Summary
The indigogenic method for β-D-galactosidase of Pearson et al. (1963) with 4-Cl-5-Br-3-indolyl-β-D-galactoside was tested and evaluated.
The acid β-D-galactosidase is not firmly associated with structures and escapes from cryostat sections prepared in the usual manner into incubation solutions. This leakage cannot be prevented by a short postfixation of these sections in cold acetone or Baker's formol-calcium chloride. The leakage is negligible from frozen sections prepared from tissue blocks fixed 12–24 h in cold Baker's solution or in 3% buffered glutaraldehyde (the latter fixation is preferred). Even if this fixation causes about 70–80% inactivation of acid β-D-galactosidase it is a prerequisite for studies concerned with its localization. The brush border β-D-galactosidase of enterocytes is more firmly structurally bound. Since its activity against 4-Cl-5-Br-3-indolyl-β-D-galactoside cannot be proved after overnight fixation in cold aldehyde fixatives its demonstration is to be performed in sections prepared from specimens fixed in cold Baker's solution for 2 h at the most, or in cold microtome sections.
The localization obtained with the original method is not correct. The addition of horseradish peroxidase did not result in any improvement of the localization because the employed samples of this peroxidase contained a concomitant β-D-galactosidase activity.
A striking improvement of the localization was achieved by a mixture of ferri- and ferrocyanide which causes a 40–75% inhibition of acid β-D-galactosidase when used in concentrations of 1 · 10−3 M to 1 · 10–2 M.
A new medium was devised consisting of 0,1 M citrate phosphate buffer pH 3,5–5,5, 8 · 10−4M 4-Cl-5-Br-3-indolyL-β-D-galactoside, and 3,1 · 10−3M potassium ferri- and ferrocyanide. This medium enabled to achieve a very good correlation with biochemical studies and to localize “acid” and “neutral” β-D-galactosidases “in situ”.
The acid enzyme was demonstrated first of all in lysosomes of many cells. Its activity is inhibited by galactonolactone, lactose and p-chloromercuribenzoate. The nature of the diffuse extralysosomal staining cannot be decided at present. The distribution pattern of this enzyme in many animal organs is given.
The “neutral” β-D-galactosidase (lactase) was localized by the improved method in the brush border of differentiated rat, human and monkey enterocytes and is inhibited by galactonolactone, lactose, gluconolactone, and cellobiose. In patients with celiac sprue this activity is very much reduced or absent. It is restituted after a gluten-free diet.
Our revised method proved also very useful in processing zymograms and immunoprecipitation lines of β-D-galactosidase(s) with homologous antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.
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Lojda, Z. Indigogenic methods for glycosidases. Histochemie 23, 266–288 (1970). https://doi.org/10.1007/BF00306428
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DOI: https://doi.org/10.1007/BF00306428