Summary
The histochemical demonstration of hetero-β-galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.
The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with β-D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of α-naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.
6-Br-2-naphthyl-β-D-glucoside is the most specific substrate for hetero-β-galactosidase. It is not split by lactase and acid β-galactosidase. Only lysosomal β-glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero-β-galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.
The simultaneous azocoupling method with α-naphthyl-β-D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero-β-galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero-β-galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal β-glucosidase does not seem to interfere.
Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero-β-galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero-β-galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal β-galactosidase and possibly of lysosomal β-glucosidase (depending on the glycoside used) is always to be considered.
Hetero-β-galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunner's glands the activity was lower. The occurrence of a low activity of hetero-β-galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.
A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.
In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero-β-galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.
In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.
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References
Ahlquist, J.: Observations on the effect of potassium ferri- and ferrocyanides on the formation of indigo from indoxyl acetate split by alkali and liver homogenates. Acta path. microbiol. scand. 57, 353–360 (1963)
Asp, N.-G.: Small intestinal β-galactosidases. Characterization of different enzymes and application to human lactase deficiency. Dissertation, Lund, 1971, p. 1–54
Asp, N.-G., Berg, N. O., Dahlqvist, A., Jussila, J., Salmi, H.: The activity of three different small-intestinal β-galactosidases in adults with and without lactase deficiency. Scand. J. Gastroent. 6, 755–762 (1971)
Asp, N.-G., Dahlqvist, A., Koldovský, O.: Human small intestinal β-galactosidases. Separation and characterization of one lactase and one hetero-β-galactosidase. Biochem. J. 114, 351–359 (1969)
Gossrau, R.: Über den histochemischen Nachweis der β-Glucosidase mit 1-Naphthyl-β-glucopyranosid. Histochemie 34, 163–176 (1973)
Gray, G. M., Santiago, N. A.: Intestinal β-galactosidases. I. Separation and characterization of three enzymes in normal human intestine. J. clin. Invest. 48, 716–728 (1969)
Gray, G. M., Santiago, N. A., Colver, E. H., Genel, M.: Intestinal β-galactosidases. II. Biochemical alteration in human lactase deficiency. J. clin. Invest. 48, 729–735 (1969)
Johnson, J. D.: Neutral hetero-β-galactosidase from rabbit small intestine. Biochim. biophys. Acta (Amst.) 302, 382–392 (1973)
Lojda, Z.: Some remarks concerning the histochemical detection of disaccharidases and glucosidases. Histochemie 5, 339–360 (1965)
Lojda, Z.: Indigogenic methods for glycosidases. I. An improved method for β-D-glucosidase and its application to localization studies of intestinal and renal enzymes. Histochemie 22, 347–361 (1970a)
Lojda, Z.: Indigogenic methods for glycosidases. II. An improved method for β-galactosidase and its application to localization studies of the enzymes in the intestine and in other tissues. Histochemie 23, 266–288 (1970b)
Lojda, Z.: Aktuelle Probleme der Cytochemie der lysosomalen Hydrolasen. Acta morph. Acad. Sci. hung. 20, 269–293 (1972)
Lojda, Z.: Histochemical methods ofr acid β-galactosidase: Technics for semipermeable membranes. Histochemie 37, 375–378 (1973)
Lojda, Z.: Topochemistry of β-glycosidases in the aortae and coronary arteries of rats, guineapigs and rabbits under normal conditions and after cholesterol feeding. Čs. Patol. 10, 1–9 (1974)
Lojda, Z., Havránková, E.: Indigogenic methods for aminopeptidase and other hydrolases. The use of phenazonium methosulfate and tetrazolium salts as oxidation agents. Histochemie (1974) (in press)
Lojda, Z., Kraml, J.: Indigogenic methods for glycosidases. III. An improved method with 4-Cl-5-Br-3-indolyl-β-D-fucoside and its application in studies of enzymes in the intestine, kidney and other tissues. Histochemie 25, 195–207 (1971)
Lojda, Z., Slabý, J., Kraml, J., Kolínská, J.: Synthetic substrates in the histochemical demonstration of intestinal disaccharidases. Histochemie 34, 361–369 (1973)
Lorenz-Meyer, H., Blum, A. L., Haemmerli, H. P., Semenza, G.: A second enzyme defect in acquired lactase deficiency: Lack of small-intestinal phlorizin-hydrolase. Europ. J. clin. Invest. 2, 326–331 (1972)
Malathi, P., Crane, R. K.: Phlorizin hydrolase: a β-glucosidase of hamster intestinal brush border membrane. Biochim. biophys. Acta (Amst.) 173, 245–256 (1969)
Meijer, A. E. F. H.: Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I. Acid phosphatase. Histochemie 30, 31–39 (1972)
Meijer, A. E. F. H., Vloedman, A. H. T.: Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. II. Non-specific esterase and β-glucuronidase. Histochemie 34, 127–134 (1973)
Nordström, C., Dahlqvist, A.: Quantitative distribution of some enzymes along villi and crypts of human small intestine. Scand. J. Gastroent. 8, 407–416 (1973)
Öckerman, P. A.: Identity of β-glucosidase, β-xylosidase and one of the β-galactosidase activities in human liver when assayed with 4-methyl-umbelliferyl-β-D-glycosides. Studies in cases of Gaucher's disease. Biochim. biophys. Acta (Amst.) 165, 59–62 (1968)
Patel, V., Tappel, A. L.: Identity of β-glucosidase and β-xylosidase activities in rat liver lysosomes. Biochim. biophys. Acta (Amst.) 191, 86–94 (1969a)
Patel, V., Tappel, A. L.: β-glucosidase and β-yxlosidase of rat kidney. Biochim. biophys. Acta (Amst.) 191, 653–662 (1969b)
Price, R. G., Dance, N.: The cellular distribution of some rat kidney glycosidases. Biochem. J. 105, 877–883 (1967)
Raychaudhuri, C., Desai, I. D.: Lysosomal β-glucosidase and β-xylosidase of rat intestine. Int. J. Biochem. 3, 684–690 (1972)
Robinson, D., Price, R. G., Dance, N.: Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney. Biochem. J. 102, 525–532 (1967)
Swaminathan, N., Radhakrishnan, A. N.: Characterization of two hetero-β-galactosidases from monkey small intestine. Arch. Biochem. Biophys. 135, 288–295 (1969)
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Lojda, Z., Havránková, E. & Slabý, J. Histochemical demonstration of the intestinal hetero-β-galactosidase (glucosidase). Histochemistry 42, 271–286 (1974). https://doi.org/10.1007/BF00492660
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DOI: https://doi.org/10.1007/BF00492660