First-order Loss Research Articles

Pharmacokinetics of Cyfluthrin in Spodoptera littoralis (Boisd.). I. In vivo Distribution and Elimination of [ 14C]Cyfluthrin in Susceptible and Pyrethroid-Resistant Larvae

Pharmacokinetics of [ 14C]cyfluthrin were studied for a period of 32 hr following the topical application of 2.5 ng/larva (LD 20 for the susceptible strain) to fourth instar susceptible and pyrethroid-resistant S. littoralis larvae. Penetration rates were determined by rinsing the outer surface of the larvae with hexane. The amounts of 14C-radioactivity in the washed larvae, the feces, and the feeding medium were obtained by catalytic combustions. A compartmental model with curve-fitting was used in order to quantify the rates of penetration, distribution, and elimination of 14C-radioactivity for the 32 hr following treatment. Penetration of [ 14C]cyfluthrin showed identical biphasic kinetics in resistant and susceptible strains, with a rapid increase of the internal cyfluthrin concentration during the first 6 hr followed by a slow first-order loss of penetrated material from the insect body. Kinetics of 14C elimination by the larvae and of 14C recovery in the feces were different in resistant and susceptible larvae. Fitting to a three-compartment model showed that these differences result from changes in the relative capacities of the distributive phase (hemolymph) and the important regurgitation of gut contents in susceptible insects. However, the internal exposure to cyfluthrin was similar in both strains. Resistance did not therefore arise from pharmacokinetic mechanisms, but is more likely to result from modifications of the target sites for pyrethroids.

Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. Selective modification of an active-site lysine.

Incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE) results in a pseudo first-order loss of enzyme activity. The pH dependence of the inactivation rate exhibits an inflection around pH 10, and the enzyme is protected from inactivation by glucose 6-phosphate. Loss of enzyme activity corresponds with the formation of one carbonyl function per enzyme subunit and the appearance of a lysine-HNE adduct. The data presented in this paper are consistent with the view that the epsilon-amino group of a lysine residue in the glucose 6-phosphate-binding site reacts with the double bond (C3) of HNE, resulting in the formation of a stable secondary amine derivative and loss of enzyme activity. We have described a mechanism by which HNE may, in part, mediate free radical damage. In addition, a method for the detection of the lysine-HNE adduct is introduced.

Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.

A three-dimensional numerical model for water flow and transport of chemically reactive solute through porous media under variably saturated conditions

A three-dimensional numerical model is developed for the simulation of water flow and chemical transport through variably saturated porous media. The nonlinear flow equation is solved using the Galerkin finite element technique with the Picard iteration scheme and a continuous velocity field is obtained by separate application of the Galerkin technique to the flux equation. A two-site adsorption-desorption model with a first-order loss term is used to describe the chemical behavior of the reactive solute. The advective part of the transport equation is solved with one-step backward particle tracking while the dispersive part is solved by the regular Galerkin finite element technique. Preconditioned conjugate-gradient-like method is used for the iterative solution of the systems of linear simultaneous equations to save on computer memory and execution time. The model is applied to a few situations and the numerical results are compared with observed and analytic values. The model is found to work quite well, even near very sharp fronts.

Unidimensional solute transport incorporating equilibrium and rate-limited isotherms with first-order loss. 3. Approximate simulations of the front propagating after a step input : Lassey, K R Water Resour ResV25, N11, Nov 1989, P2357–2366

Unidimensional solute transport incorporating equilibrium and rate-limited isotherms with first-order loss. 3. Approximate simulations of the front propagating after a step input : Lassey, K R Water Resour ResV25, N11, Nov 1989, P2357–2366

Herpes simplex virus type 1 DNA polymerase: Mechanism of inhibition by acyclovir triphosphate

Abstract Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the next nucleotide) from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.

Rate Constants for the Reactions t-C4H9+DX→i-C4H9D+X(X = Br,I), 295T(K) < 384: Heat of Formation of the tert-Butyl Radical

Absolute values for the rate constants of the metathesis reactions of DX (X = Br, I) with tert-butyl, generated by 351-nm photolysis of 2,2{prime}-azoisobutane, were determined in a low-pressure Knudsen cell reactor using the VLP{Phi} (very low pressure photolysis) technique. For X = Br, the values are 10{sup {minus}8}k (M{sup {minus}1} s{sup {minus}1}) = 0.9 and 2.3, and for X = I the values are 2.1 and 3.1 at 295 and 384 K. The latter are in good agreement with earlier measurements from this laboratory. The former values are lower by a factor of 50 compared to those recently reported at 295 K and fail to show the negative activation energy found in that work. Correcting by {radical}2 for the primary isotope effect and combining with the rate constants for the reverse reactions lead to {Delta}H{sub f}{degree} {sub 298}(tert-butyl) (kcal mol{sup {minus}1}) = 9.2 {plus minus}0.5 in both cases. First-order heterogeneous loss of tert-butyl radicals on the Teflon-coated surface employed is small (< 2 s{sup {minus}}1). 17 refs., 9 figs., 3 tabs.

Sticking coefficient of the SiH2 free radical on a hydrogenated silicon-carbon surface

The sticking coefficient of SiH2 on a hydrogenated silicon-carbon surface is measured in a low-pressure pulsed-photolysis experiment. Thermal and vibrationally excited SiH2 are created by infrared multiphoton decomposition of n-butylsilane. The first-order wall loss rates of the radicals are determined from the time dependence of the resonance-enhanced multiphoton ionization signal. The sticking coefficients of SiH2 (∼0.1) and vibrationally hot SiH2 (&amp;gt;0.5) are determined from the measured first-order loss rate constants and the calculated wall collision rate constant.

In Vitro Kinetic Studies of the Reaction of Hydralazine and its Acetone Hydrazone with Pyruvic Acid

To understand the reaction between hydralazine (HP) or its acetone hydrazone (HAH), a metabolite of HP and pyruvic acid, a new selective HPLC method for simultaneous determination of HP, HAH, and hydralazine pyruvic acid hydrazone (HPH) was developed. In vitro degradation of HAH and formation of HP and HPH were investigated at pH 7.4 and 37 C in the presence or absence of pyruvic acid. Hydralazine degraded slowly according to an apparent first-order rate (7.46 × 10−2 h−1). The degenerative reaction of HAH, accompanied by simultaneous hydrolysis to the parent drug HP, was also subject to apparent first-order loss (3.00 × 10−1 h−1). In addition, HAH was partly converted to HP and HPH in the presence of pyruvic acid. For the formation pathway of HPH, a model that included the direct reaction of HAH with pyruvic acid and the secondary formation mediated by back-conversion to HP gave a better fit to the experimental data than the model consisting of the latter reaction only. About 10% of the HPH formed was generated by the direct reaction of HAH with pyruvic acid, based on the rate constants estimated. These results suggest that the formation of HPH is not all accomplished through back-conversion to HP.

Physical stability of a recombinant alpha 1-antitrypsin injection.

Physical stability of a recombinant alpha 1-antitrypsin (rAAT) injection solution, namely, loss of rAAT to particulate formation was studied. The rAAT injection solution (1.0 mg/ml, pH 7.5, triple buffer of phosphate, Tris, and glycine, 0.075 M each) was filtered through a 0.2-micron filter and packaged in individual vials with rubber stoppers and aluminum seals. The vials were stored at 90, 80, 60, 45, 35, and 25 degrees C, and samples taken at predetermined time intervals for each storage condition. Each sample was then filtered through a 0.2-micron filter to remove particulates. The filtrate was then assayed for total protein by the biuret-phenol method. A typical first-order loss of rAAT was observed. Data were fitted to a first-order kinetics, and the shelf life (defined as t90, the time for the product to reach 90% remaining) was calculated from the resultant rate constant. The shelf lives were plotted against reciprocals of storage temperatures (a modified Arrhenius plot). A linear regression line (r = 0.9831) was drawn through the data points and extrapolated to 4 degrees C. At 4 degrees C, the predicted shelf life of the rAAT solution is 5.1 months. In real life, a batch of the rAAT solution showed that 61% of the rAAT remained in the filtrate after 18 months of storage at 4 degrees C. The observed stability compares fairly well with the predicted value of 69% for the same duration of storage at 4 degrees C. From the slope of the linear regression line, the activation energy for the particulate formation of the rAAT was calculated to be 19 kcal. This value is comparable to the activation energy of 20 kcal for ovalbumin denaturation reaction reported by Simpson and Kauzmann.

Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus.

ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.

Unimolecular decomposition of NO3 to form NO and O2 and a review of N2O5/NO3 kinetics

The nitrate free radical NO3 has been observed by several investigators in the troposphere at night. It increases during the night much less than predicted in terms of known processes, and there has been a search for an unknown NO3 scavenger. In this article we report evidence in the chemical literature for the thermal decomposition of NO3 to form nitric oxide and molecular oxygen, where the reaction w is NO3 → NO + O2, and the rate is such that this reaction is proposed as a scavenging process in the troposphere. By straightforward reinterpretation of three room temperature kinetic studies which used reactors of widely different volumes, we find the first‐order rate constant at one atmosphere pressure to be w = (3 ± 2) × 10−3 s−1; in these systems the otherwise unexplained first‐order loss of NO3 accounted for 5–74% of the observed rate. Using high‐temperature shock tube experiments, Schott and Davidson [1958] evaluated rate constants for reaction e (NO2 + NO3 → NO + O2 + NO2) and reaction g (2NO3 → 2NO2 + O2), but these values are much higher than the extrapolated results of Graham and Johnston [1978]. This difference is ascribed to the unrecognized contribution of reaction w in Schott and Davidson's system; with this assumption we evaluated by two methods the rate constants for w at high temperatures, which combine with the room temperature values to give the Arrhenius expression w = 2.5 × 106 exp (−6.1 × 103/T) s−1. This relation implies that the unimolecular reaction has a low frequency factor and a low activation energy, which are ascribed to a forbidden transition to the low‐lying excited electronic state (2E2″) of NO3. The rate of this channel of decomposition is compared with the much slower process leading to NO2 and O as products. Although this article gives evidence that the reaction NO3 → NO + O2 occurs, the quantitative values of rate coefficients are uncertain, and there are no data concerning the effect of foreign gas pressure. This article points out two methods (equations (14) and (17)) whereby deliberate studies of this reaction can be used to establish its rate coefficients and to find its M gas dependence. The recommended rate expression for reaction e is 2.05 × 1013 exp (−1.24 × 104/T)/Keq and that for g is 8.5 × 10−13 exp (−2.45 × 103/T) cm3 molecule−1 s−1, where Keq refers to the equilibrium N2O5 = NO3 + NO2.

Momentum-dependent diffusive particle acceleration in modified shock fronts

In the presently derived analytic solutions of the steady transport equation for diffusive particle acceleration in a modified, planar shock front having free escape boundaries, the fluid velocity profile through the shock transition decreases monotonically between the upstream and downstream boundaries. The spatial diffusion coefficient's spatial dependence is linked to that of the fluid velocity profile. Attention is given to the solution corresponding to monoenergetic particle injection at the shock front, with free particle escape at finite distances both upstream and downstream of the shock. The accelerated particle spectrum is dominated at high energies by an exponential cutoff, due to the competition between acceleration by the first-order Fermi mechanism and particle loss through the free escape boundaries.

In vitro interaction of aminoglycosides with beta-lactam penicillins.

The aminoglycosides are used clinically in combination with beta-lactam antibiotics. The combined use, however, produces an interaction and inactivation of the antibiotics. A study was designed to investigate the kinetics of the interaction in vitro. Four concentrations of aminoglycosides (5 to 20 micrograms of gentamicin and tobramycin per ml) and penicillins (100 to 600 micrograms of carbenicillin and ticarcillin per ml) were incubated in plasma (3 days, 37 degrees C). Samples taken at 12-h intervals were analyzed for both aminoglycosides (radioimmunoassay) and penicillin (high-pressure liquid chromatography). In controls, degradation of all four antibiotics were by first-order reactions. In incubation mixtures of two antibiotics, the rate of loss of the aminoglycosides was greater than that in the controls, whereas the rate of loss of penicillins was not significantly increased. The loss of penicillins in incubation mixtures still appeared to be by first-order reactions. However, semilogarithmic plots of aminoglycoside concentrations were curvilinear, suggesting a second-order reaction. Aminoglycoside concentrations in incubation mixtures were fitted by computer to a model incorporating a second-order interaction between aminoglycosides and penicillins and the first-order loss of penicillin from the mixture. The interaction rate constant averaged 2.2 X 10(-4) (micrograms/ml h)-1 for interaction of both carbenicillin and ticarcillin with gentamicin and 1.6 X 10(-4) (micrograms/ml h)-1 for interaction of the penicillins with tobramycin. The effect of the interaction in vivo was examined by computer simulation using the kinetic parameters determined in vitro.

Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum

Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40–50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 spm −1s −1 Malate (250 m m) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg 2+ present. Partial protection (50%) is afforded by either NAD + (1 m m) or Mg 2+ (50 m m). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10–13 histidine residues/subunit are acylated concomitant with loss of activity while 1–2 tyrosines are modified prior to any activity loss. The presence of Mg 2+ and malate at saturating concentrations protect 1–2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has a p K of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1 100 the rate of the unprotonated histidine. The results suggest that 1–2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.

Isolation, characterization, and mechanism of action of rat beta 1H.

beta 1Hrat was purified to homogeneity from fresh rat plasma by precipitation with 28.6% ammonium sulfate followed by sequential chromatography on DEAE-Sephacel, Biorex-70, and gel filtration on Sephacryl-S300. The final material was homogeneous on SDS-PAGE analysis and had an apparent m.w. of 150,000. Reduction with dithiothreitol did not affect its m.w., suggesting that the molecule is composed of one polypeptide chain. The recovery of beta 1H was approximately 10%. A monospecific antibody against beta 1Hrat was obtained from immunized rabbits, which recognized beta 1Hrat as a protein with beta-electrophoretic mobility upon immunoelectrophoresis of fresh rat plasma. The concentration of beta 1H in plasma of normal 4-mo-old Wistar rats was 243.5 +/- 36.3 micrograms/ml (mean +/- S.D.). beta 1Hrat in this study was detected by its capacity to inhibit formation of the P-stabilized cell-bound amplification C3/C5 convertase composed of cell-bound C3bhu and Bbhu. Purified beta 1Hrat produced a dose-related, first-order loss of convertase function and release of 126I-Bbhu from the P-stabilized C3bhuBbhu convertase, indicating a mechanism of action by decay-dissociation of Bbhu from the complex C3bhuBbhuP. beta 1Hrat was at least four times less effective than beta 1Hhu in release of 125I-Bbhu from the homologous convertase composed of C3bhu and Bbhu. On the other hand beta 1H was twice as effective in releasing 125I-Bbrat from the convertase composed of C3bratBbratP when compared to beta 1Hhu. These differences are presumably dependent upon the species-specific affinity of beta 1H from humans or rats for C3bhu or C3brat, respectively.

Analysis of field-weathered lures containing (E)-10-dodecen-1-yl acetate, a sex attractant for the pea moth,Cydia nigricana (F.)

Natural rubber stoppers treated with 1, 3, or 10 mg of (E)-10-dodecen-1-yl acetate, an attractant for male pea moth,Cydia nigricana (F.), were exposed in the field during the summer of 1978 and samples analyzed at intervals. The results for all three doses fitted well to first-order loss curves with half-lives of 63.5, 64.7, and 67.3 days, respectively. Thus, lures with an initial dose of 3 mg of the attractant retained approximately 1 mg after 3 months of field exposure; they should therefore maintain a constant level of attractiveness throughout this period because previous field studies showed that moths were equally responsive to fresh lures containing between 1 and 10 mg of the attractant. There was no loss of attractant during the preparation of lures or after 4 months at -15 ° C and only 13% was lost during 3 months of storage at room temperature.

Relaxation studies of the seeded emulsion polymerization of styrene initiated by γ-radiolysis

Results are reported for the relaxation kinetics of the emulsion polymerization of styrene in seeded systems initiated by γ-radiolysis. The decay in the rate of polymerization after removal from the radiation field was followed dilatometrically. The data were interpreted using an extended Smith—Ewart theory. This confirmed values for the rate coefficients for both the first-order loss of free radicals from the particles (k) and the entry of thermally generated free radicals into the particles (pρ) obtained in a separate chemically initiated study.Temperature-dependent studies showed that these two rate coefficients, for particles of radius 48 ± 2 nm, could be expressed as log(k/s–1)=(3.8 ± 0.5)–(42–10kJ mol–1/2.303 RT), and log(pρ/S–1)=(10.4 ± 1.0)–(87 ± 10 kJ mol–1/2.303 RT).

Seeded emulsion polymerization of styrene

The kinetics of the seeded emulsion polymerization of styrene, with the swollen radii of the seed ranging from 44 to 79 nm, have been measured dilatometrically. The conversion against time curves displayed an increase in the instantaneous rate of polymerization, followed by an apparent steady state domain. These measurements permitted for the first time the direct evaluation of the kinetic parameters that govern both the entry of free radicals into the latex particles and the first-order loss of free radicals from the particles. The latter rate coefficient was found to vary with the inverse square of the swollen particle radius, as expected for the diffusion controlled exit of free radicals from the particles. The magnitudes of the observed entry rate coefficients, when coupled with their dependence upon the initiator concentration, suggest that free radical capture by the seed particles was relatively inefficient. The capture efficiency increased significantly, however, with decreasing initiator concentration if the number of seed latex particles was held constant. The results obtained show clearly that the average number of free radicals per particle can be < 1/2 for styrene emulsion polymerizations under suitable conditions (e.g., low initiator concentrations and/or small particle sizes). Thus styrene can follow Smith—Ewart case 1 kinetics.The validity of the interpretation of the data is strongly supported by the excellent agreement with literature values obtained for various kinetic parameters.A background initiation process that causes the emulsion polymerization of styrene to proceed in the absence of added initiator was detected. This appears to be the emulsion polymerization analogue of the thermally induced bulk polymerization of styrene.

Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release.

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (3H2DIDS) at 4 degrees C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23 degrees C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (tau 1/2 = 360 min) component representing 33% of the radioactivity and a fast (tau 1/2 = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 microgram/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinetics of release to that of a single component (tau 1/2 = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously lost in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.