Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys 152 and Ile 153 . The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorsoventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid.

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO 2 -grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO 2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to ␤-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO 2 -binding site appeared to be unique among the ␤-CAs so far reported.

The marine diatom, Phaeodactylum tricornutum Bohlin, is probably one of the most extensively studied marine alga with respect to carbon acquisition and assimilation mechanisms. However, quantitative analyses of HCO 3 utilization and the detailed process of acclimation of cells from high CO 2 to limited CO 2 are yet to be done extensively. Suitable molecular markers for this acclimation process are not established, either. Recently, it became clear that the rate of CO 2 formation in artificial seawater is about eight times slower than that in freshwater, and that P. tricornutum cells utilize HCO 3 quite efficiently. Despite their great capacity to take up HCO 3 -, the signal controlling photosynthetic affinity for dissolved inorganic carbon has been shown to be CO 2 in the medium. Furthermore, light seems to be required for this process.Internal carbonic anhydrase (CA) activity has been shown to be crucial for high-affinity photosynthesis in a number of algae, including marine diatoms. Internal β-type CA, which has been isolated in one strain of P. tricornutum, was clearly shown to be a low-CO 2 inducible enzyme. This review paper additionally includes data showing that this CA occurs generally in P. tricornutum species.

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