Chapter 7
The Mammalian Cytosolic Broad-Specificity ß-Glucosidase 1
1,3
2
Robert H . Glew , Venkatakrishnan Gopalan , George W. Forsyth , and Dorothy J. VanderJagt 1
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1
Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, NM 87131-5221 Veterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N0W0, Canada
2
Mammals contain two ß-glucosidases: lysosomal glucocerebrosidase and a cytosolic ß-glucosidase which has a very broad-specificity and is most active at neutral p H . The physiologic function of the cytosolic ß-glucosidase is obscure, but it does hydrolyze toxic plant glycosides found in the diet of man. We summarize insights which pertain to the enzyme's structure, properties, and function and discuss the issues of tissue distribution, isolation schemes, and catalytic properties, including transglucosylation. Finally, we suggest avenues of research to be taken which are likely to lead to a better understanding of the function and pathophysiologic significance of the cytosolic ß-glucosidase. Glycosidases are widespread in nature and are responsible for the catabolism of a variety of carbohydrate-containing compounds. These hydrolases catalyze chemical transformations at the C - l position of carbohydrates, and a number of metabolic processes depend on these enzymes for their efficiency, selectivity and regulation. Alpha- and P-glycosidases catalyze the hydrolysis of a- and Pglycosidic bonds that occur in polysaccharides, glycoproteins, and glycolipids (1). In some mammalian tissues there is a bimodal distribution of P-glucosidase activity between the lysosomal and cytosolic compartments. In the earliest studies P-glucosidase activity was found in both the lysosome-rich and the unsedimentable (cytosolic) fractions of rat kidney (2, 3 ). The lysosomal P-glucosidase, glucocerebrosidase, has a p H optimum in the acidic range, whereas the cytosolic P-glucosidase is most active in the p H range 6.0 - 7.0. It is now well established that the lysosomal P-glucosidase is the enzyme responsible for the hydrolysis of the glycosphingolipid, glucocerebroside, to glucose and ceramide (4). The 3
Current address: Department of Biology, Yale University, 844 Kline Biology Tower, New Haven, CT 06511
0097-6156/93/0533-0083$08.50/0 © 1993 American Chemical Society
In ß-Glucosidases; Esen, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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deficiency of glucocerebrosidase is the biochemical basis for the sphingolipidosis called Gaucher disease (5, 6). Comprehensive information on a range of topics from purification of glucocerebrosidase to cloning of the gene is the subject of recent reviews (4,7). The other glucohydrolase is a cytosolic P-glucosidase with broad substrate specificity. In in vitro assays it catalyzes the hydrolysis of p-Dglucosides, P-D-galactosides, a-L-arabinosidesand P-D-xylosides conjugated to the aglycones p-nitrophenol or 4-methylumbelliferone (8, 9 ). The function and pathophysiological significance of this neutral p H optimum glycohydrolase remain uncertain. In animal models of renal disease, the levels of urinary cytosolic Pglucosidase were found to be elevated long before proteinuria was apparent (1012 ). Soon thereafter, it was appreciated that the increased excretion of this enzyme in urine could be used as a diagnostic marker for the detection of renal damage and to forewarn of renal-transplant rejection (13). This finding provided the initial impetus to charaterize the cytosolic P-glucosidase. Also, since the various clinical phenotypes observed in patients with Gaucher disease could not be explained on the basis of a deficiency in glucocerebrosidase activity alone, many investigators have compared the two mammalian P-glucosidases in an effort to reveal possible evolutionary or functional similarities and also to determine if the cytosolic enzyme has any role in the etiology of Gaucher disease. Until recently, research on the cytosolic enzyme had been limited primarily to its purification from various sources and characterization of its kinetic properties. The use of amphipathic compounds to probe the physical and chemical nature of hydrophobic effector sites on the guinea pig liver cytosolic P-glucosidase has furnished clues about possible physiological substrates and details of the reaction mechanism of the cytosolic enzyme (14 ). In addition, studies on the hydrolysis of aryl P-D-glucosides by the cytosolic P-glucosidase have provided insights regarding the rate-limiting step and the nature of the intermediates generated during catalysis. Finally, recent investigations have revealed that some naturally occurring toxic P-D-glucosides in the human food chain are substrates of the cytosolic P-glucosidase (15 ). In this review we attempt to condense the current fund of knowledge on the mammalian cytosolic P-glucosidase and also deliberate on new ideas and approaches to address the evasive question of the metabolic and pathophysiologic role of this enigmatic P-glucosidase. The Cytosolic Broad Specificity P-Glucosidase Standard P-Glucosidase Assays. Two particular nonphysiologic substrates have been widely utilized to measure P-glucosidase activity (Figure 1). The first is 4methylumbelliferyl-p-D-glucoside (4-MUGlc) which when cleaved yields the aglycone 4-methylumbelliferone (4-MU) that is highly fluorescent at alkaline p H (16, 17 ). Alternatively, p-nitrophenyl-P-D-glucoside has been used as a Pglucosidase substrate in a spectrophotometric assay in which the release of the pnitrophenol is estimated based on the absorbance of the phenolate ion at 400 nm (18 ). One can also measure the rate of glucose release from the glucoside substrates in a coupled assay system using hexokinase and glucose 6-phosphate dehydrogenase (19).
In ß-Glucosidases; Esen, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
7. GLEW ET AL.
The Mammalian Cytosolic Broad-Specificity P-Glucosidase
A continuous spectrophotometric assay for the P-glucosidase and one that opens the possibility for measuring pre-steady state kinetics has been reported recently (20 ). L a i and associates demonstrated that the neutral p H optimum cytosolic P-glucosidase catalyzes the hydrolysis of the plant glucosides L-picein (phydroxy-acetophenone- P-D-glucoside) and prunasin (D-mandelonitrile- p-Dglucoside) (Figure 2). The marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile permit continuous monitoring of the P-glucosidase catalyzed release of p-hydroxyacetophenone from L-picein and mandelonitrile from prunasin. K and V values obtained from the continuous spectrophotometric assays agreed well with values reported previously based on discontinuous assay procedures. m
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m a x
Tissue Distribution of Mammalian Cytosolic P-Glucosidase. There are wide differences between species and tissues in the distribution and content of the cytosolic P-glucosidase. In general, the richest source of the P-glucosidase in most vertebrates appears to be the liver. On the basis of assays performed using pnitrophenyl-P-D-galactoside as the substrate, Distler and Jourdian (21) found the following specific activities of the broad-specificity P-glucosidase in the livers of various species: bovine - 39.0; porcine - 24.2; human - 15.3; guinea pig - 6.5; rabbit - 6.3; rat - 0.88; beaver - 0.83: and mouse - 0.20 nmole/min/mg. They also reported that the livers of birds, reptiles, and fish were completely devoid of this activity. In the cow, the broad-specificity p-glucosidase is abundant in liver, kidney, and intestinal mucosa, but absent from spleen, skeletal muscle, testes, and serum. In rabbits, the enzyme is predominantly distributed in the cytosol of liver and kidney (22, 23). In the guinea pig, using Western blot analysis, we found that the cytosolic P-glucosidase is present in the largest amounts in the liver, intestine, stomach, and spleen (Table I) (S. Macko and R. H . Glew, unpublished observation). Surprisingly, there is very little cytosolic p-glucosidase activity in mouse and rat liver; however, rat kidney is a rich source of the enzyme (24). This specific distribution pattern may relate to a metabolic role of the cytosolic Pglucosidase. Isolation and Purification. The cytosolic p-glucosidase has been purified to homogeneity from the livers of man (8), calf (25), and guinea pig (19) and also from porcine kidney (26 ). In general, the purification schemes have taken advantage of the acidic and hydrophobic properties of the enzyme. Purification procedures developed in three different laboratories are reviewed in this section. The current protocol used in our laboratory for isolating guinea pig liver cytosolic P-glucosidase is described below (27 ). Liver tissue is minced and homogenized in 10 m M sodium phosphate buffer (pH 6.0) supplemented with the protease inhibitors aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride. The 100,000 x g (lh) supernatant is subjected to DEAE-cellulose and hydroxyapatite chromatography. The enzyme preparation is then applied to an octyl-Sepharose column that has been pre-equilibrated with 0.5 M ( N H ) S 0 to enhance the interaction between the cytosolic P-glucosidase and the hydrophobic affinity resin. The enzyme can be eluted from the affinity column using 60% (v/v) 4
In ß-Glucosidases; Esen, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
2
4
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^-GLUCOSIDASES: BIOCHEMISTRY AND MOLECULAR BIOLOGY
glucose
glucose-O
-N0 \
2
/
B-glucosidase
4-nitrophenyl-fi-D-
\ VN0
2
4-nitrophenol
glucopyranoside
Downloaded by UNIV OF OTTAWA on November 23, 2014 | http://pubs.acs.org Publication Date: July 27, 1993 | doi: 10.1021/bk-1993-0533.ch007
// HO-^
+ -O-glucose
V
^*r^'^ N^
-OH
B-glucosidase
CH 4-methylumbelliferyl-B-D-
3
4-methylumbelliferone
glucopyranoside
Figure 1. Two aryl-P-D-glucoside substrates used to measure P-glucosidase activity.
glucose />- C H - O-fl-D-glucose
\
>
/
+
CN B-glucosidase
Prunasin (D-mandelonitrile-8-D-glucoside)
D-mandelonitrile glucose +
O CH,- C-
»—O-B-D-glucose
O II
CH,-C-
