SummaryThe clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein in¯uences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5 H cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein speci®cally interacts with the 3 H untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also in¯uences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of`mature' final, brush border lactase-phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.z 1998 Federation of European Biochemical Societies.

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

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Investigations were performed on the structural features responsible for kinetic thermal stability of a thermostable carboxypeptidase from the thermoacidophilic archaebacterium Sulfolobus solfataricus which had been purified previously and identified as a zinc metalloprotease [Colombo, D'Auria, Fusi, Zecca, Raia and Tortora (1992) Eur. J. Biochem. 206, 349-357]. Removal of Zn2+ by dialysis led to reversible activity loss, which was promptly restored by addition of 80 microM ZnCl2 to the assay mixture. For the first-order irreversible thermal inactivation the metal-depleted enzyme showed an activation energy value of 205.6 kJ.mol-1, which is considerably lower than that of the holoenzyme (494.4 kJ.mol-1). The values of activation free energies, enthalpies and entropies also dropped with metal removal. Thermal inactivation of the apoenzyme was very quick at 80 degrees C, whereas the holoenzyme was stable at the same temperature. These findings suggest a major stabilizing role for the bivalent cation. Chaotropic salts strongly destabilized the holoenzyme, showing that hydrophobic interactions are involved in maintaining the native conformation of the enzyme. However, the inactivation rate was also increased by sodium sulphate, acetate and chloride, which are not chaotropes, indicating that one or more salt bridges concur in stabilizing the active enzyme. Furthermore, at the extremes of the pH-stability curve, NaCl did not affect the inactivation rate, confirming the stabilizing role of intramolecular ionic bonds, as a pH-dependent decrease in stability is likely to occur from breaking of salt bridges involved in maintaining the native conformation of the protein.

Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme.Mammalian metallocarboxypeptidases play key roles in major biological processes, ranging from digestive-protein degradation, as effected by carboxypeptidases A and B (9, 13), to specific proteolytic processing, as in the maturation of biologically active peptides. This latter is the role, for instance, of carboxypeptidase N and enkephalin convertase (10,17).Little is known, however, regarding microbial carboxypeptidases. Recent studies led to the cloning and crystallization of a metallocarboxypeptidase from Thermoactinomyces vulgaris (27,29), which was shown to possess a dual substrate specificity, namely, the capacity to cleave both hydrophobic and basic amino acid residues, combining the activities of carboxypeptidases A and B, respectively. Sequence comparison revealed a sequence similarity of approximately 30% and a three-dimensional structure very similar to those of both of these mammalian enzymes (27,29). Another microbial carboxypeptidase with broad substrate specificity was also isolated recently from the thermophilic eubacterium Thermus aquaticus (16). However, no sequence data are so far available for this enzyme.The lack of any knowledge regarding archaebacterial carboxypeptidases led us to purify and characterize one such enzyme from Sulfolobus solfataricus (3), an extreme thermoacidophilic archaebacterium, isolated from volcanic hot springs, that grows optimally at 87ЊC (7). We found that this protein is a zinc metalloprotease endowed with a unique substrate specificity in that it could release basic, acidic, aromatic, and, to a lesser extent, aliphatic amino acids from artificial substrates. Also, it could withstand temperatures of up to 85ЊC and some organic solvents at up to 40ЊC. Furthermore, we found that salt bridges and the zinc ion play major roles in the kinetic thermal stabilization of this molecule (30). Here, we report the molecular cloning and complete nucleotide sequence of the carboxypeptidase-encoding gene. We show that despite the lack of overall sequence homology with known carboxypeptidases, seven blocks of homology with known metallocarboxypeptidases, spanning the Zn-coordinating histidines, can be identified. We also show that S. solfataricus carboxypeptidase expressed in Escherichia coli is enzymatically...

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