diffusion equation for oxygen, established in the preceding paper. After a tetanus, deltaQO2 reached a peak within 45-90 s, then declined exponentially, and could be well fit by deltaQO2(t) = QO + Q1(epsilon -k1t - epsilon-k2t). tau2 (= 1/k2), which characterized the rise of deltaQO2, was a decreasing function of tetanus duration (range: from 1.1 +/- 0.28 min [nu = 5] for a 0.1-s tetanus, to 0.34 +/- 0.05 min [nu = 8] for a 1.0-sec tetanus). tau1 (= 1/k1), which characterized the decline of deltaQO2, was not dependent on tetanus duration, with mean 3.68 +/- -.24 min (nu = 46). A forthcoming paper in this series shows that these kinetics of deltaQO2 are the responses to impulse-like changes in the rate of ATP hydrolysis. The variation of tau2 with tetanus duration thus indicates the involvement of a nonlinear process in the coupling of O2 consumption to ATP hydrolysis. However, the monoexponential decline of deltaQO2(t), with time constant independent of tetanus duration, suggests that during this phase, the coupling is rate-limited by a single reaction with apparent first order kinetics.     

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.     

Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase.

1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis-Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/nu) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis-Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.     

Binding of positive charged ligands to DNA. The effect of ionic strength, charge and size of the ligand

The electrostatic interaction of extended cationic ligands with DNA has been considered on the basis of the analytical solution of a simplified Poisson--Boltzmann equation for the charged polyion cylinder. The control numerical solution of rigorous Poisson--Boltzmann equation shows that the assumption about the absence of coions in the vicinity of the highly charged polyion cylinder does not significantly influence the accuracy of solution and DNA electrostatic free energy evaluation. It was found that the basic contribution to the free energy of electrostatic ligand-DNA interaction is the mixing entropy change due to release of counterions from the vicinity of DNA. The equation for the dependence of the ligand to DNA binding constant K upon ionic strength c has been derived without introduction of any empirical parameters. This equation is consistent with the experimental data and can be used for the determination of a number of ligand--DNA ionic contacts in a wide range of salt concentrations. The main consequences of Manning and Record et al. theories can be considered as limiting cases of the theory presented. In particular the equation d(lnK)/d(lnc) = -0.88 N by Record et al. has a restricted range of application and it can be used only for a relative approximate estimation of the number of electrostatic bonds in ligand-DNA complexes. The analysis of electrostatic interaction of DNA with ligands which neutralize only part of phosphate groups in the binding site of DNA was also performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfhydryl groups and the interaction between the hemes in hemoglobin

1. Dialysis of a hemoglobin solution against slightly alkaline buffer (pH 8.68) causes a decrease in heme-heme interaction. The value of n in Hill's equation drops from 2.9 to 2.0, while the oxygen affinity increases slightly. Addition of glutathione largely reverses the effects of dialysis (n rises from 2.0 to 2.5 to 2.6). 2. Addition of the sulfhydryl reagent, p-chloromercuribenzoate (1.8 x 10(-3)M), results in a large though not complete blocking of heme-heme interaction. The value of n drops from 2.9 to 1.4. This effect is largely reversed with glutathione (n rises to 2.6).     

Effects of tobacco glycoprotein (TGP) on the immune system. I. TGP is a T-independent B cell mitogen for murine lymphoid cells

It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.     

Evaluation of the sliding distance in shortening muscles and in polymerizing actin from Hill's force-velocity equation

The relationship derived earlier between the sliding distance, Deltal(m), and a/P(0), the characteristic parameter of Hill's force-velocity equation for muscle contraction, was re-formulated in order to get a more general relationship which can be applied also to other biological mechano-chemical energy converters: alpha x Deltal(m)=phi (0)(a/P(0))Deltal(m)=-Deltag where Deltag is the free energy change accompanying the hydrolysis of one ATP molecule while alpha and phi (0) are, respectively, the average forces developed by a myosin head-actin complex which are responsible for shortening and for isometric tension generation. These two molecular forces are different in magnitude and in nature and it is demonstrated that alpha , not phi (0), is the true contractile force. The values of alpha and of phi (0) have been calculated for three muscles. The equation has been successfully applied to actin polymerization-based motility. The value of Deltag in different muscles under different environmental conditions can be easily determined from this equation with the value of Deltal(m) derived experimentally.     

Hydration of human nails investigated by NIR-FT-Raman spectroscopy.

The human nail, although it is usually stable against outer influences, becomes soft and flexible after soaking in water. Frequent washing increases brittleness of nails. Hydration of nails is thought to be the most important factor influencing the physical properties of nails and possibly acts through changes in keratin structure. Here NIR-FT-Raman has been used to examine molecular structural changes of intact moisten nails. Raman spectra were obtained both in vitro from nail samples and in vivo before and after soaking in water. The water uptake of normal nail samples during the first 15 min was reflected in the increasing intensity ratio of the nu(OH)/nu(CH(2)) bands. A saturating effect appeared soon after 10 min which is explained by a defined water holding capacity. R(nu) representation of the low frequency range of the Raman spectra showed that mainly bound water is found both in dry and in wet nails. This implies water-protein interaction. Protein backbone vibration at 932 cm(-1) indicating alpha-helical proteins increased in intensity in the wet nails. The nu(S-S) which is sensitive to changes in conformation of proteins showed a 4% higher intensity. Additional protein-water interactions could lead to a slight change of the dihedral angle of the C-S-S-C bonds and to geometric changes in coiling behavior of the alpha-helical protein. Suggesting a separation between matrix proteins and fiber proteins giving them a greater freedom of flexibility. The in vivo spectra detected from the distal part of the nail resembled spectra in vitro. Raman spectra of the proximal part of the nail showed that it was fully saturated with water. The proximal part of the nail did not show changes in water content and protein structure during nail moisturizing in the Raman spectra. Our results suggest that the softening of the nail following hydration may be due to changed matrix protein molecular structure induced by water.     

Reduced models of networks of coupled enzymatic reactions

The Michaelis-Menten equation has played a central role in our understanding of biochemical processes. It has long been understood how this equation approximates the dynamics of irreversible enzymatic reactions. However, a similar approximation in the case of networks, where the product of one reaction can act as an enzyme in another, has not been fully developed. Here we rigorously derive such an approximation in a class of coupled enzymatic networks where the individual interactions are of Michaelis-Menten type. We show that the sufficient conditions for the validity of the total quasi-steady state assumption (tQSSA), obtained in a single protein case by Borghans, de Boer and Segel can be extended to sufficient conditions for the validity of the tQSSA in a large class of enzymatic networks. Secondly, we derive reduced equations that approximate the network's dynamics and involve only protein concentrations. This significantly reduces the number of equations necessary to model such systems. We prove the validity of this approximation using geometric singular perturbation theory and results about matrix differentiation. The ideas used in deriving the approximating equations are quite general, and can be used to systematize other model reductions.     

Induction of Adult T-Cell Leukemia-Like Lymphoproliferative Disease and Its Inhibition by Adoptive Immunotherapy in T-Cell-Deficient Nude Rats Inoculated with Syngeneic Human T-Cell Leukemia Virus Type 1-Immortalized Cells

Human T-cell leukemia virus type 1 (HTLV-1) has been shown to be the etiologic agent of adult T-cell leukemia (ATL), but the in vivo mechanism by which the virus causes the malignant transformation is largely unknown. In order to investigate the mechanisms of HTLV-1 leukemogenesis, we developed a rat model system in which ATL-like disease was reproducibly observed, following inoculation of various rat HTLV-1-immortalized cell lines. When previously established cell lines, F344-S1 and TARS-1, but not TART-1 or W7TM-1, were inoculated, systemic multiple tumor development was observed in adult nude (nu/nu) rats. FPM1 cells, newly established from a heterozygous (nu/+) rat syngeneic to nu/nu rats, caused transient tumors only at the injection site in adult nu/nu rats, but could progressively grow in newborn nu/nu rats and metastasize in lymph nodes. The derivative cell line (FPM1-V1AX) serially passed through newborn nu/nu rats acquired the potency to grow in adult nu/nu rats. These results indicated that only some with additional changes but not all of the in vitro HTLV-1-immortalized cell lines possessed in vivo tumorigenicity. Using the syngeneic system, we further showed the inhibition of tumor development by transferring splenic T cells from immunized rats, suggesting the involvement of T cells in the regression of tumors. This novel and reproducible nude rat model of human ATL would be useful for investigation of leukemogenesis and antitumor immune responses in HTLV-1 infection.