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Study on aminopeptidase A

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Summary

A comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 3.4.11.7) 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:

  1. a)

    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.

  2. b)

    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.

  3. c)

    Brush border of cells of proximal tubules in all species.

  4. d)

    Cells of glomeruli (mainly podocytes and mesangial cells) of mouse, rat and human kidney. In the guinea-pig no activity in glomeruli was found.

  5. e)

    Muscle cells of media of arteries of mouse, rat and human coronary arteries, of rat and mouse esophagus, urinary bladder, ductus deferens and uterus.

  6. f)

    Cellular membranes of hepatocytes facing bile capillaries in the mouse and rat liver, sinusoidal lining of guinea-pig liver.

  7. g)

    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.

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Supported by Deutsche Forschungsgemeinschaft (SFB 105)

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Lojda, Z., Gossrau, R. Study on aminopeptidase A. Histochemistry 67, 267–290 (1980). https://doi.org/10.1007/BF00692761

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Keywords

  • Coupling Agent
  • Brush Border
  • Naphthylamine
  • Histochemical Demonstration
  • Ductus Deferens