Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

Thermodynamics of stabilization of RNA pseudoknots by cobalt(III) hexaammine

Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH₃) have been determined, relative to divalent Mg²⁺, for the folded, partially folded (K_f), and fully unfolded (K_u) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two‐state unfolding transitions. Trivalent Co(NH₃) was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a K_f of 5–8 × 10⁴ M⁻¹ in a background of 0.10 M K⁺, or 3‐ to 5‐fold larger than the K_f obtained for two model RNA hairpins and hairpin unfolding intermediates, and ≈ 40–50‐fold larger than K_f for Mg²⁺. The magnitude of K_f was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with K_f reaching 1.2 × 10⁵ M⁻¹ in 50 mM K⁺. Two RNA hairpins were found to have affinities for Co(NH₃) and Ru(NH₃) of 1–2 ×10⁴ M⁻¹, or ≈ 15‐fold larger than the K_f of ∼ 1000 M⁻¹ observed for Mg²⁺. Additionally, the K_u of 4,800 M⁻¹ for the trivalent ligands is ≈ 8‐fold larger than the K_u of 600 M⁻¹ observed for Mg²⁺. These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg²⁺ and Co(NH₃) binding of significantly higher affinity than a “duplexlike” delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2. © 1999 John Wiley & Sons, Inc. Biopoly 50: 443–458, 1999     

The Significance of the Internal Nutrient Load to Hypertrophic Shallow Waters

In two carp ponds with a long water residence time, the seasonal changes in concentration and internal mass-flow of inorganic nutrients have been estimated together with oxygen production P_N of phytoplankton and community respiration. The results were compared with the findings from laboratory microcosms (LM) with a high recycle rate between a) euphotic and b) aphotic + sediment compartment. In the LM's, a dark period of at least 12 hours was necessary for optimum remobilization of CO₂, PO⁻ and NH. Very high values of P_N were measured even if external nutrient sources were missing. Low nutrient concentrations could be compensated by high vertical fluxes. A cycle of nocturnal mixing and of thermal stratification with anoxia at the mud water interface which increases nutrient regeneration seems to be the principal mechanism which maintains very high P_N in hypertrophic shallow waters.     

Biopolymer–surfactant interaction. I. Kinetics of binding of cetyltrimethyl ammonium bromide with carboxymethyl cellulose (Na Salt)

The kinetics of binding of the cationic surfactant cetyltrimethyl ammonium bromide with the Na salt of carboxymethyl cellulose was studied by the electrometric method using cetyltrimetlyl ammonium⁺ (CTA⁺) ion-selective polyvinyl chloride membrane electrode. The binding process followed the first-order kinetics and occurred in three stages. Its affinity increased with increasing CTA bromide concentration and decreased with ionic strength. The activation process comprised moderate E and ΔH^≠ and negative ΔS^≠ for all three stages with a ΔH^≠ < TδS^≠ trend proving it to be entropy controlled. The ΔG^≠ values followed the trend ΔG < ΔG < ΔG (in accordance with k₁ > k₂ > k₃). The enthalpies (ΔH^≠) and entropies (ΔS^≠) of activation followed a systematic and interdependent trend. The multiple-stage binding kinetics is grossly comparable with the kinetics of binding of proteins to solid surfaces. © 1995 John Wiley & Sons, Inc.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Conformational analysis of acetyl-L-phenylalanine p-acetyl and p-valeryl anilides

Empirical force-field calculations and ir and ¹H-nmr spectra indicate that five-membered (C₅) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C₅/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.     

Compressibility studies of three proteins in CsCI solutions in the analytical ultracentrifuge

The compositional buoyant densities, ρ;, of human γ-immunoglobulin, bovine serum mercaptalbumin, and egg albumin have been measured in CsCl solutions in the analytical ultracentrifuge as a function or pressure. Standard pressure coefficients, ψ⁰, and standard partial specific volumes of the solvated proteins, υ ,0, have been computed from these data. The ψ⁰ values obtained are strikingly different from each other and from the only other pressure coefficients which have been measured, those values obtained for nucleic acids and nucleoproteins. The ψ value for γ-immunoglobulin is negative, the first nonpositive value obtained, and suggests an unusual internal structure for this protein. The pressure coefficient of mercaptalbumin is not constant. A second-order relation is derived and utilized to interpret these data. The slope of the ρ(P) plot for egg albumin was constant and negative and yielded values of ψ⁰ which are about 20% as large as those reported for DNA. Evaluation of published isopiestic data for egg albumin in CsCl solutions provided the dependence of preferential hydration on water activity. This quantity, (dΓ′/da) as well as α, were found to be negative. The values of ψ⁰ and α were used to compute the effective density gradient from which the correct molecular weight of egg albumin was obtained. The apparent specific volume of egg albumin in a buoyant CsCl solution was measured using the Mettler-Paar densimeter.     

Enantioselective interactions of inversion-labile trigonal iron(II) complexes upon binding to DNA

We studied the interactions of the substitution-inert inversion-labile complexes Fe(bipy) and Fe(phen) [and the inversion-stable complex Ru(bipy)] with DNA. The association of these complexes to DNA is mainly electrostatic, and Fe(phen) shows a more effective binding to DNA than the two bipyridyl complexes, possibly owing to a different binding mode. The interactions are enantioselective, leading to a Pfeiffer shift in the diastereomeric inversion equilibria and an excess of the Δ-enantiomer of Fe(phen) and Fe(bipy), which is directly monitorable through CD. The partition constants for the inversion equilibrium range from 1.3 to 2.0 for Fe(bipy) and Fe(phen), depending on ionic conditions. From flow LD information about the orientation of the complexes on DNA was obtained: it is consistent with a fit of the Δ-enantiomer in the major groove of the right-handed DNA helix. The mechanisms of interaction are discussed against equilibrium, spectroscopic, and kinetic data.     

Studies on ventilation of culture broths. I. Behavior of CO2 in model systems

The rate of dissolution and dehydration of CO₂ in a liquid model system was investigated. Components in the model system established the main conditions which may exist, in the extracellular space of a microbiological culture liquid. The charge in voltage of a glass electrode was measured which indicated the formation of H⁺ ions in the H₂CO₃ ? HCO H⁺ reaction. The rate of CO₂ hydration increased with the increase of temperature from 0 to 40°C. Likewise the equilibrium of the reaction was shifted towards the forward reaction. Similar results were observed when the tip velocity of the impeller was increased. Data suggest that agitation promotes the dissolution of CO₂ in the culture liquid through the reduction of gas-liquid film resistance in the diffusion of this gas. The rate of hydration of CO₂ into the bulk of the liquid was independent of pCO₂ above the surface of the liquid but depended on pCO₂ in the gas bubble within the liquid. The concentration of HCO was, furthermore, influenced by the buffer components, buffer capacity, and the viscosity of the system. Since pCO₂ and the HCO concentration in the extracellular space depend on both physical and chemical factors, the ventilation of a culture liquid necessitates both exhaust of CO₂ from the gas bubbles of the culture broth and shift of the H₂CO₃ ? HCO + H⁺ reaction towards the backward direction.     

Apparent molar volumes of some amino acids and peptides in aqueous urea solutions

Densities of solutions of several α-amino acids and peptides in 3 and 6m aqueous urea solvents have been determined at 298.15 K. These data have been used to evaluate the infinite-dilution apparent molar volumes of the solutes and the volume changes due to transfer (V ) of the α-amino acids and peptides at infinite dilution from water to aqueous urea solutions. The sign and magnitude of the V values have been rationalized in the framework of Friedman's cosphere-overlap model. The V values for the glycyl group (? CH₂CONH? ) and alkyl side chains have been estimated.     

Regulation of the Na+-K+(NH)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH₄Cl, and the variations of the intracellular concentration of calcium ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured using fura-2 and 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH₄Cl significantly increased the [Ca²⁺]_i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH₄Cl, the cells acidified immediately. At 30 mmol/l, NH₄Cl first alkalinized the cells and the pH_i subsequently dropped. This drop reflects the uptake of NH ions that dissociate to NH₃ and H⁺ in the cytosol. These protons are exchanged for extracellular sodium by the Na⁺/H⁺ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH₄Cl. Ouabain partly blocked the uptake of NH. In the combined presence of ouabain and bumetanide (an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter), 1 mmol/l NH₄Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na⁺-K⁺-2Cl⁻ cotransporter in the uptake of NH was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β-adrenergic agonist. ATP increased the release of ³H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na⁺-K⁺-2Cl⁻ cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley-Liss, Inc.     

Conformation of poly(L-arginine). I. Effects of anions

The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P₂O, P₃O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH^?, Cl^?, F^?, H₂PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).     

Gel electrophoresis measurement of counterion condensation on DNA

We used agarose gel electrophoresis to measure the effective charge neutralization of DNA by counterions of different structure and valence, including Na⁺, Mg²⁺, Co(NH₃), and sperinidine³⁺, which competed for binding with an excess of Tris acetate buffer. Linear DNA molecules ranged in size from 1 to 5 kilobases, and supercoiled plasmid pUC18 was also measured. In all cases, the results were in good agreement with theoretical predict ions from counterion condensation theory for two-counterion mixtures. © 1995 John Wiley & Sons, Inc.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Physical association of two simple alkylators to some DNA sequences

Molecular mechanics calculations have been used to determine the preferred physical association sites of the known alkylating agent dimethyl aziridinium ion (Az⁺) and a CH prototype test probe with B-form, tetrameric DNA sequences. Electrostatic interactions are most important in determining these preferential physical association sites. In turn, the intermolecular energy minima depend on the charge distribution assigned to the DNA sequence. However, for three reported DNA charge distributions, only two distinct sets of energy minima were obtained for the CH-like ion interacting with (G-C)₄, (A-T)₄, and [(G-C)·(A-T)]₂ deoxyribonucleic acids. These minima correspond to physical association geometries in which the CH-like ion is near known alkylation sites. The results of the Az⁺ … [(G-C)·(A-T)]₂ interaction are virtually identical to those found for the CH-like ion. Aqueous solvation energetics have little effect on the physical association of Az⁺ with [(G-C)·(A-T)]₂.     

Number of binding sites of DNA and polynucleotides with tris(2,2'-dipyridyl)ruthenium(II) cations

The binding of tris(2,2′-bipyridyl)ruthenium(II) cations [Ru(bpy)] with single- and double-stranded (ss and ds) DNA, and the polynucleotides poly(A), poly(C), poly(G), poly(I), poly(I) · poly(C), and poly(U), was studied in aqueous solution. Steady-state electrical conductivity measurements with the polynucleotides, ssDNA, and dsDNA reveal that approximately three nucleotides offer one binding site. This may be compared with the ratio [nucleotide]/[Mg²⁺] of 2.4 : 1 for dsDNA. After laser excitation (353 nm), the luminescence of Ru(bpy) bound to nucleic acids shows two decay components. The contribution of the fast component, which is interpreted as resulting from quenching processes of the absorbed ruthenium complex, exhibits a maximum with increasing [nucleotide]/[Ru(bpy)] at a ratio of about three to one. Bound Ru(bpy) can be released from the strand by addition of NaClO₄ [half-concentration: 2.5 and ≤ 10 mM for poly(U) and dsDNA, respectively].     

Intrinsic viscosity of native and single-stranded T7 DNA and its relationship to sedimentation coefficient

The intrinsic viscosity and sedimentation coefficient, of native and single-stranded T7 DNA have been determined at 25°C as a function of ionic strength in neutral and alkaline NaCl. The relationship between [η] and S_,w is well represented by the Mandelkern-Flory equation over the entire range of conditions between 0.0013 and 1M Na⁺. An apparent discrepancy between the two methods at moderate to high ionic strengths is probably due to a change in V with ionic strength. It appears that [η] is a more sensitive and reliable measure of molecular expansion for native DNA, S_,w but is a better index of conformational change in single strands, since [η] becomes too small to measure conveniently at high ionic strengths. At moderate to high ionic strengths, denaturation leads to a decrease in [η], although unfolded single strands retain considerable viscosity. At sufficiently low ionic strength, the intrinsic viscosity of the single strands becomes higher than that of native DNA, and the effective volume of a single strand approaches that of the native molecule.     

Bromophenol red as a probe of lysozyme-active site environment: a temperature-jump study.

Spectrophotometric techniques have been employed to study the binding of bromophenol red (BPR) to hen egg white lysozyme and the consequent inhibition of enzyme activity. Experimental evidence is given from the dye binding studies in the presence of hexasaccharide and from the studies on activity that BPR binds at a site outside the proposed cleft region (A–F) in such a way that it inhibits the lytic activity towards cell walls but does not inhibit the activity towards hexasaccharide. These observations are consistent with the kinetics of binding [studied using temperature-jump (T-jump)] in the presence of Co⁺⁺ or chitotriose in large concentrations and the experiments with acetylated lysozyme which suggest that the binding site of BPR is closer to a lysine residue near the cleft. It is suggested that the binding site of BPR could be important in positioning the peptide segment of the cell walls, which are cleaved in the cleft. Evidence for the statement that this binding takes place at least by a two-step process, in which the bimolecular step is followed by a slower monomolecular step, is given from the observations of two types of 1:1 complexes at 24°C in equilibrium studies and from the concentration dependence of the relaxation observed at 605 nm in the T-jump experiments. The binding process is examined by analyzing the T-jump data obtained between 18 and 33°C in the pH range 5.2–9.2 and ionic strength 0.01–01. The ionic strength and pH dependences of the equilibrium constant associated with the bimolecular step k₂/k₁ and the forward rate constant associated with monomolecular step k₃ have been given as evidence for the suggestion that a Coulombic interaction is involved in the first step of binding. However, the final state of binding is hydrophobic in nature. The enthalpy of activation ΔH and the entropy of activation ΔS associated with k_f[= k₃(k₁/k₂)] showed compensation behavior with pH variation, with maxima around pH ～ 7.5 in H₂O. This has been interpreted as a maximal disordering of water structure in a region of the enzyme at this pH during the monomolecular step. However, the binding of chitotriose or Co⁺⁺ in the cleft reduces the ΔH and ΔS associated with the monomolecular step of BPR binding, probably by disordering the structured water during their binding in the cleft. The differences in the kinetic parameters obtained in H₂O and in D₂O probably arise due to subtle differences in the conformation of the enzyme in the two solvents and apart from isotope effects. The correlation between the pH (or pD) dependence of the “intrinsic activity” towards cell walls and ΔH or ΔS indicates that ordered water structure could be playing a role in controlling the catalytic activity. It is also suggested that this factor is associated with the rate constant k₃^s of the monomolecular step leading to the formation of the final bound state of the substrate in cell lysis, which is also a factor controlling k_cat.