Phosphylation kinetic constants and oxime-induced reactivation in acetylcholinesterase from fetal bovine serum, bovine caudate nucleus, and electric eel.

Kinetic constants for selected phosphonate and phosphinate inhibitors of fetal bovine serum acetylcholinesterase (FBS AChE; EC 3.1.1.7), bovine caudate nucleus AChE (BCN AChE), and eel AChE have been determined. Oxime reactivation of the phosphylated enzymes has also been evaluated. In general, a rank order with respect to organophosphorus compound (OP) inhibition of the enzymes was observed: soman (pinacolyl methylphosphonofluoridate) was found to be the most potent inhibitor, and 4-nitrophenyl methyl(phenyl)phosphinate (PMP) the least potent. On average the bimolecular rate constant for soman inhibition of eel AChE was nearly twofold greater (9.3 x 10(7) M-1 s-1) than that for FBS AChE (5.5 x 10(7) M-1 s-1) and nearly fourfold greater than that for BCN AChE (2.2 x 10(7) M-1 s-1). In addition, 4-nitrophenyl chloromethyl(phenyl)phosphinate (CPMP) inhibition of eel AChE on average was nearly 10-fold greater than FBS AChE and three orders of magnitude greater than BCN AChE. The oxime HI-6 reactivated soman phosphonylated enzymes to a considerably greater extent than other oximes, and FBS AChE was notably more responsive to HI-6 than to other oximes. The individual mean values of the ki for each inhibitor in each class (phosphonate or phosphinate) were different with respect to each AChE, which may be a reflection of differences in enzyme configuration, whereas the general rank order of inhibitor potency within each class, reflected by the ki, was similar with respect to each AChE, which may be related to similar active centers. In general, oxime potency and some rank order varied with each inhibitor and with each AChE, although there was some similarity in oxime rank order between the two mammalian AChEs. Overall, the data support the selection of FBS AChE as the enzyme of choice for in vitro testing of OP inhibitors and reactivators.

Cardiotonic drugs inhibit purified mammalian acetylcholinesterase.

Oxime- and non-oxime-related drugs, as well as cardiotonic drugs (CDs), have been used to treat the effects of organophosphorus (OP) poisoning. We conducted our experiments to determine what effects CDs may have on acetylcholinesterase (AChE), and how CDs interact with other treatment drugs as well as with OP-inhibited AChE. True AChE (EC 3.1.1.7) was purified from fetal bovine serum, and enzyme activity was measured according to Ellman et al. The CDs coumingine, cassaine, proscillaridin and convallatoxin were incubated with AChE at 550 microM at pH 7.6 and 25 degrees C. The CD ouabain was incubated with AChE at 500 microM. The CDs inhibited AChE by 97%, 89%, 10%, 7% and 6%, respectively. The mean AChE activities for these experiments, except for ouabain, were significantly different (P = 0.05) from their controls, as determined by the two-tailed Student's t-test. In a separate experiment, the oxime TMB-4.2Br (100 microM), which did not inhibit AChE, increased the inhibitory effect of proscillaridin from 4% to 11% (a 3.7-fold increase). When AChE was inhibited 39% with 37 nM VX, the addition of proscillaridin increased the inhibition to 51% (a 1.3-fold increase). When TMB-4 was added to the proscillaridin- and VX-inhibited AChE mixture, the inhibition decreased from 50% to 32% (a 0.37-fold decrease), whereas TMB-4 alone added to VX-inhibited AChE decreased the inhibition from 39% to 24% (a 0.38-fold decrease). The results show that TMB-4 increases the inhibition of AChE by proscillaridin. However, TMB-4 decreases the inhibition of AChE by VX and proscillaridin combined.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxime-induced reactivation of acetylcholinesterase inhibited by organophosphinates.

The comparative potency of oximes for reactivation of inhibited eel acetylcholinesterase (AChE) in vitro is dependent on the organophosphinate inhibitor. Some of the data, dealing with a reference organophosphonate, support the conclusion of other investigators that the oxime potency order is also dependent on the inhibiting phosphonate. This work was done to identify more clearly the nature of phosphinylated AChE with regard to oxime reactivation potency and the potential of phosphinates as pretreatment drugs to protect AChE against organophosphonate poisoning. We have determined the reactivation potency of four oximes--2-PAM, HI-6, TMB-4 and toxogonin--against four phosphinates--4-nitrophenyl methyl(phenyl)phosphinate (PMP), 4-nitrophenyl chloromethyl(phenyl)phosphinate (CPMP), 4-nitrophenyl trifluoromethyl(phenyl)phosphinate and 4-nitrophenyl bis(2-thienyl)phosphinate. For comparison, the phosphonate sarin (GB, isopropyl methylphosphonofluoridate) was included. Incubation of the inhibited enzyme (I-AChE) at 25 degrees C was with 0.30 microM oxime for PMP, 3.0 microM oxime for sarin and CPMP and 100 microM oxime for the two remaining phosphinates. AChE activity was assayed spectrophotometrically for 3.0 min at 272.5 nm at 25 degrees C in 0.10 M MOPS buffer (pH 7.60) using phenyl acetate as substrate. When sarin was the inhibitor (0% spontaneous recovery after a 2-h incubation), the order of oxime reactivation was 2-PAM (46%) greater than or equal to toxogonin (33%) = TMB-4 (31%) greater than HI-6 (9%) after 2-h incubations. For PMP (12% spontaneous recovery after a 2-h incubation) the oxime order was toxogonin (67%) greater than TMB-4 (53%) greater than 2-PAM (40%) after 2-h incubations.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase.

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin.

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.

Small intestinal epithelial renewal in the Syrian hamster exposed to cholera enterotoxin.

Epithelial renewal of the small intestine was measured in the Syrian hamster utilizing tritiated thymidine by standard autoradiographic and a scintillation counting technique. Scintillation counting of intestinal replicates proved to be as accurate as standard autoradiography. Average mucosal cell turnover was 71 +/- 3.0 hr in jejunum and 79 +/- 4.2 hr in ileum. Scintillation counting was utilized to study the effect of a maximum cholera enterotoxic secretory stimulus on small intestinal mucosal cell turnover. No significant change in epithelial cell migration occurred during cholera enterotoxin (CT)-induced fluid and electrolyte secretion. The rate of decline in radioactivity as a measure of cell turnover in CT-exposed animals was no different from controls. Epithelial cell proliferation 1 to 42 hr after CT exposure showed no difference from controls. Intestinal fluid and electrolyte secretion persisted for 24 hr after CT exposure. It is concluded that (1) the small intestinal epithelial cell migration was unaltered by this metabolic secretory stimulus, and (2) the data are consistent with the concept that epithelial migration after CT exposure was one factor, although not necessarily the major determinant of the progressive decline in intestinal secretory activity.

Purification of tritium-labeled cholera toxin.

Cholera toxin was labeled with tritium by the Wilzbach technique, and highly purified radiolabeled toxin was obtained by Sephadex column chromatography and disc gel electrophoresis. 3H-labeled cholera toxin retained its biological activity and chemical stability and had a specific activity of 405.9 muCi/mumol. The methods utilized in extraction and purification of 3H-labeled toxin may be advantageous for preparation of other biologically active radiolabeled proteins.