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Recently, asparaginyl endopeptidase was purified to homogeneity from jack bean (Canavalia ensiformis) seeds, and its NH2-terminal amino acid sequence was determined for 25 residues [Abe, Y. et al. (1993) J. Biol. Chem. 268, 3525-3529]. On the basis of this sequence information, we searched for seed cDNAs encoding this enzyme. Seven clones were obtained and sequenced. By combining four of them, we obtained a cDNA for a precursor of the enzyme containing the reported NH2-terminal sequence. The other three clones seemed to be for precursors of its isozymes. When the deduced amino acid sequences of these enzyme precursors were compared with those in the GeneBank, EMBL, and NBRF databases, only one protein was found with a homologous sequence. It was a precursor of hemoglobinase from a blood fluke (Schistosoma mansoni). Significant homology was observed only in the range of sequence for the mature form. Although hemoglobinase and asparaginyl endopeptidase behave as cysteine proteinases, their protein natures are distinct from either papain-type proteinases or clostripain. Various plant seeds have been reported to contain asparaginyl endopeptidases. This may be the first report, however, that deals with the primary structure of such a proteinase.

A cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3.5.1.14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney lambda gt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (M(r) 45,260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.

Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] have been isolated from porcine liver cDNA lambda gt10 cDNA libraries and sequenced. Sequence analyses of the cDNA and several Achromobacter protease I-digested peptides of the purified protein revealed that porcine liver AARE consists of four identical subunits, and each comprising a single chain of 732 amino acids with acetylmethionine at the N-terminus.