Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase which is highly homologous to angiotensin-converting enzyme (ACE). ACE2 produces vasodilator peptides angiotensin 1-7 from angiotensin II. In the present study, we synthesized various internally quenched fluorogenic (IQF) substrates (fluorophore-Xaa-Pro-quencher) based on the cleavage site of angiotensin II introducing N-terminal fluorophore N-methylanthranilic acid (Nma) and C-terminal quencher N ε -2,4-dinitrophenyl-lysine [Lys(Dnp)]. The synthesized mixed substrates "Nma-Xaa-Pro-Lys(Dnp)" were hydrolyzed by recombinant human (rh) ACE2. The amount of each product was determined by liquid chromatography mass spectrometry (LC-MS) with fluorescence detection and it was found that Nma-His-Pro-Lys(Dnp) is the most suitable substrate for rhACE2. The K m , k cat , and k cat /K m values of Nma-His-Pro-Lys(Dnp) on rhACE2 were determined to be 23.3 μM, 167 s −1 , and 7.17 μM −1 s −1 , respectively. Using the rhACE2 and the newly developed IQF substrate, we found rhACE2 inhibitory activity in soybean and isolated the active compound soybean ACE2 inhibitor (ACE2iSB). The physicochemical data on the isolated ACE2iSB were identical to those of nicotianamine. ACE2iSB strongly inhibited rhACE2 activity with an IC 50 value of 84 nM. This is the first demonstration of an ACE2 inhibitor from foodstuffs.

Abstract. The aim of the present study was to compare the effects of full-length rat kisspeptin (rKp-52) with C-terminal decapeptide (Kp-10) of rat or human kisspeptin on LH release in intact male rats. Plasma LH profiles were determined by frequent blood sampling at 6-min intervals for 3 h after central or peripheral injection of kisspeptins. Intracerebroventricular (icv) injection of rKp-52 (0.1 nmol) induced a gradual increase in the plasma LH level, which remained high for the rest of the sampling period. On the other hand, icv injection of rKp-10 did not increase the plasma LH level at the same dose (0.1 nmol). A 10-times higher dose (1 nmol) of rKp-10 and hKp-10 increased the plasma LH level, but the increase was lower than that of rKp-52 icv injection. Intravenous (iv) injection of kisspeptins also stimulated LH release at 10 or 100 nmol/kg. In rKp-52 (10 nmol/kg)-treated animals, the plasma LH level reached a peak within 30 min and remained high until 60 min postinjection. The rKp-10-and hKp-10-injected animals showed a more rapid decline in plasma LH level after the peak found at around 30 min after the injections at both middle (10 nmol/kg) and high (100 nmol/kg) doses. The present study indicates that full-length kisspeptin is more effective in stimulating LH release compared with Kp-10 in male rats. The difference in LH-releasing activity may be the result of a difference in degradation of the peptides, but it is still worth determining whether an active domain other than the Cterminal decapeptide is present in full-length kisspeptin. Key words: Gonadotropin-releasing hormone (GnRH), Gpr54, Kiss1, Metastin (J. Reprod. Dev. 55: [378][379][380][381][382] 2009) isspeptin (also known as metastin) [1], a hypothalamic C-terminal amidated peptide, plays a key role in regulating reproductive function in mammals [2][3][4][5][6][7][8][9][10]. Previously deduced amino acid sequences of rat kisspeptin show that the C-terminal amidated 10-amino-acid sequence (rat Kp-10, YNWNSFGLRY-NH2) is identical to other mammalian species examined except for primates that have phenylalanine at the C-terminal [1,11]. Ohtaki et al. [1] showed that the C-terminal Kp-10 sequence is involved in in vitro receptor activation. The C-terminal decapeptide is more effective than full-length kisspeptin in hOT7T175 (nearly identical to KISS1R, kisspeptin receptor)-expressing cells, as shown by FLIPR assay. The C-terminal nonapeptide is less effective compared with full-length kisspeptin, indicating that the decapeptide is mostly involved in receptor interaction [1].Earlier in vivo studies have shown that both full-length kisspeptin and its C-terminal decapeptide stimulate gonadotropin release when injected peripherally or centrally in rats [12][13][14][15][16], mice [17,18] and monkeys [19]. The stimulatory action of kisspeptins on gonadotropin release appears to be mediated by gonadotropinreleasing hormone (GnRH) release, as GnRH antagonist prevents this response [13,17]. The GnRH neuronal terminal in the median eminence, which has no ...

SummaryThe phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C 4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-speci®c antibodies to the C 4 -form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that¯anking the speci®c site of regulatory phosphorylation (Ser15) and subsequently puri®ed by af®nity-chromatography. Speci®city of the resulting antibodies to the C 4 -form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the ®eld, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C 4 -form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.