Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis.

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.

Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase.

Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of protein farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4-6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Brønsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Role of metals in the reaction catalyzed by protein farnesyltransferase.

Protein farnesyltransferase catalyzes the posttranslational farnesylation of several proteins involved in signal transduction, including Ras, and is a target enzyme for antitumor therapies. Efficient product formation catalyzed by protein farnesyltransferase requires an enzyme-bound zinc cation and high concentrations of magnesium ions. In this work, we have measured the pH dependence of the chemical step of product formation, determined under single-turnover conditions, and have demonstrated that the prenylation rate constant is enhanced by two deprotonations. Substitution of the active site zinc by cadmium demonstrated that one of the ionizations reflects deprotonation of the metal-coordinated thiol of the peptide "CaaX" motif, pK(a1) = 6.0. These data provide additional evidence for the direct involvement of a metal-coordinated sulfur nucleophile in catalysis. The second ionization was assigned to a hydroxyl on the pyrophosphate moiety of farnesyl pyrophosphate, pK(a2) = 7.4. Deprotonation of this group is important for binding of magnesium. This second ionization is not observed for catalysis in the absence of magnesium or when the substrate is farnesyl monophosphate. These data indicate that the maximal rate constant for prenylation requires formation of a zinc-coordinated thiolate nucleophile and enhancement of the electrophilic character at C1 of the farnesyl chain by magnesium ion coordination of the pyrophosphate leaving group.

Mechanistic studies of rat protein farnesyltransferase indicate an associative transition state.

Protein farnesyltransferase is a zinc metalloenzyme that catalyzes the transfer of a 15-carbon farnesyl group to a conserved cysteine residue of a protein substrate. Both electrophilic and nucleophilic mechanisms have been proposed for this enzyme. In this work, we investigate the detailed catalytic mechanism of mammalian protein farnesyltransferase by measuring the effect of metal substitution and/or substrate alterations on the rate constant of the chemical step. Substitution of cadmium for the active site zinc enhances peptide affinity approximately 5-fold and decreases the rate constant for the formation of the thioether product approximately 6-fold, indicating changes in the metal-thiolate coordination in the catalytic transition state. In addition, the observed rate constant for product formation decreases for C3 fluoromethyl farnesyl pyrophosphate substrates, paralleling the number of fluorines at the C3 methyl position and indicating that a rate-contributing transition state has carbocation character. Magnesium ions do not affect the affinity of either the peptide or the isoprenoid substrate but specifically enhance the observed rate constant for product formation 700-fold, suggesting that magnesium coordinates and activates the diphosphate leaving group. These data suggest that FTase catalyzes protein farnesylation by an associative mechanism with an "exploded" transition state where the metal-bound peptide/protein sulfur has a partial negative charge, the C1 of FPP has a partial positive charge, and the bridge oxygen between C1 and the alpha phosphate of FPP has a partial negative charge. This proposed transition state suggests that stabilization of the developing charge on the carbocation and pyrophosphate oxygens is an important catalytic feature.

Zinc-catalyzed sulfur alkyation:insights from protein farnesyltransferase.

Zinc metalloenzymes catalyze many important cellular reactions. Recently, the involvement of zinc in the catalysis of alkylation of sulfur groups has gained prominence. Current studies of the zinc metalloenzyme protein farnesyltransferase have shed light on its structure and catalytic mechanism, as well as the general mechanism of zinc-catalyzed sulfur alkylation.

Ultraviolet irradiation (UVB) interrupts calcium cell signaling in lens epithelial cells.

A preliminary study was undertaken to establish whether low-dose UV irradiation (UVB) affects calcium cell signaling in rabbit lens epithelia. In a suspension of lens epithelial cells (line NN1003A), changes in intracellular Ca2+ were measured by Fura-2 fluorescence in response to exogenously added ATP. The cellular response to ATP, referred to as the calcium signal, is characterized by a brief increase and subsequent decrease in cytosolic Ca2+ levels. Ultraviolet B irradiation (1.8-9 mJ/cm2) was found to reduce the magnitude of the Ca2+ signal in a dose-dependent manner. A 5 min UVB exposure (9 mJ/cm2) completely altered the biphasic nature of the calcium signal, causing only an immediate and steady rise in cytosol Ca2+ levels. Lower fluences of UVB irradiation (2 min exposure times or 3.6 mJ/cm2) induced a 50% reduction in the calcium signal. When irradiated cells were returned to culture for 3 h after irradiation, calcium signals induced by ATP were normal. In view of the photooxidative nature of UVB irradiation, the oxidative state of cells was assessed by measuring glutathione (GSH) levels. Ultraviolet B irradiation caused a rapid 20% decline in GSH levels that returned to near-control values after a 3 h postirradiation incubation. The results of this study indicate that fluences lower than previously found to be cataractogenic can perturb calcium cell signaling in cultured lens epithelial cells.

H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the microM range (KD = 4 microM) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pKa values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pKa of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pKa of the thiol resulting in formation of a bound thiolate at physiological pH.

The relationship between osmotic stress and calcium elevation: in vitro and in vivo rat lens models.

Both in vivo and in vitro models were employed in the present study to assess the relative contribution of osmotic stress and increasing calcium levels to the development of sugar cataracts. In galactose cataract obtained from galactosemic weanling rats, the concentration of total calcium increased by nearly 10% at the first sign of visible opacification observed on the fourth day post-galactose feeding. After 7 days of galactose feeding, calcium levels continued to rise, to 0.8 mM. During the first 10 days, loss of lens transparency and calcium elevation was gradual and steady, with precipitous changes occurring on days 11 and 12. In groups of rats where galactose feeding was stopped after 7 days, cataract reversal was followed during the next 5 weeks. During the initial first week of recovery, calcium influx and elevation in the lens continued but began to decline steadily thereafter. After 3 weeks of recovery, lens transparency had returned to almost normal. Calcium levels continued to decline and reached normal levels between day 34 and 42, nearly 4 weeks after removal of the galactose diet. The relationship between osmotic stress and calcium elevation was investigated more directly by culturing normal rat lenses in hypoosmotic medium (280 mOsm) to create osmotic gradients similar to that in galactosemic lenses. The results showed that during the first day of culture (12 hr), osmotically stressed lenses gained 3 mg of water, became opaque and gained excess calcium (7 mM compared to 0.7 mM). Microscopic vacuoles appeared to accompany the process of opacification and contributed to increased light scattering and the loss of lens transparency. Additional experiments were designed to further distinguish between the effects of osmotic stress and calcium elevation on the opacification process. Thus, lenses were incubated in control and high-calcium medium (20 mM) at 300 mOsm. Within 12 hr of incubation, calcium elevation progressed to 1.37 mM, nearly doubling the normal value. Although opacification was observed in these lenses, no sign of vacuoles was evident. Collectively, the findings from this study support the premise that an early influx of calcium is brought about by osmotic stress and is responsible for the observed loss in transparency in osmotic (sugar) cataract.

The role of calcium in UVB-induced damage in irradiated ocular lenses.

The purpose of this study was to evaluate the role of altered calcium homeostasis in the development of irreversible membrane damage in the UVB-irradiated ocular lens. In particular, experiments were designed to determine whether restricting calcium influx could prevent membrane damage that typically leads to ion imbalances and lens opacification following short-term exposure to ultraviolet light (UVB). The influx of calcium was reduced by culturing lenses in a low-calcium culture medium containing 0.3 mM Ca2+ rather than physiological concentrations of 1.6 mM. This low-calcium protocol retarded calcium accumulation in UVB-irradiated lenses for 2 days of culture, and opacification was delayed by 24 h. Loss of transparency did occur during the second day of culture, but more slowly than in irradiated lenses cultured in normal-calcium medium. Membrane damage was assessed by evaluating loss in cation transport activity, assessed by measuring 86Rb uptake into cultured lenses. Uptake was markedly inhibited in UVB-irradiated lenses and low-calcium culture did not prevent this inhibition of cation transport, a finding that explains why low-calcium protocol did not help maintain sodium homeostasis in irradiated lenses. Inhibition of cation transport and sodium accumulation eventually caused lens hydration and light scattering during extended culture in the absence of significant calcium elevation. Additional experiments were done to establish whether initial damage sustained by membranes could be repaired through the biosynthesis of new membrane proteins. Incorporation of 14C-histidine in membranes of the UVB-exposed lens was measured to assess membrane synthesis essential for repairing membrane damage. The rate of membrane protein synthesis, assessed by measuring incorporation of labeled amino acids, declined in UVB cataract, despite the prevention of calcium accumulation. These results suggest that one explanation for irreversible gain in sodium and calcium content accompanying opacification is the inability of lenses to replenish damaged membrane proteins comprising ion channels or transporters.

Subunit movement during catalysis by F1-F0-ATP synthases.

The catalytic portion (F1) of ATP synthases have the subunit composition alpha 3, beta 3, gamma, delta, epsilon. This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual alpha beta pairs with the small subunit must change as binding sites properties alternate. A rotation of the gamma subunit within the alpha 3 beta 3 hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete gamma subunit during ATP hydrolysis is not mandatory for activity. The gamma subunit of chloroplast F1 may be cleaved into three large fragments that remain bound to F1. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of alpha 3 beta 3 have been shown to have significant ATPase activity in the absence of gamma. Mg2+ATP affects the interaction of gamma with the different beta subunits, and induces other changes in F1, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF1 by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.

Influence of nucleotides on the cold stability of chloroplast coupling factor 1.

Chloroplast coupling factor 1 (CF1), a cold-labile enzyme, contains six nucleotide binding sites. These sites are located at the alpha/beta interfaces of the alpha 3 beta 3 heterohexamer. The cold lability of CF1 is decreased by the presence of nucleotides in the medium. We have studied the influence of both different nucleotides and different binding sites on the cold dissociation of CF1. To monitor the dissociation of CF1 during cold treatment, 8-anilino-1-naphthalenesulfonic acid (ANS) was employed. The increase in ANS fluorescence during cold treatment is the result of increased accessibility of intersubunit hydrophobic regions as the complex dissociates. Mg(2+)-adenosine triphosphates, tightly bound to CF1, markedly stabilize the enzyme in the cold. ADP only protects CF1 from dissociating in the cold when it is bound to the loose sites or when it is bound in conjunction with Mg2+. CF1 that contained 2 mol of ADP/mol and little bound Mg2+ was nearly as cold labile as CF1 that contained just 0.2 mol of ADP/mol. When about one of the two bound ADPs was replaced with adenylyl beta, gamma-imidodiphosphate (AMP-PNP), some protection from cold dissociation was observed. These results show that the site(s) occupied, as well as the nucleotides they contain, strongly influence(s) the structural stability of CF1.

Proteolytic cleavage within a regulatory region of the gamma subunit of chloroplast coupling factor 1.

The gamma subunit of chloroplast coupling factor 1 (CF1) is susceptible to selective proteolysis when the enzyme is in solution and the epsilon subunit is removed [CF1(-epsilon)]. In spinach thylakoid membranes, rapid cleavage of gamma is dependent on the generation of an electrochemical proton potential. The tryptic cleavage sites within the gamma of oxidized CF1 in illuminated thylakoids as well as of reduced CF1(-epsilon) in solution were determined by N-terminal amino acid sequencing. Two large gamma fragments of 27 000 (gamma27) and 10 000 (gammma10) molecular weight were generated by trypsin treatment of membrane-bound CF1. THe N-terminal gamma27 contains amino acids 1-215, and the C-terminal gamma10 contains 232-323. These polypeptides were tightly associated with the trypsin-resistant core of the enzyme. In contrast, three large gamma fragments were produced by trypsinolysis of reduced CF1(-epsilon). These polypeptides, which were also tightly associated with the trypsin-resistant core, have molecular weights of 7 900(gamma8), 14 850 (gamma15), and 10 000 (gamma10). These fragments contain residues 1-70, 71-204, and 232-323, respectively. The C-terminal gamma10 fragment generated by trypsin treatment of membrane-bound and soluble CF1 are identical. These results suggest that the gamma subunit of CF1 in illuminated thylakoids resembles that of CF1(-epsilon) with respect to accessibility to proteolytic cleavage. Cleavage of gamma between residues 215 and 232 is sufficient to fully activate the ATPase activity of the enzyme without reduction of the gamma disulfide. In addition, cutting within this region is responsible for loss of affinity for the inhibitory epsilon subunit.

Structural stability of chloroplast coupling factor 1 determined by differential scanning calorimetry and cold inactivation.

At least part of the gamma subunit of the catalytic portion of the chloroplast ATP synthase (CF1) is present in the middle of the alpha3beta3 heterohexamer. Interactions of the alpha/beta subunits with the gamma subunit stabilize the hexameric structure. Surprisingly, neither reduction of the gamma disulfide nor selective proteolysis of alpha, beta and gamma affects the thermal stability of EDTA-treated CF1 preparations, as determined by differential scanning calorimetry. Dissociation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF1 subunit depleted of its epsilon subunit [CF1(-epsilon)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF1(-epsilon) treated with trypsin in solution was much faster than that CF1(-epsilon)(8.1 versus 38.7 min, respectively, for 50% loss of activity). The increased cold liability of the trypsin-treated enzyme was not a consequence of the cleavage of the gamma. CF1 incubated with trypsin under conditions in which gamma is not cleaved was as cold labile as CF1 with cleaved gamma. Instead, loss of the delta subunit and a few residues from the C-terminal of the beta subunits were responsible for the increased cold liability of the enzyme.

The role of the lens epithelium in development of UV cataract.

In view of renewed interest in the lens epithelium as the initiation site for cataract development, it seemed timely to review recent studies which appear to establish UV damage in the lens epithelium as the cause of UV cataract. While UV photons can and do interact with lens proteins in the cortex and nucleus, experimental results from cultured lenses and tissue cultured epithelial cells also demonstrate both mutagenic and cytotoxic effects in the epithelium. This minireview examines UV-induced changes in lens physiology that appear to follow epithelial cell damage, including inactivation of critical enzymes of transport and metabolic processes. Changes in membrane function include altered cation transport, increased permeability, and altered biosynthesis. One potential scenario for the propagation of damage from the epithelium to the underlying fiber cells includes calcium elevation, an early event in cataract development and critical to many physiological processes.

Lens epithelium: a primary target of UVB irradiation.

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

Membrane damage in UV-irradiated lenses.

The purpose of this study was to investigate three possible causes of membrane damage following UV irradiation: photooxidation of membrane thiol (SH) groups, peroxidation of membrane lipids and inhibited synthesis of membrane proteins. Thiol loss was not observed. Thin-layer chromatography showed a four-fold increase in several primary lipid peroxidation products such as hydroperoxyl lipids in the epithelial membrane preparations isolated from irradiated lenses. The formation of new hydroxyl lipid bands not seen in control preparations was also observed in isolated membranes from irradiated lenses. Irradiation in the presence or absence of oxygen produced lipid peroxidation products. Aerobic irradiation produced small, but statistically significant increases in lipid hydroxyls and hydroperoxyls relative to controls. Repair of initial damage might be compromised by the observed 60% reduction in rate of protein synthesis measured in lens membranes following irradiation. Synthesis was affected by means other than depleted potassium or elevated calcium levels.

Selenite-induced damage to lens membranes.

The present study focuses on three aspects of membrane damage to explain selenite-induced loss of ion homeostasis: membrane transport processes, i.e. cation pump; biosynthesis of membrane proteins and membrane permeability. Cation pump activity, assessed by both 86Rb uptake and Na-K-ATPase activity in the epithelium, was observed to decline gradually after exposure to selenite and subsequent culture for 2 days in a selenite-free medium. In fact, the major loss of transport and ATPase activity occurred during culture of lenses after transfer from selenite to a selenite-free medium. The delay between selenite presentation to the lens and final inhibition of the cation pump (47%) corresponds to the delay in the observed loss of Na-K-ATPase activity (50%). Initial loss of cation transport and Na-K-ATPase activity may be due to the oxidative capacity of selenite. Oxidation, however, might not explain the delayed, progressive loss of transport activity after selenite removal. A plausible cause for this sustained loss might be a depleted supply of Na-K-ATPase due to impaired biosynthesis. Evidence for such a possibility comes from the observation that the rate of synthesis of total membrane protein is impaired by 44% in selenite-treated lenses. Membrane permeability to Na+ was not affected at the end of day 1, a conclusion based on the following observation: ouabain-treated lenses exposed to selenite did not gain any more Na+ than did ouabain-treated lenses. With the pump blocked by ouabain in both groups of lenses, the passive influx of Na+ was unchanged by selenite, indicating little damage to membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

A review of the evidence that ultraviolet irradiation is a risk factor in cataractogenesis.

There are two approaches to the question of whether solar radiation contributes to human cataract. The first, epidemiological studies, investigates correlations between man's environmental UV dose and cataract frequency. The second, animal models, investigates the effects of varying UV strengths and spectra on lens opacification in vivo or in vitro. While the latter approach typically provides for direct evidence, the data must still be extrapolated to human lenses. Results of physiological studies suggest that UV photons interact with proteins of the epithelial cell membranes, in particular tryptophan residues, transport ATPases and cytoskeletal proteins. One hypothesis is that damage to ion pumps and channels accumulates over the years as repair processes incompletely restore membrane function. Peroxidative damage is likely in view of the formation of UV-induced lipid peroxides in the lens epithelial membranes. Loss of homeostatic control of ions, particularly Ca++, leads to crystallin disorder in small regions of the underlying fiber cells. In our diabetic cataract studies, intracellular Ca++ electrodes detected large shifts in intracellular Ca++ before bulk-lens changes were apparent. Similar occurrences likely characterize UV cataract. Our lab is one of few studying lens physiology and how it is altered following transient exposures to UV-B and UV-A, both of which pass through the cornea. Some changes include: loss of epithelial cell GSH; elevated Ca++; loss of membrane voltage; impaired transport of Na+; increased permeability to ions and water; inhibition of critical enzymes; and a decrease in the rate of membrane synthesis.