A comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 18.104.22.168) was carried out. α-Glu-1NA, α-Glu-2NA, α-Glu-MNA and Asp-2NA were used as substrates, Fast Blue B (FBB), hexazotized new fuchsin (HNF) and hexazonium-p-rosaniline (HPR) as coupling agents. Biochemical determination of APA activity was performed in whole homogenates as well as in homogenized freeze-dried cryostat sections of many rat, guinea-pig and human organs. α-Glu-2NA proved to be the best substrate for the biochemical determination of APA activity. It displays favourable kinetic properties and abilities for spectrophotometric as well as fluorometric measurements.K m of rat kidney enzyme amounts to 0.15–0.43 mM, of rat jejunum enzyme 0.35 mM, of myocardial enzyme 0.2 mM and of rat brain enzyme 0.25 mM. The enzyme is activated by Ca2+ and inhibited by EDTA (1 mM) and 1,10-phenanthroline (1 mM). E600, DFP and PCMB (1 mM) did not influence APA activity. The activity was detected in every organ examined. Great organ and species differences were demonstrated. The highest APA activity resides in the kidney and in the small intestinal mucosa.
The structural association of APA is not absolutely firm and up to 30% (depending on the organ and eventual pretreatment of sections with chloroform-acetone which impedes the leakage to some extent) escapes into the aqueous incubation solution.
The highest values were recorded in phosphate and cacodylate buffers followed by citrate phosphate, citrate, Tris-HCl and acetate buffers. There were no great differences in activities measured at pH 6.5 and 7.2.
For the histochemical demonstration of APA activity α-Glu-MNA is the substrate of choice.K m estimated biochemically amounts to 0.3–0.43 mM for the kidney enzyme, 0.35 mM for the rat jejunal enzyme and 0.5 mM for rat brain enzyme.K m estimated by microdensitometric measurements of the reaction product in the brush border of cells of proximal tubules and of jejunal enterocytes at the sites of villi amount to 0.6 and 0.4 mM respectively. The best results were obtained using 2–4 mM concentration of α-Glu-MNA, and satisfactory results using 0.36–0.72 mM concentration. FBB appears to be the best coupling agent in the routine. The most sensitive way to depict APA in sites with low and moderate activity is to use chloroform-acetone pretreated cryostat sections adherent to semipermeable membranes. Sites with high enzyme activity are best revealed upon incubation of chloroform-acetone pretreated cryostat sections adherent to slides. Of the unsubstituted naphthylamine derivative of α-Glu or Asp α-Glu-2NA is superior to α-Glu-1NA or Asp-2NA. In this case HPR or HNF are to be used as coupling agents in cryostat sections adherent to slides.
Due to a higher decomposition rate and higher inhibitory influence of diazonium salts on APA activity in the alkaline pH range it is advisable to carry out the reaction at pH 6.5–7.
The most important binding sites of APA are:
Endothelial cells of the capillary bed in all organs (in some organs its venous portion). In the majority of organs this localization represents the unique or at least prevalent binding site of APA. There are differences in the degree of activity in individual organs of the same species and also interspecies differences in the same organ.
Brush border of small intestinal enterocytes. The activity is present in crypt enterocytes and reaches its maximum in enterocytes of villi. It appears in the duodenum, increases in the aboral direction and after reaching its maximum in the jejunum it declines in the distal ileum. The APA activity is reduced in patients suffering florid coeliac sprue.
Brush border of cells of proximal tubules in all species.
Cells of glomeruli (mainly podocytes and mesangial cells) of mouse, rat and human kidney. In the guinea-pig no activity in glomeruli was found.
Muscle cells of media of arteries of mouse, rat and human coronary arteries, of rat and mouse esophagus, urinary bladder, ductus deferens and uterus.
Cellular membranes of hepatocytes facing bile capillaries in the mouse and rat liver, sinusoidal lining of guinea-pig liver.
Intracellularly APA occurs in the Golgi apparatus of jejunal enterocytes and in the cytoplasm of B cells of Langerhans islets in the guinea-pig.
The functional significance of APA is discussed in relation to the degradation of angiotensin II (endothelium, kidney) and to other brush border peptidases releasing amino acids which are absorbed (enterocytes). The histochemical demonstration is indispensable for a correct interpretation of biochemical data.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Barrett AJ (1977) Introduction to the history and classification of tissue proteinases. In: Barrett AJ (ed) Proteinases in mammalian cells and tissues. North-Holland, Amsterdam New York Oxford, pp 1–55
Blenk R, Gossrau R (in press 1980) Biochemische und quantitativ-histochemische Untersuchungen von Proteasen in der Glandula submandibularis. Acta Histochem Suppl
Caspary WF (1973) Interaction of dipeptide absorption and transport of free amino acids. In: Rommel K, Goebbel H (eds) Biochemical and clinical aspects of peptide and amino acid absorption. F.K. Schattauer, Stuttgart/New York, pp 29–40
Denker H-W, Stangl R (1976) Versuche zur Lokalisierung und Abgrenzung verschiedener Aminosäure-Arylamidasen in Uterus und Blastozyste des Kaninchens. Acta Histochem Suppl 16:249–257
Douglas WW (1975) Polypeptides-angiotensin, plasma kinins, and other vasoactive agents; prostaglandins. In: Goodman LS, Gilman A, Gilman AG, Koelle GB (eds) The pharmacological basis of therapeutics. McMillan Publ. Co. New York, pp 630–652
Glenner GG, Folk JE (1961) Glutamyl peptidases in rat and guinea-pig kidney slices. Nature (London) 192:338–340
Glenner GG, McMillan PJ, Folk JE (1962) A mammalian peptidase specific for the hydrolysis of N-terminal α-L-glutamyl and aspartyl residues. Nature (London) 194:867
Gossrau R (1979) Peptidasen II. Zur Lokalisation der Dipeptidylpeptidase IV (DPP IV). Histochemische und biochemische Untersuchung. Histochemistry 60:231–248
Hartmann K, Gossrau R (1978) Postnatale Entwicklung des Dünndarmepithels beim Meerschweinchen. Prog Histochem Cytochem 11:1–67
Hess R (1965) Arylamidase activity related to angiotensinase. Biochim Biophys Acta 99:316–324
Johnson AR, Boyden NT (1977) Proteases in cultured human endothelial cells. In: Haberland GL, Rohen JW, Suzuki T (eds) Kininogenases 4. F.K. Schattauer, Stuttgart New York, pp 113–118
Keatinge WR (1979) Blood vessels. Br Med Bull 35:249–254
Kenny AJ (1977) Proteinases associated with cell membranes. In: Barrett AJ (ed): Proteinases in mammalian cells and tissues. North-Holland, Amsterdam New York Oxford, pp 393–444
Khairallah PA, Hall MM (1977) Angiotensinases. In: Genest JG, Koin E, Kuchel O (eds) Hypertension. McGraw Hill, New York, pp 179–183
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666
Lojda Z (1958) Azo-coupling reactions in the histochemical demonstration of enzymes. Stát Zdrav Nakl, Praha
Lojda Z (1975) The use of hexazonium-p-rosaniline in the histochemical demonstration of peptidases. Histochemistry 44:323–335
Lojda Z (1971) Indigogenic methods for glycosidases. IV. An improved method for β-glucuronidase. Histochemie 27:182–192
Lojda Z (1979a) Studies on dipeptidyl(amino)peptidase IV (glycyl-proline naphthylamidase). II. Blood vessels. Histochemistry 59:153–166
Lojda Z (1979b) The histochemical demonstration of peptidases by natural substrates. Histochemistry 62:305–323
Lojda Z (1979c) The histochemical demonstration of brush border endopeptidase. Histochemistry 64:205–221
Lojda Z, Gossrau R (manuscript in preparation 1980) Peptidases of the capillary endothelium
Lojda Z, Nádvorník F (in press 1980) Membrane aminopeptidases in the human aorta and coronary arteries. Sb Lék 82
Lojda Z, Šmídová J (manuscript in preparation 1980) Peptidases in jejunal biopsies of patients with malabsorption syndrome
Lojda Z, Gossrau R, Schiebler TH (1976) Enzymhistochemische Methoden. Springer, Berlin Heidelberg New York
Lojda Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New York
Matsunaga M, Masson GMC (1970) Hepatic and renal neutral angiotensinases. Experientia 26:1297–1299
McDonald JK, Schwabe C (1977) Intracellular exopeptidases. In: Barrett AJ (ed) Proteinases in mammalian cells and tissues. North-Holland, Amsterdam New York Oxford, pp 311–391
Peach MJ, Chiu AT (1974) Stimulation of aldosterone biosynthesis in vitro by angiotensin II analogs. Circ Res 34 (Suppl 1):7–13
Robertson AL, Khairallah PA (1974) Effects of angiotensin II on the permeability of the vascular wall. In: Page IH, Bumpus FM (eds) Angiotensin. Springer, Berlin Heidelberg New York
Romeis B (1948) Mikroskopische Technik. Leibnitz, München
Wachsmuth ED, Donner P (1976) Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis. Histochemistry 47:271–283
Ward PE, Erdös EG (1977) Metabolism of kinins and angiotensins in the kidney. In: Haberland GL, Rohen JW, Suzuki T (eds) Kininogenases 4. F.K. Schattauer, Stuttgart New York, pp 107–110
Supported by Deutsche Forschungsgemeinschaft (SFB 105)
About this article
Cite this article
Lojda, Z., Gossrau, R. Study on aminopeptidase A. Histochemistry 67, 267–290 (1980). https://doi.org/10.1007/BF00692761
- Coupling Agent
- Brush Border
- Histochemical Demonstration
- Ductus Deferens