Biomedical subjects

G G Chang

Publications and source records attributed to G G Chang.

At least 37 records · Page 2Linked to original sources

PubMed1998

Inhibitory effect of magnesium ion on the human placental alkaline phosphatase-catalyzed reaction in a reverse micellar system.

Human placental alkaline phosphatase is a membrane-anchored protein. Entrapping the enzyme into a reverse micellar vesicle mimics the in vivo conditions and allows examination of the properties of the enzyme. Placental alkaline phosphatase is enzymatically active in Aerosol-OT/isooctane reverse micelles. Substantially different kinetic behavior of the enzyme has been observed in aqueous or reverse micellar systems. In aqueous solution, Mg2+ is a nonessential activator of the enzyme. In the experiments described in the present report Mg2+ was found to be an inhibitor for the enzyme in reverse micelles. This inhibition is presumably due to a time-dependent conformational change of the enzyme molecule, which resulted in a curvature in the recorder tracings of the enzyme assays. The Mg2+-induced conformational change of the enzyme was completely prevented by phosphate and partially reserved by EDTA. High concentrations of Zn2+ also strongly inhibited enzyme activity in both aqueous and reverse micellar solvent systems, presumably by occupying the Mg2+ (M3) site of the enzyme. However, binding of Zn2+ at the M3 site did not cause conformational change of the enzyme and the enzyme assay tracing was linear. The M3 site of the enzyme is proposed to have a modulatory role in vivo using magnesium ion as the modulator.

Alkaline Phosphatase↗

PubMed1998

Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes.

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.

Animals↗

PubMed1997

pH-induced reversible dissociation of tetrameric duck lens delta-crystallin.

Animal lenses constitute many soluble proteins, which play a prominent role in eyes' light transparency. delta2-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 pKa 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 delta2-crystallin thus possesses a double dimer structure (alpha2)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.

Animals↗

PubMed1997

Functional roles of the N-terminal amino acid residues in the Mn(II)-L-malate binding and subunit interactions of pigeon liver malic enzyme.

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-. In this work, various mutants of the enzyme with individual or combinational deletion (delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells. A major protein band corresponding to an Mr of approximately 65000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with Mr approximately 260000 or a mixture of tetramers and dimers with the exception of delta(K2K3G4) and delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed. K3A and K3E also dissociated substantially. K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers. All tetramers and dimers were enzymatically active in the gels. All mutants displayed a similar apparent Km value for NADP+. The apparent Km for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the delta(K2/K3) and the delta(1-16) mutants. The small binding affinity of delta(K2/K3) with Mn(II)-L-malate was specific. With additional deletion at positions 3 and/or 4, the delta(K2K3), delta(K2G4/K3G4) or delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type. The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation. The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface. These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions.

Animals↗

PubMed1996

Involvement of Phe19 in the Mn(2+)-L-malate binding and the subunit interactions of pigeon liver malic enzyme.

A triple mutant, F19S/N250S/L353Q, of pigeon liver malic enzyme was found to have no detectable enzymatic activity [Chou, W.-Y., Huang, S.-M., & Chang, G.-G. (1994) Arch. Biochem. Biophys. 310, 158-166]. In the present study, point mutants at these positions (F19S, N250S, and L353Q) were prepared by site-directed mutagenesis. Both N250S and L353Q have kinetic properties similar to those of the wild-type. On the other hand, the K(m)(app) values for both Mn2+ and L-malate of F19S were increased by approximately 10-fold, while the kcat value was decreased by 5-fold, which results in a decrease of the apparent catalytic efficiency (kcat/K(mNADP)K(mMal)K(mMn) by approximately 300-fold. These results clearly indicate that the F19S mutation is mainly responsible for the undetectable enzyme activity of the triple mutant. Three more Phe19 mutants (F19Y, F19G, and F19A) were then prepared. There is a direct correlation between the size of the substitutes and the affinities for Mn2+ and L-malate. The kinetic parameters for F19Y were similar to those for wild-type. Both F19A and F19G reveal a 5-fold decrease of kcat values. Two K(dMn) values for the high- and low-affinity sites, respectively, were detectable for the wild-type. On the contrary, only one K(dMn) value was detected for the F19 mutants, which was increased in the order of F19G > F19A > F19S > F19Y, with F19G being the most affected mutant. The K(mMal) values of F19G and F19A were increased 100- and 6-fold, respectively. The catalytic efficiency (kcat/K(mNADP)K(dMal)K(dMn)) of F19G was decreased to only 0.01% of that of the wild-type. The above results clearly indicate that the hydrophobic aromatic ring at position 19 plays a critical role in L-malate and Mn2+ binding. Furthermore, all mutants that have a small residue at position 19 exist as monomers. Therefore, Phe19 may locate in or near the regions for Mn(2+)-L-malate binding as well as for the subunit contact. These results are compatible with the asymmetric model for the quaternary structure of malic enzyme we proposed previously [Chang, G.-G., Huang, T.-M., Huang, S.-M., & Chou, W.-Y. (1994) Eur. J. Biochem. 225, 1021-1027]. The possible roles of the N-terminus of malic enzyme were also addressed.

Amino Acid Sequence↗

PubMed1996

Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles.

Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (omega(o)) of the system with maximum rate observed at omega(o) 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56 0.07 and 8.81 0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69 0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40-1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo is discussed.

Animals↗

PubMed1996

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell.

A cDNA coding for human breast cancer cell cytosolic NADP(+)-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3'-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994), FEBS Lett. 344, 181-186] reveals that three nucleotides in the open reading frame and the length of 3'-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally in Escherichia coli cells. Its Km value for L-malate (1.21 +/- 0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29 +/- 0.04 mM) but similar to that for the full-length recombinant enzyme (1.06 +/- 0.07 mM). The Km values for Mn2+ and NADP+ (0.26 +/- 0.03 and 0.97 +/- 0.4 microM, respectively) are similar to those for the natural enzyme (0.12 +/- 0.02 and 1.9 +/- 0.3 microM, respectively) or the recombinant wild-type enzyme (0.56 +/- 0.04 and 0.44 +/- 0.02 microM, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. The Km values for L-malate and Mn2+ of the truncated enzyme (11.2 +/- 0.9 mM and 61.2 +/- 4.6 microM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21 +/- 0.02 mM and 1.06 +/- 0.08 microM, respectively) or the recombinant wild-type enzyme (0.25 +/- 0.01 mM and 1.48 +/- 0.05 microM, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.

Amino Acid Sequence↗

PubMed1996

Metal-catalyzed oxidation and cleavage of octopus glutathione transferase by the Cu(II)-ascorbate system.

Glutathione transferase (GST) from octopus hepatopancreas was rapidly inactivated by micromolar concentration of Cu(II) in the presence of ascorbate at neutral pH and 0 degree C. Omitting the metal ion or ascorbate, or replacing the Cu(II) with Fe(II) did not result in any inactivation. Glutathione or the conjugation product of glutathione and 1-chloro-2,4-dinitrobenzene offered complete protection of the enzyme from Cu(II)-induced inactivation. 1-Chloro-2,4-dinitrobenzene, however, did not provide any protection. The inactivation was time and Cu(II) concentration dependent. The dependence of inactivation rate on Cu(II) concentration displayed saturation kinetics, which suggests that the inactivation occurs in two steps with Cu(II) binding with the enzyme first (KdCu = 260 microM), then the locally generated free radicals modify the essential amino acid residues in the active center, which results in enzyme inactivation. The Cu(II)-ascorbate system is, thus, an affinity reagent for the octopus GST. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native octopus GST has a subunit M(r) of 24,000. The inactivated enzyme was cleaved at the C-terminal domain (domain II) of the enzyme molecule and resulted in the formation of peptide fragment of M(r) 15,300, which has the identical N-terminal amino acid sequence as the native enzyme. The other half of the peptide with M(r) approximately 7700 was visible in the gels only after silver staining, which also revealed a minor cleavage site, also located at the domain II, to produce peptide fragments of M(r) approximately 11,300 and 8300. The oxygen carrier molecule in the cephalopods' blood is the copper-containing hemocyanin, which during turnover will release Cu(II). Our results indicate that Cu(II) catalyzes a site-specific oxidation of the essential amino acid residues at the C-terminus of GST causing enzyme inactivation. The modified-enzyme is then affinity cleaved at the putative metal binding site. The ability of octopus GST to bind with free Cu(II) may have important biological implications to enable cephalopods to avoid copper-induced cellular toxicity.

Animals↗

PubMed1996

Kinetic characterization of the endogenous glutathione transferase activity of octopus lens S-crystallin.

The kinetic mechanism of the endogenous glutathione transferase (GST) activity of octopus S-crystallin was investigated by steady-state kinetics. Biphasic double-reciprocal plots were obtained for both glutathione and the hydrophobic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Substrate inhibition was observed only for CDNB with Ksi value of 29.7 +/- 0.01 mM. The catalytic constant for S-crystallin was three orders of magnitude smaller than that for the digestive gland GST of the same species. The initial-velocity studies indicated that the enzyme reaction might conform to a steady-state random Bi-Bi kinetic mechanism, being similar to the reaction of GST from other sources. The pH-rate profiles also suggest that the same chemical mechanism for the nucleophilic aromatic substitution between GSH and CDNB was employed for S-crystallin. The interaction of Tyr7 with the bound GSH lowered the pKa value of the sulfhydryl group of GSH to 6.82-6.85, which is 2.32-2.35 pH unit smaller than that found in aqueous solution. This lowering of pKa value produces the thiolate anion of GSH, a better nucleophile to attack the ipso carbon of CDNB, resulting in formation of Meisenheimer complex intermediate. Removing the chloride ion from this intermediate complex produces the conjugate product. Using the method devised by Wang and Srivastava (Anal. Biochem. 216, 15-26, 1994), the functional unit of the dimeric S-crystallin was estimated to be a monomer. The possible biological implications of the endogenous detoxification ability of cephalopods S-crystallin are discussed.

Animals↗

PubMed1995

Selective oxidative modification and affinity cleavage of pigeon liver malic enzyme by the Cu(2+)-ascorbate system.

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe2+ in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at the chemical bond between Asp258 and Ile259 (Wei, C.H., Chou, W.Y., Huang, S.M., Lin, C.C., and Chang, G.G. (1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C.H., Chou, W.Y., and Chang, G.G. (1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu2+ was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe2+ did. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465. Mn2+ and L-malate synergistically protect the enzyme from Cu2+ inactivation at acidic pH. Cu2+ is also a competitive inhibitor versus Mn2+ in the malic enzyme-catalyzed reaction with Ki value 70.3 +/- 5.8 microM. The above results indicated that, in addition to the previously determined Asp258 at neutral pH, Asp141, Asp194, and Asp464 are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu(2+)-ascorbate system proceed in the following sequence. First, Cu2+ binds with the enzyme at the Mn2+ binding site and reduces to Cu+ by ascorbate. Next, the local oxygen molecules are reduced by Cu+, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.

Amino Acid Sequence↗

PubMed1995

Octopus S-crystallins with endogenous glutathione S-transferase (GST) activity: sequence comparison and evolutionary relationships with authentic GST enzymes.

S-Crystallin is a major protein present in the lenses of cephalopods (octopus and squid). To facilitate the cloning of this crystallin gene, cDNA was constructed from the poly(A)+ mRNA of octopus lenses, and amplified by PCR for nucleotide sequencing. Sequencing of 10 of 15 positive clones coding for this crystallin revealed three distinct S-crystallin isoforms with 61-64% identity in nucleotide sequences and 42-58% similarity in amino acid sequences when compared with homologous crystallins in squid lenses. These charge-isomeric crystallins also show between 26 and 33% amino acid sequence identity to four major classes of glutathione S-transferase (GST), a major detoxification enzyme present in most mammalian tissues. For further analysis, expression of one of the S-crystallin cDNAs was carried out in the bacterial expression system pQE-30, and the S-crystallin protein produced in Escherichia coli was purified to homogeneity to determine the enzymic properties. We found that the expressed octopus S-crystallin possessed much lower GST activity than the authentic GSTs from other tissues. Sequence comparison and construction of phylogenetic trees for S-crystallins from squid and octopus lenses and various classes of GSTs revealed that S-crystallins represent a multigene family which is structurally related to Alpha-class GSTs and probably derived from the ancestral GST by gene duplication and subsequent multiple mutational substitutions.

Amino Acid Sequence↗

PubMed1995

Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase.

The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction.

Animals↗

PubMed1995

Identification of Asp258 as the metal coordinate of pigeon liver malic enzyme by site-specific mutagenesis.

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1994

Dissociation of pigeon-liver malic enzyme in reverse micelles.

Pigeon-liver malic enzyme has a tendency to aggregate at a large concentration of protein. The larger aggregates (hexamer and octamer) were demonstrated to be enzymically active with specific activity similar to that of the tetramer. When the enzyme was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl)-sulfosuccinate (AOT) in isooctane, the tetrameric enzyme dissociated into monomers. The dissociated monomers were also enzymically active but with diminished specific activity relative to the activity in aqueous media. The decreased enzyme activity in reverse micelles was due to interactions of surfactant with the enzyme molecules, suggesting that the cytosolic malic enzyme is located near the plasma membrane. When the dissociation was monitored by altering the degree of hydration of the system (represented by the ratio [H2O]/[AOT]), the detergent and organic solvent slightly affected KTD, the dissociation constant of tetramer to dimers (T <--> 2 D), but increased KDM, the dissociation constant of dimer to monomers (D <--> 2 M), by 1-2 orders of magnitude; this change caused a 2-3 orders of magnitude increase in the overall dissociation constant KTM (T <--> 4 M). The dissociation of the tetrameric malic enzyme to monomers was favored by approximately 16 kJ/mol in AOT/isooctane reverse micelles versus aqueous media. We propose water-shell and induced-fit models for the enzyme in AOT/isooctane reverse micelles at large and small [H2O]/[AOT] ratios to explain this data, respectively. The asymmetric quaternary structure of the enzyme [Lee, H. J. & Chang, G. G. (1990) FEBS Lett. 277, 175-179] was re-evaluated in terms of the subunit interactions and various interconvertible enzyme forms.

Animals↗

PubMed1994

Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line.

Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.

Alkaline Phosphatase↗

PubMed1994

Affinity cleavage at the putative metal-binding site of pigeon liver malic enzyme by the Fe(2+)-ascorbate system.

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

Amino Acid Sequence↗

PubMed1994

Possible kinetic mechanism of human placental alkaline phosphatase in vivo as implemented in reverse micelles.

Human placental alkaline phosphatase is an integral membrane protein. It catalyzes the hydrolysis of phosphoester linkage of a broad-range substrate. We have embedded the enzyme in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. Linear Lineweaver-Burk and Eadie-Hofstee plots for the substrate and linear Arrhenius plot for the temperature-dependent enzyme reaction were obtained in reverse micelles suggesting that the substrate diffusion limitation was not a rate-limiting step in the system and exchange of materials between reverse micelles was very rapid. The catalytic constant (kcat) of the enzyme was decreased, and the Km for the substrate was increased in reverse micelles, both in an exponential way with the [H2O]/ AOT] ratio (omega O). The enzyme was more stable in reverse micelles than in aqueous solution at 30 degrees C but was unstable at higher temperature (65 degrees C). The activation energy of the enzyme in reverse micelles was 46.5 +/- 2.6 kJ/mol, which was about 20 kJ/mol higher than that in aqueous solution and reflected in the lower Kcat value at low omega O. The binding affinity between the substrate 4-nitrophenyl phosphate and the enzyme in reverse micelles was decreased as implemented by the higher Km and higher Ki for phosphate values. In aqueous solution, the log kcat/pH plot suggested that amino acid residues with pKa values of 9.03 +/- 0.03 and 11.37 +/- 0.10 are involved in catalysis. The former should be deprotonated and the latter should be protonated for the reaction to proceed. In reverse micelles, both the above-mentioned pKa values were detected. However, both groups have to deprotonated to give the optimum catalytic function. In aqueous solution, the enzyme's specificity was highly dependent on pH and buffer. The pKa value of the amino acid residues involved in substrate binding was found to be 8.69 +/- 0.07 in carbonate buffer, but changed to 9.80 +/- 0.06 in Tris buffer. Our results suggested that the rate-limiting step of the enzyme-catalyzed reaction may be changed from phosphate releasing in aqueous solution to another critical step in the reverse micelles.

Alkaline Phosphatase↗

PubMed1994

Purification and kinetic mechanism of the glutathione S-transferase from C6/36, an Aedes albopictus cell line.

Glutathione S-transferase from an Aedes albopictus cell line, C6/36, was purified to apparent homogeneity by a single glutathione-Sepharose 4B affinity chromatography, with an overall yield of 66% and 226-fold purification. The enzyme is presumably a homodimer with subunit M(r) 23,000, which is similar to the enzyme isolated from other sources. Under native conditions, the enzyme exhibited multiple forms upon isoelectric focusing or polyacrylamide gel electrophoresis, and all these forms retained enzymatic activity. The pI value of the purified enzyme was distributed between approximately 4.7 and 5.4 with major form at 4.9. The purified enzyme lost 63% activity at -20 degrees C within 1 week. Stability of the purified enzyme was greatly improved by the addition 10 mg/ml of bovine serum albumin, under which only 7% activity was lost after 1 week at -20 degrees C. Activation energy for the enzyme-catalyzed conjugation of glutathione with 1-chloro-2,4-dinitrobenzene was found to be 49.1 kJ/mol. The enzyme could utilize 1-chloro-2,4-dinitrobenzene and ethacrynic acid as substrate, but not bromosulfophthalein, cumene hydroperoxide, or trans-4-phenyl-3-buten-2-one. The initial-velocity and product-inhibition studies indicated that the enzyme reaction conformed to a steady-state random Bi-Bi kinetic mechanism, which was similar to the glutathione S-transferase from other sources. The kinetic data also indicated a synergistic effect between the binding of glutathione G-site and that of organic electrophilic substrate in the H-site of the enzyme active center. The biological significance of the conjugation reaction is discussed.

Aedes↗