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The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1–2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation.

The expression and regulation of alkaline phosphatase (AP) was studied in the human gastric cancer cell line TMK-1. Biochemical analysis, reverse transcription-polymerase chain reaction, and Northern blot analysis demonstrated that the cells express placental, germ cell, and intestinal AP isozymes constitutively. Dexamethasone (Dex), a synthetic glucocorticoid, was shown to specifically induce the placental AP activity to about 10-fold and sodium butyrate (NaBu) induced germ cell AP activity to about 4-fold, respectively. In contrast, these two agents showed little effect on the level of intestinal isozymes. Dex and NaBu also differentially induced the mRNA levels of the placental and germ cell APs. Northern blot analysis of the placental AP transcript in the presence of the transcription inhibitor, 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole, revealed that the half-life of placental AP mRNA is about 27 h for both the Dex-treated and untreated cells. Nuclear run-on transcription analysis indicated an apparent increase in the rate of placental AP gene transcription in Dex-treated cells. These results indicated that the effect of Dex occurred primarily by activation of the placental AP gene transcription in the cells. In order to study the direct Dex and NaBu effect on AP gene expression, the proximal promoter regions of AP genes were fused to luciferase reporter vectors. Despite the high similarity in nucleotide sequences of these two genes, transient transfection analysis demonstrated that Dex and NaBu exerted a specific stimulation only through the respective placental and germ cell AP gene promoter. Taken together, this study indicates that the expression of PAP and GCAP isozymes have specific regulatory mechanisms that can be differentially controlled by signals including glucocorticoid and NaBu.

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.

A full-length 1927-base-pair cDNA of pigeon liver malic enzyme was obtained by utilizing the screening of the cDNA library and polymerase chain reaction techniques. The cDNA contained one open reading frame coding for 557 amino acid residues, flanked by 86 and 167 nucleotides at the 5' and 3' termini, respectively, and was successfully cloned and expressed in Escherichia coli cells. Functionally active recombinant malic enzyme was purified from the cells. This recombinant enzyme has a Km value for L-malate of 160 +/- 30 microM, which is almost identical to that for the natural enzyme (150 +/- 17 microM). The Km value for Mn2+ (4.2 +/- 0.3 microM) is higher than that for the natural pigeon malic enzyme (1.4 +/- 0.2 microM), while the Km value for NADP+ (3.8 +/- 0.3 microM) is lower than that for the natural enzyme (10.8 +/- 0.1 microM). The catalytic constant (kcat) for the recombinant enzyme is decreased by 3.6-fold, but the substrate inhibition constant for L-malate is increased by about 40-fold. Change in the quaternary structure of the recombinant enzyme was revealed in the pH perturbation examination. A truncated pigeon liver malic enzyme, lacking the first 13 amino acid residues, and a recombinant protein, mutated at F19S, N250S, and L353Q, showed no enzymatic activity. Both abortive recombinant mutant proteins were still able to bind with 2',5'-ADP agarose; however, the fluorescence emission spectrum of the protein bound NADPH did not show a blue shift as the natural enzyme. In accordance with these observations, we suggest that the adenosine 2',5'-bisphosphate binding domain of the NADP+ binding site in the beta alpha beta motif may still be retained in these mutant proteins. However, the local hydrophobic environment for the binding of the nicotinamide moiety of the coenzyme molecule may be altered. Therefore, the lack of catalytic activity of the mutant proteins could be attributed to an improper orientation of the bound NADP+.

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.

The kinetic mechanism of the cytosolic NADP(+)-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP+ as the leading substrate followed by L-malate. The products are released in the order of CO2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to L-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.