Investigation of the nature of o-phthalaldehyde reaction with octopine dehydrogenase.

The effect of o-phthalaldehyde on octopine dehydrogenase inactivation has been studied. o-Phthalaldehyde binds to the proximal cysteine and lysine residues of the enzyme leading to the formation of isoindole derivative. Double inhibition studies with o-phthalaldehyde and p-chloromercuricphenyl sulfonic acid have indicated that o-phthalaldehyde does not bind to the functional cysteine present at the active site. Protection experiments have shown that L-arginine prevented o-phthalaldehyde inactivation. This could be only due to the reaction of the amino group of L-arginine with o-phthalaldehyde as per the mechanism proposed elsewhere since L-arginine cannot bind to the enzyme prior to NADH. Other substrates such as pyruvate oR NADH could not prevent the o-phthalaldehyde reaction with the enzyme. Fluorescence spectral studies demonstrated that in the presence of externally added amino acid no isoindole derivative formation occurs. However, a characteristic isoindole derivative is formed in the presence of beta-mercaptoethanol although the enzyme does not lose its activity. This indicated that o-phthalaldehyde can bind with lysine of the enzyme and thiol of externally added beta-mercaptoethanol. Pyridoxal 5'-phosphate, a lysine specific reagent also binds to the enzyme giving the characteristic absorption and fluroescence peak at 325 nm and 395 nm respectively. However, no loss of enzyme activity was observed. On the basis of these experiments we would suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

pCMPS-induced changes in lens membrane permeability and transparency.

PURPOSE: To investigate the role of externally facing membrane protein sulfhydryl groups in controlling lens permeability and transparency using the impermeant sulfhydryl complexing agent p-chloromercuriphenyl sulfonate (pCMPS). METHODS: Membrane permeability changes were studied by measuring lens voltage and resistance. Ion movements were monitored by measuring 22Na+ and 45Ca2+ fluxes, and internal free calcium was monitored by ion-selective microelectrodes. Opacification was quantified by photographing and analyzing back-scattered light. RESULTS: pCMPS, at concentrations above 1 microM, produced a depolarization of membrane potential and decrease in membrane resistance. These changes were accompanied by a marked stimulation in 22Na+ and 45Ca2+ influxes into the lens. There was a concomitant loss of lens transparency, mainly in the bow region. The pCMPS-induced electrical changes could be prevented by substituting N-methyl-D-glucamine for Na+ in the external medium. Na(+)-free solution alone increased 45Ca2+ influx, and the addition of pCMPS further stimulated the influx. Quinine (300 microM) was found to reduce the pCMPS-induced stimulation of 22Na+ and 45Ca2+ influxes and also to reduce opacification. CONCLUSIONS: pCMPS at low concentrations induces many of the cation permeability changes previously found to occur with age and cataract in the lens. The fact that quinine can ameliorate pCMPS-induced changes in ion movements and opacification suggests a novel approach for membrane-based anticataract strategies.

D600 increases the resistance associated with the equatorial potassium current of the lens.

The effects of D600, the methoxy analog of verapamil, on a pCMPS system were studied. A major effect of D600 is to increase the resistance associated with the equatorial potassium current of the lens. The increase in resistance is statistically significant at concentrations above 200 microM. At concentrations of 25-50 microM, D600 counteracts the decrease in resistance produced by binding sulfhydryl groups with 1 microM pCMPS. This effect is similar to that produced by quinine and by a calcium-free medium, and is attributed to the prevention of an increase in the calcium-dependent conductance produced by pCMPS.

A study of the effects of p-chloromercuribenzene sulfonic acid on acetylcholine-induced responses of molluscan neurons.

The effects of the organic mercurial p-chloromercuribenzene sulfonic acid (PCMBS) on the acetylcholine receptors of two types of neurons from the marine gastropod Anisodoris nobilis were studied. Cell types were distinguished by the ion-specific conductance change resulting from applied cholinergic agents: in certain cells a conductance increase to Cl resulted (Cl-neurons); in other cells a cation conductance increase resulted (D cells). Exposure of the Cl-neurons to 1 mM PCMBS at 4 degrees C had two distinct effects: 1) the sensitivity of the membrane conductance to microelectrophoretically applied carbamylcholine was blocked in a few minutes; and 2) the membrane conductance slowly increased with an inversion potential which was the same as that of the cholinergically induced response. d-Turbocurarine did not appreciably reduce this conductance increase. However, applying carbamylcholine during the exposure period to PCMBS reduced the PCMBS response. The cholinergic response of D cells was affected to a much lesser degree by PCMBS.

Mercurial perturbation of brush border membrane permeability in rabbit ileum.

The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.