Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate

Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues.

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.     

Mechanism of the inhibition of catalase by ascorbate. Roles of active oxygen species, copper and semidehydroascorbate

Ascorbate reversibly inhibits catalase, and this inhibition is enhanced and rendered irreversible by the prior addition of copper(II)-bishistidine. In the absence of copper, the inhibition was prevented and reversed by ethanol, but not by superoxide dismutase, benzoate, mannitol, thiourea, desferrioxamine, or DETAPAC. In the presence of the copper complex mannitol, benzoate, and superoxide dismutase still had no effect, but thiourea, desferrioxamine, DETAPAC, or additional histidine decreased the extent of inactivation to that seen in the absence of copper. In the presence of copper, ethanol protected at [ascorbate] less than 1 mM, but was ineffective at [ascorbate] greater than 2 mM, even in the absence of oxygen. Although in the absence of copper, complete removal of oxygen provided full protection against inactivation by ascorbate, this protection was not seen if the catalase was briefly preincubated with H2O2 prior to flushing with nitrogen, or if copper was present. In fact, if copper was present, inactivation was enhanced by the removal of oxygen. Increasing the concentration of oxygen from ambient to 100% slowed the inactivation, whether or not copper was present. It is concluded that the initial reversible inactivation involves reaction with H2O2 to form compound I, followed by one electron reduction of compound I to compound II. In the presence of added copper, the initial (reversible) inactivation allows H2O2 to accumulate sufficiently to permit irreversible inactivation. Since in the presence of copper oxygen is not required, and neither the reversible nor the irreversible inactivation was prevented by conventional scavengers of active forms of oxygen, the inactivation is likely mediated by semidehydroascorbate, and/or it may involve site-specific generation of the damaging intermediates.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37 degrees C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H2O2 and HbO2, may be related to the pathogenesis of hemolysis in favism.     

The Ambivalent Role of Glutathione in the Protection of Dna Against Singlet Oxygen

Glutathione (GSH) was examined with respect to its ability to protect DNA against ¹O₂ damage. We have found that GSH protected, at least partly, the DNA against inactivation by ¹O₂. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of ¹O₂, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against ¹O₂-induced inactivating DNA damage.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.     

Attenuation of galactose-induced cataract by pyruvate

Data in the present paper demonstrate a significant inhibition in the progress of sugar cataract formation by systemic administration of pyruvate. The formation of the cataract was induced by feeding young rats a diet containing 30% galactose. All animals fed this diet developed nuclear lens opacity by the end of 30 days. This was delayed if the diet and water contained, in addition, 2% sodium pyruvate. The incidence of cataract in the latter group was 0% at day 30 and only 25% at day 55. Physiologically, the inhibition was associated with the prevention of lens membrane damage as reflected by its ability to maintain transport of rubidium ions against a concentration gradient; decreased tissue hydration as indexed by the lens wet weight; inhibition of protein glycation, and higher levels of ATP. Since pyruvate, being a normal tissue metabolite, is likely to be non-toxic, the findings are considered useful for further pharmacological studies with this and other similar metabolites, relevant to protection against various secondary complications of diabetes and galactosemia.     

Attenuation of galactose-induced cataract by pyruvate

Data in the present paper demonstrate a significant inhibition in the progress of sugar cataract formation by systemic administration of pyruvate. The formation of the cataract was induced by feeding young rats a diet containing 30% galactose. All animals fed this diet developed nuclear lens opacity by the end of 30 days. This was delayed if the diet and water contained, in addition, 2% sodium pyruvate. The incidence of cataract in the latter group was 0% at day 30 and only 25% at day 55. Physiologically, the inhibition was associated with the prevention of lens membrane damage as reflected by its ability to maintain transport of rubidium ions against a concentration gradient; decreased tissue hydration as indexed by the lens wet weight; inhibition of protein glycation, and higher levels of ATP. Since pyruvate, being a normal tissue metabolite, is likely to be non-toxic, the findings are considered useful for further pharmacological studies with this and other similar metabolites, relevant to protection against various secondary complications of diabetes and galactosemia.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37°C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H₂O₂ and HbO₂, may be related to the pathogenesis of hemolysis in favism.     

Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.     

Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22

Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.     

The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.     

The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases

The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.     

The molecular chaperone,alpha-crystallin,protects against loss of antigenicity and activity of esterase caused by sugars,sugar phosphate and a steroid

Previously we showed that glycation-induced inactivation and loss of antigenicity of enzymes occur simultaneously. Alpha-crystallin, a major structural protein of the mammalian lens, prevents the aggregation of other proteins and protects enzyme function against post-translational modification in vitro. However, it is not known whether alpha-crystallin can also protect against loss of antigenicity of enzymes. Esterase activity in the lens is decreased in senile cataract and diabetes. We investigated the loss of antigenicity of esterase caused by different insults and the ability of alpha-crystallin to protect. Inactivation of carboxylesterase by sugars, fructose 6-phosphate (F6P) and a steroid, prednisolone-21-hemisuccinate (P-21-H), was measured spectrophotometrically in the presence and absence of alpha-crystallin, while loss of antigenicity was monitored simultaneously using an immunoprecipitation method. The esterase was progressively inactivated by fructose, F6P, ribose, and P-21-H. Bovine alpha-crystallin fully protected against inactivation of esterase by all four compounds, and also protected against loss of antigenicity of the esterase by fructose, ribose and P-21-H at a molar ratio of 1:1. The results indicated that alpha-crystallin, under our experimental conditions, clearly exhibited the ability to prevent loss of antigenicity and inactivation of esterase. The protective effect of alpha-crystallin against loss of antigenicity indicates a novel aspect of its chaperoning function.