Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, . . ., 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.     

Inhibition of bone resorption in vitro by serine-esterase inhibitors

The effect of two synthetic serine esterase inhibitors, N-alpha-dansyl(p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (D-Phe-Pro-Arg-CH2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca2+) and inorganic phosphate (Pi). Organic matrix degradation was studied by analyzing the release of 3H from [3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 mumol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E2 and 1-alpha-hydroxyvitamin D-3. I 2581 (50 mumol/l) inhibited PTH-stimulated release of 3H from [3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 mumol/l) inhibited the release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in bones stimulated by PTH and 1-alpha-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-alpha-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 mumol/l) did not affect the basal release of 45Ca, Ca2+, beta-glucuronidase and beta-N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. D-Phe-Pro-Arg-CH2Cl (100 mumol/l) significantly inhibited PTH- and PGE2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.     

New bivalent thrombin inhibitors with N(alpha)(methyl)arginine at the P1-position

A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.     

Quantitative structure-activity relationships for the pre-steady state of Pseudomonas species lipase inhibitions by p-nirophenyl-N-substituted carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1-6) are composed of two steps: (1) formation of the non-covalent enzyme-inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme-inhibitor adduct (E-I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, KS, and the rate constant for formation of the tetrahedral E-I adduct from the E:I complex, k2 are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k(obs)) versus inhibition concentration ([I]) plot against k(obs)=k2+k2[I]/(KS+[I]). Values of pKS, and log k2 are linearly correlated with the sigma* values with the rho* values of -2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the rho* value of -2.0. The tetrahedral E-I adducts on the other hand are more negative charges than the E:I complexes due to the rho* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

Qualitative analysis of a mathematical model of a open enzyme reaction

A general case of the set of two differential equations, describing an open reaction v1 leads to S v reversible E P v2 leads to, has been considered. The requirements to the character of the functions v1([S]), v2([P]) and v([S], [P]) were formulated for the case of existence and absence of alternative steady states and sustained oscillations. The formulae were derived to determine the slope of the unstable portion of the quasi-steady state characteristic. The generalized model of Monod, Wyman and Changeux has been considered as an example of v([S], [P]). It has been shown that with monotonically decreasing v1 and monotonically increasing v2, the alternative steady states and oscillations are possible only in the presence of substrate inhibition or product activation. However, under the joint action of substrate inhibition and product activation, the system will exhibit bistability rather than an oscillatory behavior. In the case of an irreversible two-substrate reaction which can be described by a similar mathematical model, inhibition by the first and second substrate is equivalent to substrate inhibition and product activation.     

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.     

Halogenated phlorethols and fucophlorethols from the brown alga Cystophora retroflexa.

From an ethyl acetate fraction of the brown alga Cystophora retroflexa several halogenated phlorotannins were isolated. Most of the compounds are derivatives of diphlorethol penta-acetate and triphlorethol-A hepta-acetate. The majority turned out to be chlorinated and/or brominated. Only one iodinated substance, 2-iodophloroglucinol triacetate, was isolated. The structure of this derivative and the following compounds have been characterized previously: 2([D])-bromodiphlorethol penta-acetate, 3([A1])-bromodiphlorethol penta-acetate, 4([D])-bromo-diphlorethol penta-acetate, 4([D])-chlorodiphlorethol penta-acetate, 3([A1])-chlorotriphlorethol-A hepta-acetate, 4([D])-bromotriphlorethol-A hepta-acetate and 4([D])-chlorobisfucopentaphlorethol-A nonadeca-acetate. Ten halogenated phlorethols and two chlorinated fucophlorethols are described for the first time and characterized as their acetates: 2([B])-bromotriphlorethol-A hepta-acetate, 2([D])-bromotriphlorethol-A hepta-acetate, 2([B]), 2([D])-dibromotriphlorethol-A hepta-acetate, 3([A1]), 5([A1])-dichlorotriphlorethol-A hepta-acetate, 3([A1]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 3([A1])-chloro-4([D])-bromotriphlorethol-A hepta-acetate. 2([B]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 2([D]), 3([A1])-dibromotriphlorethol-A hepta-acetate, 3([A1])-bromo-2([D])-chlorotriphlorethol-A hepta-acetate, 2([D])-bromotetraphlorethol-C nona-acetate, 4([D])-chlorofucotriphlorethol-B dodeca-acetate and 4([D])-chlorobisfucotetraphlorethol-A heptadeca-acetate.     

Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism.

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513--522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.     

Evaluation of the growth rate of MCF-7 breast cancer multicellular spheroids using three mathematical models

Abstract. Growth data on 60 multicellular spheroids of MCF-7 human breast cancer cells were fitted, on an individual basis, by the Gompertz, Bertalanffy and logistic equations. MCF-7 spheroids, initiated and grown in medium containing oestrogens, exhibited a growth rate that decreased continuously as spheroid size increased. Plots of spheroid volume v. time generated sigmoid curves that showed an early portion with an approximately exponential volume increase; a middle region or retardation phase characterized by a continuously decreasing growth rate; and, finally, a late segment or plateau phase approaching zero growth rate, that permitted an estimate of the maximum spheroid size (V_max). Growth curves generated by MCF-7 spheroids under different experimental conditions (hormones, drugs and radiation exposures) can be compared after normalization. Linearized forms of the fitted Gompertz curves provided a convenient way to express differences in growth rate.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.     

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.     

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.     

Estimation of long-term effective population sizes through the history of durum wheat using microsatellite data

Estimation of long-term effective population size (N(e)) from polymorphism data alone requires an independent knowledge of mutation rate. Microsatellites provide the opportunity to estimate N(e) because their high mutation rate can be estimated from observed mutations. We used this property to estimate N(e) in allotetraploid wheat Triticum turgidum at four stages of its history since its domestication. We estimated the mutation rate of 30 microsatellite loci. Allele-specific mutation rates mu were predicted from the number of repeats of the alleles. Effective population sizes were calculated from the diversity parameter theta = 4N(e)mu. We demonstrated from simulations that the unbiased estimator of theta based on Nei's heterozygosity is the most appropriate for estimating N(e) because of a small variance and a relative robustness to variations in the mutation model compared to other estimators. We found a N(e) of 32,500 individuals with a 95% confidence interval of [20,739; 45,991] in the wild ancestor of wheat, 12,000 ([5790; 19,300]) in the domesticated form, 6000 ([2831; 9556]) in landraces, and 1300 ([689; 2031]) in recent improved varieties. This decrease illustrates the successive bottlenecks in durum wheat. No selective effect was detected on our loci, despite a complete loss of polymorphism for two of them.     

Hexafluoroisopropanol and acid destabilized forms of apomyoglobin exhibit structural differences

The conformational properties of partially folded states of apomyoglobin have been investigated using an integrated approach based on fluorescence spectroscopy and hydrogen/deuterium exchange followed by mass spectrometry. The examined states were those obtained: (i) by adding 4% v/v hexafluoroisopropanol to native myoglobin, HFIP-MG(N); (ii) by adding 4% v/v hexafluoroisopropanol to acid unfolded myoglobin, HFIP-MG(U); (iii) at pH 3.8, I-1 state; and (iv) at pH 2.0-0.2 M NaCl, A state. Proteolytic digestion of the hydrogen/deuterium exchanged proteins showed that, in I-1 state, the helices C, D, E, and F incorporate more deuterium, whereas in HFIP-MG(N) the exchange rate is similar for all protein regions. These results suggest that I-1 contains the ABGH domain in a native-like organization, whereas HFIP-MG(N) loses a large number of tertiary interactions, thus acquiring a more flexible structure. The fluorescence data are consistent with the above picture. In fact, the tryptophan/ANS energy transfer is much less efficient for the ANS-HFIP-MG(N) complex than for the other complexes, thus suggesting that the distances between the fluorophores might be increased. Moreover, fluorescence polarization measurements indicated that the rotational motion of HFIP-MG(N) occurs on a longer time scale than the other partially folded states, thus suggesting that the volume of this state could be larger. The overall results indicate that addition of hexafluoroisopropanol to native myoglobin results in the formation of a true molten globule where tertiary interactions are reduced, while the secondary structure and the globular compactness are conserved.     

Inhibition and activation of enzymes. The effect of a modifier on the reaction rate and on kinetic parameters

A combined analysis of enzyme inhibition and activation is presented, based on a rapid equilibrium model assumption in which one molecule of enzyme binds one molecule of substrate (S) and/or one molecule of a modifier X. The modifier acts as activator (essential or non-essential), as inhibitor (total or partial), or has no effect on the reaction rate (v), depending on the values of the equilibrium constants, the rate constants of the limiting velocity steps, and the concentration of substrate ([S]). Different possibilities have been analyzed from an equation written to emphasize that v = f([X]) is, in general and at a fixed [S], a hyperbolic function. Formulas for Su (the value of [S], different from zero, at which v is unaffected by the modifier) and v(su) (v at that particular [S]) were deduced. In Lineweaver-Burk plots, the straight lines related to different [X] generally cross in a point (P) with coordinates (Su, v(su)). In certain cases, point P is located in the first quadrant which implies that X acts as activator, as inhibitor, or has no effect, depending on [S]. Furthermore, we discuss: (1) the apparent Vmax and Km displayed by the enzyme in different situations; (2) the degree of effect (inhibition or activation) observed at different concentrations of substrate and modifier; (3) the concept of Ke, a parameter that depends on the concentration of substrate and helps to evaluate the effect of the modifier: it equals the value of [X] at which the increase or decrease in the reaction rate is half of that achieved at saturating [X]. Equations were deduced for the general case and for particular situations, and used to obtain computer-drawn graphs that are presented and discussed. Formulas for apparent Vmax, Km and Ke have been written in a way making it evident that these parameters can be expressed as pondered means.     

Fracture Faces in the Cell Envelope of Escherichia coli

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.     

Kinetic properties of derepressible acid phosphatase from the yeast from of Yarrowia lipolytica

(1) An acid phosphatase from depressed cells of the yeast form of Yarrowia lipolytica has been characterized kinetically by studies on specificity, inhibition, rate equation forms and modelling of the enzyme mechanisms. (2) The study on specificity revealed that the acid phosphatase is a rather unspecific phosphohydrolase that has similar activity on several different phosphate esters. A very weak transphosphorylating activity was also detected. (3) Among the reversible inhibitors, phosphate and vanadate were outstanding, whereas EDTA behaved as an activator. (4) v vs. [S] studies with o-carboxyphenyl phosphate as substrate show that the acid phosphatase of Y. lipolytica exhibits non-Michaelian behaviour, a minimum degree of 2:2 being detected for the rate equation in [S]. (5) The inhibition by phosphate and vanadate seems to have the same pattern of partial inhibition with a certain non-competitive nature, 1:1 being the minimum degree of the rate equation detected in (I).     

Ectopic expression of inhibitors of protein phosphatase type 1 (PP1) can be used to analyze roles of PP1 in Drosophila development

We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGF beta superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.