Mikio Fujii

Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides derived from hemoglobin

From proteolytic digest of swine hemoglobin, we isolated four peptide, E-1 (Phe-Gln-Lys-Val-Val-Ala), E-2 (Phe-Gln-Lys-Val-Val-Ala-Gly), peptide 30-3 (Phe-Gln-Lys-Val-Val-Ala-Lys) and H-1 (Gly-Lys-Lys-Val-Leu-Gln). These peptides inhibited angiotensin I-converting enzyme activity with an IC50 of 5.8, 7.4, 2.1 and 1.9 microM, respectively. Oral administration of 50 mg/kg E-1 and 50 mg/kg H-1 decreased blood pressure in spontaneously hypertensive rats. In normotensive rats, oral administration of 500 mg/kg E-1 and 500 mg/kg H-1 inhibited the pressor effect of i.v. administrated 300 ng/kg angiotensin I, possibly by inhibiting its conversion to angiotensin II. These results suggest that these peptides are orally effective inhibitors of angiotensin I-converting enzyme that have a hypotensive effect.

Antidiabetic effect of orally administered conophylline-containing plant extract on streptozotocin-treated and Goto-Kakizaki rats.

Conophylline, a vinca alkaloid from Ervatamia microphylla, is known to induce the differentiation of pancreatic precursor cells to insulin-producing cells. In the present research we examined the antidiabetic effects of this alkaloid in vivo by oral administration. Crude conophylline preparations were prepared from the leaves of Tabernaemontana divaricata collected in Okinawa Prefecture, Japan. Conophylline was orally absorbed and showed an increase in its plasma level in both normal and streptozotocin-induced diabetic Sprague-Dawley rats. The plasma conophylline concentration reached its maximum from 1.5 to 3h after a single oral administration and gradually decreased in 24h. The alkaloid decreased the blood glucose level and increased the plasma insulin level in streptozotocin-induced diabetic rats after repetitive administration for 15 days. Fasting blood glucose levels in rats treated orally with conophylline at 0.11 and 0.46 mg/kg/day were 411+/-47 and 381+/-65 mg/dl, respectively; whereas the glucose level of the control rats was 435+/-46 mg/dl. Conophylline also decreased the fasting blood glucose level in Goto-Kakizaki rats in a dose-dependent manner after repetitive administration for 42 days. These results suggest that the extract from conophylline-containing leaves may be useful as a functional food for the treatment of type 2 diabetes mellitus.

Cloning of the pepX gene of Lactobacillus helveticus IF03809 encoding salt-tolerant X-prolyl dipeptidyl aminopeptidase and characterization of the enzyme.

X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactobacillus helveticus IF03809 expressed nearly full activity under high salt conditions, such as 2 M NaCl. We cloned and sequenced the pepX gene for X-PDAP. The calculated M, of deduced X-PDAP (803 amino acids) was 90,847 and the protein was distantly related (35 to 44% identity) to known X-PDAPs of Lactobacillus sp. including L. helveticus CNRZ32 (40% identity). Native and recombinant X-PDAP were purified to homogeneity from both L. helveticus IF03809 and Escherichia coli DH5alpha harboring the pepX gene on a plasmid, respectively. The native enzyme appeared to be a dimer of 220 kDa, as estimated by gel filtration column chromatography. It hydrolyzed an X-prolyl-linkage, but not prolyl- or X-prolyl-X-peptide bonds, and tolerated up to 2 M NaCl as well as some other chlorides of monovalent cations. Determination of the flanking sequences revealed two divergent genes. The upstream region of the pepX gene encodes oppA gene for a putative oligopeptide permease, while the downstream region encodes tnp gene specifying a possible transposase of the IS3 family. The oppA gene shares a 176 bp-promoter region with pepX in the intergenic region, implying a relationship between this oligopeptide transport system and X-PDAP.

Aminopeptidase from Sphingomonas capsulata.

A novel aminopeptidase with unique substrate specificity was purified from a culture broth ofSphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min−1 for glutamate. At pH 7.5 (pH optimum) and 25 °C, the kinetic parameters for alaninepara-nitroanilide were found to bek cat = 7600 min−1 andK m = 14 mm. For alanine β-naphthylamide, they were k cat = 860 min−1 and K m = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.

CLONING OF A NOVEL PROLIDASE GENE FROM AUREOBACTERIUM ESTERAROMATICUM

The prolidase gene from Aureobacterium esteraromaticum was cloned and expressed in Escherichia coli. The cloned enzyme had the same enzymatic properties as the wild-type enzyme. Kinetic analysis of the enzyme indicated that the best substrate was Pro-Hyp, which was not hydrolyzed by other prolidases. Interestingly, there was no homology between the deduced amino acid sequence of A. esteraromaticum prolidase and those of the other sources such as human E. coli and Lactobacillus. However, homology was seen with the yeast hypothetical protein YJL213w, the function of which is unknown. These findings indicate that the A. esteraromaticum prolidase is a novel enzyme different from other prolidases reported to date.

Novel Aminopeptidase Specific for Glycine from Actinomucor elegans

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly>>Ala>>Met>Arg>Ser>Leu. The K m value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40°C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.