Purification and Properties of Aminoendopeptidase from Rat Epidermis

Ito, Yoshimasa; Fukuyama, Kimie; Yabe, Kazuo; Epstein, William L.

doi:10.1111/1523-1747.ep12340333

Cited by 24 publications

(7 citation statements)

References 26 publications

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“…22) Its activity was reported to be higher (5 times) in rats than in humans, although these experiments were independently carried out by other investigators. 23,24) Therefore, aminopeptidase exhibits a tendency similar with respect to species difference as esterases in cutaneous metabolism, suggesting the possibility of using cultured keratinocytes for studying cutaneous metabolism.…”

Section: Discussionmentioning

confidence: 99%

Age Dependency of Esterase Activity in Rat and Human Keratinocytes

Ngawhirunpat¹,

Kawakami²,

Hatanaka

et al. 2003

Biological & Pharmaceutical Bulletin

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The age and species dependent characteristics of cutaneous esterase activity were examined in cultured keratinocytes of neonatal and adult humans and of rats at the age of 1, 3, 10, and 50 d. The existence of esterases was characterized using fluorescein-5-isothiocyanate diacetate under a confocal laser scanning microscope. In vitro hydrolysis of ethyl nicotinate (EN), an esterified prodrug of nicotinic acid, was investigated in homogenate of cultured keratinocytes, and the Michaelis-Menten parameters (V(max) and K(m)) of EN were evaluated. Together with development and growth of rats and humans, V(max) and V(max)/K(m) increased drastically, suggesting that esterases in keratinocytes develop markedly during the growth process. The affinity parameter, K(m), was almost the same among the ages in each species. These findings in cultured keratinocytes corresponded with our previous report using dissected skin specimens. Species differences in V(max), V(max)/K(m) and K(m) were also observed, and these parameters of EN hydrolysis in rats were significantly higher than that in humans. In conclusion, cultured keratinocytes can be an advantageous method with which to estimate cutaneous activation of ester prodrugs in humans and during the growth process.

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Section: Discussionmentioning

confidence: 99%

Age Dependency of Esterase Activity in Rat and Human Keratinocytes

Ngawhirunpat¹,

Kawakami²,

Hatanaka

et al. 2003

Biological & Pharmaceutical Bulletin

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“…Ito's aminoendopeptidase might be the epidermal thiol protease (ETP) in this paper, because this enzyme was also inhibited by NEM, monoiodo-acetic acid, and o-phenanthrolin and located in the stratum granulosum (6). If the substance activated in the above process is indeed a thiol protease, there must be some change in the granular cells after the proteolytic function of this enzyme or enzymes.…”

Section: Discussionmentioning

confidence: 85%

“…In order to examine the denaturing effects on in situ protease activation, the following fixatives and fixation conditions were used: [1] 98%,cold ethanol (-20°C) for 4 minutes; [2] -20°C acetone for 4 minutes; [3] 10%formaldehyde solution for 2 hours; [4] 2.5% glutaraldehyde solution at 4°C for 1 hour; [5] acetic acid-98% ethanol solution (1:3 by volume) for 10 minutes; [6] boiling water (l00°C) for 5 seconds and [7] a control (unfixed sections).…”

Section: Effects Of Thefixationmentioning

confidence: 99%

“…In the skin, the plasminogen activator has been proven to play an important role in pathogenesis of pemphigus vulgaris (1, 2), kallikrein-like protease was found to hydrolyze keratin in psoriasis vulgaris (3,4) and cysteine-proteinase and its inhibitor were found in squamous cell carcinoma (5). Epidermal thiol protease (6,7) and the thiol protease inhibitors (8-10) have been extracted and purified from the 2-day-old…”

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confidence: 99%

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The Substrate of Epidermal Proteases

Takahashi

Tezuka

1989

The Journal of Dermatology

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In newborn rat skin, disulfide bonds exist in the cell membrane of the stratum corneum and in the cytoplasm of the stratum granulosum. However, in the latter region, they can be detected after ethanol fixed epidermis remains for 2 weeks at 4 degrees C. Thus, the idea arose that the visualization of the disulfide bonds in the stratum granulosum may be caused by proteases. Therefore, the in situ effects by both activation and inhibition of epidermal proteases were examined in tissue sections. Cryostat sections which were fixed in cold ethanol (-20 degrees C, 98%) were incubated either in PBS, pH 7.4, containing 10mM 2-ME as a control or in an epidermal homogenate (15,000 x g supernatant fraction containing 2-ME) at 37 degrees C for 30 minutes and 4 hours, respectively, in order to activate the epidermal proteases and then reacted with NEM, 2-ME, and DACM in steps. Many minute fluorescent particles were seen in the stratum granulosum under both of the above conditions. However, with incubation in PBS without 2-ME, no fluorescent particles were seen. In addition, when the tissue was treated with NEM and zinc chloride before incubation, no fluorescent particles were seen. O-phenanthrolin remarkably inhibited the appearance of these fluorescent particles. In contrast, leupeptin and antipain only slightly inhibited the appearance of these fluorescent particles, and pepstatin and phenylmethylsulfonyl fluoride didn't inhibit at all. In general, as NEM and zinc chloride strongly inhibit thiol proteases, these findings suggest that presence of the minute fluorescent particles in the stratum granulosum could be due to the effects of proteases, especially thiol proteases.

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“…The standard assay for other dipeptides and tripeptides was performed in the same way. The catalytic activity of the enzymes on amino acid-,/-naphthylamide derivatives was measured by the method of Ito et al (1984). One unit of enzyme activity was defined as that which hydrolysed I ,tmol of substrate/min.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Characterization of two dipeptidases purified from hepatic schistosome egg granulomas in mice. Leukotriene D4 hydrolases of granulomatous tissue

Sato

Ito

Iida

et al. 1992

Biochemical Journal

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Extracts prepared from tissue with granulomatous inflammation experimentally produced in liver of CBA-strain mice showed increased hydrolysis of leukotriene D4 (LTD4), Leu-Leu and Ala-Gly as compared with normal hepatic cells. Two dipeptidases, Leu-Leu dipeptidase and Ala-Gly dipeptidase, were purified from hepatic granulomas, and quantitative conversion of LTD4 into leukotriene E4 (LTE4) by both enzymes was demonstrated. M(r) values of the purified enzymes were 178,000 for Leu-Leu dipeptidase and 183,000 for Ala-Gly dipeptidase. The enzymes showed homogeneity, appearing as a single band on SDS/PAGE, and the M(r) values of the subunits were 56,000 and 57,000 for Leu-Leu and Ala-Gly dipeptidase respectively. The amino acid compositions of the two enzymes differed considerably from each other. The activity of Leu-Leu dipeptidase was inhibited by bestatin and captopril and stabilized with MnCl2. The Km for LTD4 was 25 microM with a V(max.) of 49.0 mumols/min per mg. In contrast, the activity of Ala-Gly dipeptidase was inhibited by cilastatin, cytinylglycine, EDTA and dithiothreitol, and also by captopril. The Km for LTD4 was 5.3 microM with a V(max.) of 50.4 mumols/min per mg. The findings indicate that the conversion of LTD4 into LTE4 by microsomal dipeptidases is elevated during granulomatous tissue reaction. This enzyme activity may become useful for biochemical quantification of the pathological tissue reaction that occurs in organized granulomas.

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Purification and Properties of Aminoendopeptidase from Rat Epidermis

Cited by 24 publications

References 26 publications

Age Dependency of Esterase Activity in Rat and Human Keratinocytes

Age Dependency of Esterase Activity in Rat and Human Keratinocytes

The Substrate of Epidermal Proteases

Characterization of two dipeptidases purified from hepatic schistosome egg granulomas in mice. Leukotriene D4 hydrolases of granulomatous tissue

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