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Gu-gang Chang - One of the best experts on this subject based on the ideXlab platform.
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pH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin
Experimental eye research, 1997Co-Authors: Gu-gang Chang, Hwei-jen Lee, Rue-huan ChowAbstract:Abstract Animal lenses constitute many soluble proteins, which play a prominent role in eyes' light transparency. δ2-Crystallin, one of the major taxon-specific Crystallins in duck lens, is a tetrameric protein consisting of four identical subunits, which contain endogenous argininosuccinate lyase activity. Under a neutral pH environment in this work, the protein was cross-linked with glutaraldehyde as tetrameric and dimeric forms with tetramer as the major form. Under acidic conditions, the protein was time-dependently dissociated into monomers with amino acid residues of pK a values 6.29±0.45 and 7.17±0.49 being involved in the monomer-monomer interactions and 6.20±0.10 and 8.88±0.07 in the dimer-dimer interactions. Duck lens δ2-Crystallin thus possesses a double dimer structure (α 2 ) 2 with stronger monomer-monomer interactions than the dimer-dimer interactions. The acidic protein solution's reneutralization caused rapid reassociation of monomers into dimers and tetramers. The tetramer-dimer-monomer dissociation-reassociation thus is a pH-dependent freely interconvertible process.
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Characterization of the Multiple Forms of Duck Lens δ-Crystallin with Endogenous Argininosuccinate Lyase Activity
Archives of biochemistry and biophysics, 1994Co-Authors: Hwei-jen Lee, Ching-chun Lin, Shyh-horn Chiou, Gu-gang ChangAbstract:Duck lens Delta-Crystallins were the translational products of two tandem arranged genes, Delta 1 and Delta 2. Two fractions of the Delta-Crystallin (Delta a and Delta b) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with pI value ranging from 5.05 to 5.45. The charge heterogeneity of Delta-Crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy approximately 12.5 +/- 1.6 kcal/mol. Delta b-Crystallins showed higher enzyme activity than Delta a-Crystallins. Slightly different kinetic parameters were observed for the multiple forms of Delta b-Crystallins. On the other hand, Delta a-Crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in kcat value between these two subgroups. Delta a-Crystallins also have lower Km values than the Delta b-Crystallins. When examined by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar M(r) of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck Delta-Crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two Delta-Crystallin forms were demonstrated to be the N-truncated Delta 2-Crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher pI values were shown to be much more thermostable than those with lower pI values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.
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Inactivation of the endogenous argininosuccinate lyase activity of duck Delta-Crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.
Biochemical Journal, 1993Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:The argininosuccinate lyase activity of duck Delta-Crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and Delta-Crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of Delta-Crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the Delta-Crystallin-L-arginine and Delta-Crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified Delta-Crystallin had minimum, if any, conformational changes as compared with the native Delta-Crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of Delta-Crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.
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Biochemical characterization and kinetic analysis of duck Delta-Crystallin with endogenous argininosuccinate lyase activity.
Biochemical Journal, 1992Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:Delta-Crystallin, the most abundant Crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens Delta-Crystallin showed only 0.4-0.8% of the enzyme activity of duck Delta-Crystallin under identical assay conditions. Biochemical comparison of Delta-Crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of Delta-Crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, Delta-Crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck Delta-Crystallin possess enzyme activity whereas that of chicken Delta-Crystallin is devoid of activity. For duck Delta-Crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the Delta-Crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck Delta-Crystallin with the formation of various abortive Delta-Crystallin-argininosuccinate-arginine, Delta-Crystallin-argininosuccinate-fumarate and Delta-Crystallin-argininosuccinate-citrulline ternary complexes.
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Screening and kinetic analysis of Delta-Crystallins with endogenous argininosuccinate lyase activity in the lenses of vertebrates.
Biochemistry international, 1991Co-Authors: Shyh-horng Chiou, Hwei-jen Lee, H Chu, T A Lai, Gu-gang ChangAbstract:Screening of lens homogenates from the representative species of five major classes of vertebrates was undertaken to search for Delta-Crystallin with argininosuccinate lyase activity. Purification and biochemical characterization of Delta-Crystallins from the avian and reptilian species revealed differences in their electrophoretic and kinetic properties in spite of their similar tetrameric structure of about 200 kDa in the native forms. Chicken Delta-Crystallin, in contrast to those obtained from duck, goose and caiman, is almost devoid of the enzymatic activity. Two-dimensional gel electrophoresis of lens homogenates indicated that in the chicken lens Delta-Crystallin is composed of a subunit with an isoelectric point of 5.9 and a subunit mass of 50 kDa whereas that of goose lenses possesses heterogeneous subunits with isoelectric points spreading in a range of 5.9 to 6.8. Immunological comparison of inactive and active Delta-Crystallins from the chicken, duck and caiman lenses established the apparent structural similarity of all Delta-Crystallins to the authentic enzyme regarding some of common surface epitopes, yet they are not completely identical. Kinetic constants for two of the active Delta-Crystallins, i.e. those from the duck and goose of the Anatidae family, were also determined and their catalyzed reaction was shown to conform to a random Uni-Bi kinetic mechanism similar to that of the argininosuccinate lyase from the bovine liver.
Shyh-horng Chiou - One of the best experts on this subject based on the ideXlab platform.
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Taxon-specific lens Crystallins with endogenous enzymic activities : some perspectives on synthetic applications
Biotechnology and applied biochemistry, 1994Co-Authors: Shyh-horng ChiouAbstract:A systematic and general approach has been carried out to provide basic structural and mechanistic information on epsilon- and Delta-Crystallin with lactate dehydrogenase and argininosuccinate lyase activities respectively from representative species of birds. A detailed kinetic analysis of these two Crystallins revealed considerable differences in the enzymic activities among different species of birds. Especially noteworthy is the abundant presence of high activity in duck and goose epsilon- and Delta-Crystallins in contrast with the total lack of epsilon-Crystallin and diminished activities of Delta-Crystallins in chicken and pigeon lenses. Sequence comparison of inactive pigeon Delta-Crystallin and active duck Crystallin revealed several sequence variations and replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain birds. Bio-organic synthesis based on epsilon- and Delta-Crystallins isolated from duck lenses provides an efficient and practical tool for asymmetric or enantioselective synthesis of some pharmaceutically important chiral intermediates which may open new avenues and potentials in Crystallin research.
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Inactivation of the endogenous argininosuccinate lyase activity of duck Delta-Crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.
Biochemical Journal, 1993Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:The argininosuccinate lyase activity of duck Delta-Crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and Delta-Crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of Delta-Crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the Delta-Crystallin-L-arginine and Delta-Crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified Delta-Crystallin had minimum, if any, conformational changes as compared with the native Delta-Crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of Delta-Crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.
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Biochemical characterization and kinetic analysis of duck Delta-Crystallin with endogenous argininosuccinate lyase activity.
Biochemical Journal, 1992Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:Delta-Crystallin, the most abundant Crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens Delta-Crystallin showed only 0.4-0.8% of the enzyme activity of duck Delta-Crystallin under identical assay conditions. Biochemical comparison of Delta-Crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of Delta-Crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, Delta-Crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck Delta-Crystallin possess enzyme activity whereas that of chicken Delta-Crystallin is devoid of activity. For duck Delta-Crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the Delta-Crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck Delta-Crystallin with the formation of various abortive Delta-Crystallin-argininosuccinate-arginine, Delta-Crystallin-argininosuccinate-fumarate and Delta-Crystallin-argininosuccinate-citrulline ternary complexes.
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Structural analysis of pigeon lens Crystallins by near-infrared Fourier transform Raman spectroscopy.
Biochemistry international, 1992Co-Authors: Shyh-horng Chiou, Chen WAbstract:Crystallins from pigeon eye lenses were isolated and purified by gel-permeation and anion-exchange chromatographies and characterized by gel electrophoresis, amino acid analysis and Raman spectroscopy. alpha- and beta-Crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of Delta Crystallin from the beta-Crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of gel filtration column. Structural analyses of total crude soluble proteins and purified alpha-, beta- and Delta-Crystallin fractions were made with respect to their amino acid compositions and characteristic near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structures of alpha- and beta-Crystallins are mainly anti-parallel beta-pleated sheet in nature as judged by the Raman signals at 1242 (amide III) and 1669-1670 cm-1 (amide I) whereas Delta-Crystallin consists of a significant content of alpha-helices as evidenced by the Raman signal at 1657-1660 cm-1 (amide I). The low intensity of S-S disulfide stretching vibration at 508-510 cm-1 coupled with the presence of S-H stretching at 2560-2580 cm-1 for alpha-, beta- and Delta-Crystallin pointed to the fact that sulfhydryl groups in most Crystallins are resistant to air oxidation during the process of homogenization and protein extraction. It is also found that the relative Raman signal intensities of Tyr, Phe, and Trp residues in purified Crystallins correlate very well with the data obtained from amino acid analysis. Especially noteworthy is the demonstrated usefulness of applying Raman techniques in the detection of the microenvironments of the aromatic amino acids such as Tyr and Trp in the native Crystallins, which may prove useful in the study of contribution of these aromatic residues to Crystallin packing and stability.
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Ostrich Crystallins. Structural characterization of Delta-Crystallin with enzymic activity.
Biochemical Journal, 1991Co-Authors: Shyh-horng Chiou, Chien-ying Chang, Takehito Itoh, Hiroyuki Kaji, Tatsuya SamejimaAbstract:Lens Crystallins from the African ostrich (Struthio camelus) were isolated and characterized. Four Crystallin fractions corresponding to alpha-, Delta/beta- and beta-Crystallins similar to those of duck Crystallins were isolated, but epsilon-Crystallin was found to be absent. The native molecular masses and subunit structures of the purified fractions were analysed by gel filtration. SDS/PAGE and isoelectric focusing, revealing various extents of heterogeneity in each orthologous Crystallin class. An ion-exchange chromatographic method was used for the large-scale preparation of Delta-Crystallin suitable for structural and enzymic studies. It was unexpectedly found that the purified native Delta-Crystallin of ostrich lens possessed high argininosuccinate lyase activity, in contrast with chicken Delta-Crystallin. The c.d. spectra indicated a predominant beta-sheet structure in alpha- and beta-Crystallins, and a significant contribution of alpha-helical structure in the Delta-Crystallin fraction. The estimate of secondary structures from c.d. spectroscopy for each Crystallin class bears a resemblance to that of duck Crystallins, except that ostrich Delta-Crystallin possesses much less helical content than duck Delta-Crystallin. Comparison of Crystallin compositions and structures from aquatic and terrestrial birds revealed distinct differences.
Hwei-jen Lee - One of the best experts on this subject based on the ideXlab platform.
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The effect of N-terminal truncation on double-dimer assembly of goose Delta-Crystallin.
The Biochemical journal, 2005Co-Authors: Hwei-jen Lee, Young-hsang Lai, Yu-hou ChenAbstract:Delta-Crystallin is a soluble structural protein in avian eye lenses that confers special refractive properties. In the presence of GdmCl (guanidinium chloride), tetrameric Delta-Crystallin undergoes dissociation via a dimeric state to a monomeric molten globule intermediate state. The latter are denatured at higher GdmCl concentrations in a multi-state manner. In the present study, the X-ray structure of goose Delta-Crystallin was determined to 2.8 A (1 A=0.1 nm). In this structure the first 25 N-terminal residues interact with a hydrophobic cavity in a neighbouring molecule, stabilizing the quaternary structure of this protein. When these 25 residues were deleted this did not produce any gross structural changes, as judged by CD analysis, but slightly altered tryptophan fluorescence and ANS (8-anilino-1-naphthalenesulphonic acid) spectra. The dimeric form was significantly identified as judged by sedimentation velocity and nondenaturing gradient gel electrophoresis. This mutant had increased sensitivity to temperature denaturation and GdmCl concentrations of 0.3-1.0 M. This protein was destabilized about 3.3 kcal/mol (1 kcal=4.184 kJ) due to N-terminal truncation. After incubation at 37 degrees C N-terminal truncated proteins were prone to aggregation, suggesting the presence of the unstable dimeric conformation. An important role for the N-terminus in dimer assembly of goose Delta-Crystallin is proposed.
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pH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin
Experimental eye research, 1997Co-Authors: Gu-gang Chang, Hwei-jen Lee, Rue-huan ChowAbstract:Abstract Animal lenses constitute many soluble proteins, which play a prominent role in eyes' light transparency. δ2-Crystallin, one of the major taxon-specific Crystallins in duck lens, is a tetrameric protein consisting of four identical subunits, which contain endogenous argininosuccinate lyase activity. Under a neutral pH environment in this work, the protein was cross-linked with glutaraldehyde as tetrameric and dimeric forms with tetramer as the major form. Under acidic conditions, the protein was time-dependently dissociated into monomers with amino acid residues of pK a values 6.29±0.45 and 7.17±0.49 being involved in the monomer-monomer interactions and 6.20±0.10 and 8.88±0.07 in the dimer-dimer interactions. Duck lens δ2-Crystallin thus possesses a double dimer structure (α 2 ) 2 with stronger monomer-monomer interactions than the dimer-dimer interactions. The acidic protein solution's reneutralization caused rapid reassociation of monomers into dimers and tetramers. The tetramer-dimer-monomer dissociation-reassociation thus is a pH-dependent freely interconvertible process.
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Characterization of the Multiple Forms of Duck Lens δ-Crystallin with Endogenous Argininosuccinate Lyase Activity
Archives of biochemistry and biophysics, 1994Co-Authors: Hwei-jen Lee, Ching-chun Lin, Shyh-horn Chiou, Gu-gang ChangAbstract:Duck lens Delta-Crystallins were the translational products of two tandem arranged genes, Delta 1 and Delta 2. Two fractions of the Delta-Crystallin (Delta a and Delta b) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with pI value ranging from 5.05 to 5.45. The charge heterogeneity of Delta-Crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy approximately 12.5 +/- 1.6 kcal/mol. Delta b-Crystallins showed higher enzyme activity than Delta a-Crystallins. Slightly different kinetic parameters were observed for the multiple forms of Delta b-Crystallins. On the other hand, Delta a-Crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in kcat value between these two subgroups. Delta a-Crystallins also have lower Km values than the Delta b-Crystallins. When examined by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar M(r) of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck Delta-Crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two Delta-Crystallin forms were demonstrated to be the N-truncated Delta 2-Crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher pI values were shown to be much more thermostable than those with lower pI values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.
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Inactivation of the endogenous argininosuccinate lyase activity of duck Delta-Crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.
Biochemical Journal, 1993Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:The argininosuccinate lyase activity of duck Delta-Crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and Delta-Crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of Delta-Crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the Delta-Crystallin-L-arginine and Delta-Crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified Delta-Crystallin had minimum, if any, conformational changes as compared with the native Delta-Crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of Delta-Crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.
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Biochemical characterization and kinetic analysis of duck Delta-Crystallin with endogenous argininosuccinate lyase activity.
Biochemical Journal, 1992Co-Authors: Hwei-jen Lee, Shyh-horng Chiou, Gu-gang ChangAbstract:Delta-Crystallin, the most abundant Crystallin in the avian species, was isolated and purified from duck lenses. It was shown to possess endogenous argininosuccinate lyase activity catalysing the reversible cleavage of argininosuccinate to give fumarate and arginine with an equilibrium constant of 1.8 +/- 0.23 mM. In contrast, chicken lens Delta-Crystallin showed only 0.4-0.8% of the enzyme activity of duck Delta-Crystallin under identical assay conditions. Biochemical comparison of Delta-Crystallins from these two species revealed distinct differences in their structural and kinetic properties. An activity-staining method was developed for the easy detection of endogenous enzyme activity of Delta-Crystallin from crude lens extracts of different avian species. Two-dimensional gel electrophoresis of lens homogenates indicated that, in the chicken lens, Delta-Crystallin is composed mainly of a subunit with a pI of 5.9 and a subunit mass of 50 kDa, whereas that of duck lens possesses various 50 kDa subunits in a pI range of 5.9-6.8. Activity staining corroborated the fact that all charge isoenzymes of duck Delta-Crystallin possess enzyme activity whereas that of chicken Delta-Crystallin is devoid of activity. For duck Delta-Crystallin, variation of the enzyme activity with argininosuccinate concentration in the forward reaction followed saturation kinetics with an apparent Michaelis constant for the substrate of 17 +/- 5 microM. In the reverse reaction, initial-velocity studies showed intersecting patterns. Inhibitions of the forward reaction by products (fumarate and arginine) were both non-competitive with respect to argininosuccinate. Citrulline, an analogue of arginine, inhibited the enzyme activity in both directions and was competitive with respect to arginine but non-competitive with respect to fumarate or argininosuccinate. Succinate, which inhibited the bovine argininosuccinate lyase, did not affect the Delta-Crystallin enzyme activity in a concentration range between 1 and 300 mM. These results suggest a random Uni Bi kinetic mechanism for the argininosuccinate lyase activity of duck Delta-Crystallin with the formation of various abortive Delta-Crystallin-argininosuccinate-arginine, Delta-Crystallin-argininosuccinate-fumarate and Delta-Crystallin-argininosuccinate-citrulline ternary complexes.
D. I. De Pomerai - One of the best experts on this subject based on the ideXlab platform.
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A choice between glycogen and δ-Crystallin accumulation is made in glial cells and not influenced by overlying neurons
Biochemical Journal, 1992Co-Authors: S A Karim, D. I. De PomeraiAbstract:Chick-embryo neuroretinal cells convert extensively into lens under low-glucose conditions, but this transdifferentiation process is blocked by high-glucose media. We have previously observed an inverse relationship between the levels of glycogen (a marker of normal retinoglial differentiation) and of Delta-Crystallin (a lens marker) in such cultures. However, most of the glycogen accumulated under high-glucose conditions is apparently localized in those glial (G) cells underlying clusters of neurons (N cells). We here show that glial-enriched cultures (largely depleted of N cells) both accumulate glycogen and fail to transdifferentiate in high-glucose media. Moreover, glycogen localization in groups of glial cells is unaffected by the absence of N cells. Thus the choice between normal and foreign differentiation pathways is made autonomously within the retinoglial-cell population and is not influenced significantly by the presence or absence of N cells.
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A choice between glycogen and Delta-Crystallin accumulation is made in glial cells and not influenced by overlying neurons.
The Biochemical journal, 1992Co-Authors: S A Karim, D. I. De PomeraiAbstract:Chick-embryo neuroretinal cells convert extensively into lens under low-glucose conditions, but this transdifferentiation process is blocked by high-glucose media. We have previously observed an inverse relationship between the levels of glycogen (a marker of normal retinoglial differentiation) and of Delta-Crystallin (a lens marker) in such cultures. However, most of the glycogen accumulated under high-glucose conditions is apparently localized in those glial (G) cells underlying clusters of neurons (N cells). We here show that glial-enriched cultures (largely depleted of N cells) both accumulate glycogen and fail to transdifferentiate in high-glucose media. Moreover, glycogen localization in groups of glial cells is unaffected by the absence of N cells. Thus the choice between normal and foreign differentiation pathways is made autonomously within the retinoglial-cell population and is not influenced significantly by the presence or absence of N cells.
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Expression in non-lens tissues of an enzyme activity related to the 'lens-specific' protein, Delta Crystallin.
Development (Cambridge England), 1991Co-Authors: D. I. De Pomerai, M. Mclaughlin, K.c. PerryAbstract:When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, Delta Crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the Delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a Delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick Delta 1 Crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related Delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable Delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-Delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick Delta Crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the Delta-related protein band. So-called ‘ectopic’ expression of Delta Crystallin in embryonic NR (and other tissues) may thus involve the Delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.
K.c. Perry - One of the best experts on this subject based on the ideXlab platform.
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Expression in non-lens tissues of an enzyme activity related to the 'lens-specific' protein, Delta Crystallin.
Development (Cambridge England), 1991Co-Authors: D. I. De Pomerai, M. Mclaughlin, K.c. PerryAbstract:When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, Delta Crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the Delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a Delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick Delta 1 Crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related Delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable Delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-Delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick Delta Crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the Delta-related protein band. So-called ‘ectopic’ expression of Delta Crystallin in embryonic NR (and other tissues) may thus involve the Delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.