Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations

Single molecule assays were performed on streptavidin-β-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule β-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min^?1, which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be ?1.32 × 10^?4 ± 0.19 × 10^?4 cm²V^?1s^?1. The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-β-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.     

Free solution mobility of oligomeric DNA

The free solution electrophoretic mobility of a charged oligomer in an ionic solvent that approximately takes into account relaxation field effects, screening of the velocity field, and the hydrodynamic interactions resulting from motions of the charges due to an electric field is described. For double‐stranded DNA, the free solution electrophoretic mobility under ionic strengths determined by the buffer and pH conditions relevant to capillary electrophoresis increases with increasing molecular weight up to few hundred base pairs. © 1999 John Wiley & Sons, Inc. Biopoly 49: 209–214, 1999     

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.     

Dopant-free Starlike Molecular Hole Conductor with Ordered Packing for Durable all-Inorganic Perovskite Solar Cells

All-inorganic n-i-p perovskite solar cells (PSCs) using doped Spiro-OMeTAD as hole transport material (HTM) suffer from photothermal stability due to ionic diffusion and radical-induced degradation by the dopants. In this article, dopant-free starlike molecule (N2, N2-bis(4-(bis(4-methoxyphenyl)amino)phenyl)-N5,N5-bis(4-methoxyphenyl)pyridine-2,5-diamine (BD)) is synthesized to engineer the stacking properties and delivered higher hole mobility than doped Spiro-OMeTAD (3.2 × 10⁻⁴ versus 1.76 × 10⁻⁴ cm² V⁻¹ s⁻¹) as dopant-free HTM. Starlike BD HTM has a twisted acceptor unit and strong dipole, forming crystalline and ordered packing film to ensure intramolecular charge transfer and improve mobility. The BD CsPbI₃ PSCs deliver the maximum efficiency of 19.19%, which is the highest performance for all-inorganic PSCs based on dopant-free HTMs. Meanwhile, the ordered molecules-packing blocks the migration channel of I⁻ ions to metal electrodes and improves the device stability. BD-based devices maintain more than 93% and 80% of the initial efficiency after 85 °C storage for 35 days and maximum power point (MPP) tracking at 85 °C for 1000 h, respectively.     

DNA and buffers: the hidden danger of complex formation

The free solution electrophoretic mobility of DNA differs significantly in different buffers, suggesting that DNA-buffer interactions are present in certain buffer systems. Here, capillary and gel electrophoresis data are combined to show that the Tris ions in Tris-acetate-EDTA (TAE) buffers are associated with the DNA helix to approximately the same extent as sodium ions. The borate ions in Tris-borate-EDTA (TBE) buffers interact with DNA to form highly charged DNA-borate complexes, which are stable both in free solution and in polyacrylamide gels. DNA-borate complexes are not observed in agarose gels, because of the competition of the agarose gel fibers for the borate residues. The resulting agarose-borate complexes increase the negative charge of the agarose gel fibers, leading to an increased electroendosmotic flow of the solvent in agarose-TBE gels. The combined results indicate that the buffers in which DNA is studied cannot automatically be assumed to be innocuous.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Electrokinetic properties of asparaginase sensitive or resistant mouse leukemic cells treated with L-asparaginase

The electrophoretic mobility of L5178Y cells in 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2, at 25°C was — 1.78 μ·s^?1·V^?1·cm^?1 while that of an L-asparaginase resistant subline, L5178Y/ASN, was — 1.11 μm·s^?1·V^?1·cm^?1. Both cell lines were characterized by terminal sialic acid residues on their surfaces. Treatment of L5178Y cells for 90 min with 10 units of L-asparaginase per ml in saline decreased the electrophoretic mobility of the cells to — 1.65 μm·s^?1·V^?1·cm^?1 while treatment in Fischer's medium decreased the mobility to — 1.25 μm·s^?1·V^?1·cm^?1; neither treatment had a significant effect on the L5178Y/ASN electrophoretic mobility. The results suggest that L-asparaginase has an immediate and specific effect on synthesis of cell surface asparaginyl glycoproteins.     

Probing soft polymeric coatings of a capillary by atomic force microscopy

Atomic force microscopy (AFM) has been used to probe the surface of a capillary after coating with “soft” polymers, notably polyacrylamides. The aim was the investigation of the efficiency of coverage of the silica surface, so as to reduce or eliminate the electroosmotic flow (EOF), particularly noxious in the separation of macromolecules. The quality of such coating is strongly dependent on two variables: temperature and pH. In the first case, progressively higher temperatures produce open silica patches, where no polymer seems to be bound. The transition from coated to largely uncoated surfaces occurs at 50°C. Also the pH of the polymerizing solution strongly affects the coating efficiency. Since in all coating procedures the monomer solution is not buffered, addition of accelerator (TEMED, N,N,N′N′-tetramethylethylendiamine) induces polymer growth at pH 10–11. These pH values generate hydrolysis of the siloxane bridge anchoring the bifunctional agent (Bind Silane, onto which the polymer chain should grow) to the wall. Thus, coating and de-coating occur simultaneously. Low temperatures during polymer growth (typically 10°C) and buffered solutions (pH 7, titrated after TEMED addition) ensure a most efficient and thorough coating, with virtual elimination of EOF: well coated capillaries exhibit residual EOF values, at pH 10, of the order of 10⁻⁷ cm² V⁻¹ s⁻¹ vs. a standard value for uncoated capillaries of the order of 10⁻⁴ cm² V⁻¹ s⁻¹. The AFM data have been fully confirmed by direct measurement of EOF in coated and uncoated capillaries under an electric field.     

Batch‐to‐Batch Variation of Polymeric Photovoltaic Materials: its Origin and Impacts on Charge Carrier Transport and Device Performances

A detailed investigation of the impact of molecular weight distribution of a photoactive polymer, poly[N‐9′‐heptadecanyl‐2,7‐carbazole‐alt‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole)] (PCDTBT), on photovoltaic device performance and carrier transport properties is reported. It is found that different batches of as‐received polymers have substantial differences in their molecular weight distribution. As revealed by gel permeation chromatography (GPC), two peaks can generally be observed. One of the peaks corresponds to a high molecular weight component and the other peak corresponds to a low molecular weight component. Photovoltaic devices fabricated with a higher proportion of low molecular weight component have power conversion efficiencies (PCEs) reduced from 5.7% to 2.5%. The corresponding charge carrier mobility at the short‐circuit region is also significantly reduced from 2.7 × 10^?5 to 1.6 × 10^?8 cm² V^?1 s^?1. The carrier transport properties of the polymers at various temperatures are further analyzed by the Gaussian disorder model (GDM). All polymers have similar energetic disorders. However, they appear to have significant differences in carrier hopping distances. This result provides insight into the origin of the molecular weight effect on carrier transport in polymeric semiconducting materials.     

Exploring the intercalation binding of tiamulin with DNA using multispectroscopy and a molecular modelling approach

The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity (η) and melting temperature (T_m) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant (K_a) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10⁴ M⁻¹) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10³ M⁻¹) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol⁻¹ using van der Waals forces as well as hydrogen bonds.     

Binding characteristics of estrone,estradiol and estriol to the human myometrial estrogen receptor

The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (K_D 0.38 × 10⁻¹⁰M) has the highest affinity to the receptor followed by estrone (K_D 0.76 × 10⁻¹⁰M) and estriol ((K_D 1.33 × 10⁻¹⁰M). The association rate constant is 2.8 × 10⁵M⁻¹s⁻¹ for estradiol, 2.1 × 10⁵M⁻¹s⁻¹ for estrone and 0.79 × 10⁵M⁻¹s⁻¹ for estriol. The dissociation constants and the association rate constants increase with temperature. The calculated thermodynamic parameters indicate a positive change in entropy for the formation of the estrogen receptor complex.The cytoplasmic estrogen receptor has a sedimentation coefficient of 4 s in low salt sucrose gradients. In buffer containing diisopropylfluorophosphate (DFP) to inhibit proteolytic activity the estrogen receptor complex sediments solely as an 8 s peak if [³H]-estradiol is added to the buffer prior to homogenization and the tissue sample is used immediately after hysterectomy. Estrogen receptor complexes that sediment at 4 s and 8 s are found if [³H]-estradiol is omitted from the homogenization buffer and instead added after the cytosol preparation. Most likely a protease is involved the activity of which is not completely inhibited by DFP.Addition of low concentrations of Cu²⁺ (10 μM) to the cytosol increases the dissociation constant and decreases the estrogen-binding capacity of the receptor. The rate of association is reduced in the presence of 20 μM Cu²⁺. The estrogen receptor complexes do not show any change in their sedimentation profiles in the presence of Cu²⁺.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA

The results of semiselective ¹H-nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D₂O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A-T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s^?1 at 25°C. Corresponding values for the G-C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A-T and G-C imino protons, resembling the effect recently observed for poly(rA)-poly(rU) and poly(rI)-poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base-pair opening rates as assumed in the past. Lower limits to the base-pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 10³ s^?1 for the A-T, and 50 s^?1 for the G-C, base paris at 62°C.     

Mechanism of reversible glycine cleavage reaction in Arthrobacter globiformis. Function of lipoic acid in the cleavage and synthesis of blycine.

The physical and chemical characterization of horse serum butyrylcholinesterase has been extended. The results show that the enzyme is a glycoprotein containing about 20% carbohydrate by weight. Mannose, glucosamine, galactose, and sialic acid are the sugar residues found. The extinction coefficient of butyrylcholinesterase, E_1cm^1% at 280 nm, was found to be 15.2 ± 0.3 by dry weight determination. The molecular weight of the protein in dilute phosphate buffer was determined to be (31.7 ± 1.2) × 10⁴ by high speed equilibrium sedimentation with a redetermined partial specific volume of 0.723 ± 0.003 ml/g. Subunit molecular weights for the dissociated protein were found to be (7.9 ± 0.4) × 10⁴ and (8.1 ± 0.1) × 10⁴, respectively, in guanidine hydrochloride and in a solution at pH 11.8. The subunit molecular weight was also estimated to be (8.8 ± 0.2) × 10⁴ by analytical sodium dodecyl sulfate-gel electrophoresis. This apparently higher subunit molecular weight from dodecyl sulfate gels is expected for glycoproteins containing significant amounts of carbohydrate. No free sulfhydryl group was detected, even though there are six half-cystines in each subunit. Therefore, it seems likely that there are three pairs of disulfide bonds per subunit. The available data indicate that native butyrylcholinesterase is a tetrameric glycoprotein consisting of subunits of equal molecular weight.     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.     

Trivalent counterion condensation on DNA measured by pulse gel electrophoresis

Pulse gel electrophoresis was used to measure the reduction of mobilities of λ-DNA-Hind III fragments ranging from 23.130 to 2.027 kilobase pairs in Tris borate buffer solutions mixed with either hexammine cobalt(III), or spermidine³⁺ trivalent counterions that competed with Tris⁺ and Na⁺ for binding onto polyion DNA. The normalized titration curves of mobility were well fit by the two-variable counterion condensation theory. The agreement between measured charge fraction neutralized and counterion condensation prediction was good over a relatively wide range of trivalent cation concentrations at several solution conditions (pH, ionic strength). The effect of ionic strength, trivalent cation concentration, counterion structure, and DNA length on the binding were discussed based on the experimental measurements and the counterion condensation theory. © 1996 John Wiley & Sons, Inc.     

Joining of bacteriophage T4D heads and tails: A kinetic study by inelastic light scattering

We have used inelastic laser light scattering to study the kinetics of the spontaneous assembly of heads and tails of bacteriophage T4D to form noninfectious tail fiberless particles. For interpretation of the kinetics, it was first necessary to determine the physical properties of the strongly scattering phage parts. For heads, these are D_{20,w = 3.60 × 10⁻⁸cm²/s, 820,w = 1025 S, M = 1.76 × 10⁸}. For tail fiberless particles, D_20,w = 3.14 × 10⁻⁸cm²/s, 8_20,w = 968 S, and M = 1.95 × 10⁸. The kinetics of the head-tail joining process was followed by measuring the time variation of the homodyne scattering autocorrelation function. This was interpreted as a sum of exponentials whose decay constants were known from the scattering angle and the diffusion coefficients, and whose amplitudes were related to the concentrations of reactants and products. Scattering experiments at 22 °C gave a bimolecular rate constant of 1.02 × 10⁷m⁻¹ s⁻¹, while infectivity assays at 30 °C gave a rate constant of 1.28 × 10⁷. Adjustment of both rate constants to 20 °C, assuming diffusion controlled reaction, gave 0.97 × 10⁷ and 0.98 × 10⁷m⁻¹ s⁻¹, respectively. This rate is about $\text{1}\text{500}$ that predicted by Smoluchowski theory for a diffusion controlled reaction between two spherical particles; the discrepancy is largely explicable from orientational factors.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.