Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

Four steroid 21-hydroxylase B [P-450(C21)B] genes (designated P.7, P.10-1, P.10-2, and P.3) from three P-450(C21)-deficient patients were isolated to analyze their structures and functions. Several base changes were observed in the sequences of the four P-450(C21)B genes as compared to that of the functional B gene. Many of these base changes were identical to those of the P-450(C21)A pseudogene. The three DNAs (P.10-1, P.10-2, and P.3) produced no P-450(C21) activity in a functional assay for P-450(C21) by the COS cell expression system, while the P.7 DNA expressed the activity.The P.10-1 and P.10-2 DNAs were shown to have a point mutation in the second intron, causing aberrant splicing. The P.3 DNA carried three clustered missense mutations in the sixth exon, which impaired P-450(C21) activity. AM these critical mutations could be seen in the corresponding site of the P-450(C21)A pseudogene. These data strongly suggest the involvement of gene conversion in this genetic disease.Steroid 21-hydroxylase [P-450(C21), steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] deficiency (1, 2) is the most frequent cause (=95%) ofcongenital adrenal hyperplasia, one of the most common inborn errors of metabolism, affecting -1 in 15,000 births. This disease is inherited as a monogenic autosomal recessive trait closely linked to HLA major histocompatibility complex (3). P-450(C21) is involved in the conversion of progesterone and 17-hydroxyprogesterone to ll-deoxycorticosterone and ll-deoxycortisol, the intermediate steps in mineralocorticoid and glucocorticoid biosynthesis, respectively (4). By the defect of the P-450(C21) gene, synthesis of aldosterone and/or cortisol is impaired and 17-hydroxyprogesterone consequently accumulates, leading to excess production of androgen. These hormonal conditions develop three types of clinical symptoms, referred to as the salt-wasting, simple-virilizing, and nonclassical forms (5).Recent gene cloning analysis showed that there are two P450(C21) genes-P-450(C21)A and P-450(C21)B-each of which is located immediately adjacent to the 3' end ofthe two genes encoding the fourth component of complement, C4A and C4B, which reside in the class III region of the HLA complex (6, 7). The B gene characterized by the 3.7-kilobase (kb) Taq I fragment of the genomic DNA is functional, whereas the A gene of the 3.2-kb Taq I fragment is a pseudogene harboring at least three deleterious mutations in the coding sequence (8, 9). Nonetheless, the two genes are extremely homologous (-98%) even in the introns and the 5'-flanking sequence, suggesting their concerted evolution by exchanging genetic information with each other (8).Genomic blot analysis of P-450(C21)-deficient patients by using cloned DNAs as probes (10-13) revealed that some patients had the B gene either totally deleted or converted to the A gene. Further analysis with oligonucleotide probes specific for the three deleterious sequences in the A gene could show that at least one alle...

Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.

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Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by double-stranded RNA-mediated interference (RNAi). Embryonic and post-embryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12, where RNAi caused no abnormality. However, synthetic suppression of Ce-Rpn10 and Ce-Rpn12 was lethal, whereas neither the combination of Ce-Rpn9 with Ce-Rpn10 nor with Ce-Rpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and post-embryonic development, although Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12 are not essential, at least under the conditions used. Ce-Rpn10 and Ce-Rpn12 are considered to compensate for the suppression of each other.