Radiation inactivation probe of membrane-bound enzymes: γ-Glutamyltranspeptidase, aminopeptidase N, and sucrase

ANALYTICALBIOCHEMISTRY

158,278-282(1986)

Radiation Inactivation Probe of Membrane-Bound Enzymes: y-Glutamyltranspeptidase, Aminopeptidase N, and Sucrase BRUCER. STEVENS,*ELLIS S.

KEMPNER,~

AND ERNEST

M. WRIGHT+

*Department of Physiology, University of Florida College of Medicine, Box J-274, Gainesville, Florida 32610; tNational Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205; and *Department of Physiology, UCLA Medical Center, Los Angeles, California 900 24

Received May 20, 1986 y-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at - 135°C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (-40 kDa) and small (-25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomahase complex. 0 1986 Academic PIW Inc. KEY WORDS: radiation inactivation; y-ghttamyltranspeptidase; aminopeptidase; sucrase;enzyme subunits; membrane enzymes.

The functional state of membrane-bound enzymes and receptors is fundamental to cell biochemical and physiological processes. Only a few techniques are useful in studying this problem. We have employed the technique of target analysis of radiation inactivation to determine the size of active membrane-bound enzymes in situ. This method provides a direct application in analyzing membrane processes. Sucrase, y-glutamyltranspeptidase, and aminopeptidase N are apical membranebound intestinal enzymes involved in the final stages of digestion. Sucrase is predominantly associated with the intestinal brush border membrane, while the other two enzymes exist in renal and intestinal membranes. The renal and intestinal forms of each have similar immunologic and biochemical properties ( l-3). Each of the three enzymes is translated from mRNA and glycosylated as a single polypeptide. Their final destination is the apical plasma membrane where they are attached by 0003-2697186

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a short N-terminal hydrophobic domain; the remaining portion of the polypeptide faces the lumen (1). After insertion into the membrane, each of these enzymes is cleaved into subunits which remain attached (4,5). There are many conflicting reports regarding the sizes and catalytic interactions of these subunits. Part of the problem is due to the diversity of techniques employed, e.g., protease or detergent extraction, denatured or native gel electrophoresis, molecular sieving of glycoproteins, immunologic diversity of partially glycosylated forms of the enzymes, etc. Furthermore, many analyses reveal the sizes of the structural units without regard to their functional activity. An alternative approach, inactivation by ionizing radiation, permits measurement of the size of the functional unit in situ. The technique depends on (i) the total loss of activity in any biologically active molecule struck by radiation and (ii) the complete lack of effect

RADIATION

INACTIVATION

PROBE OF MEMBRANE-BOUND

on all other molecules which did not interact directly with the radiation (6,7). Each molecule is thus an individual target for radiation and this independent loss of function permits several different enzymes to be analyzed in the same irradiated sample. In this report we investigate the interaction or independence of functional subunits of three distinct enzymes which are bound to purified plasma membranes derived from rabbit small intestinal epithelium brush border. MATERIALS

AND METHODS

Epithelial brush border membrane vesicles were prepared from rabbit small intestine (jejunum) as described previously (8). The enzymes in the purified membranes were enriched 25fold compared with mucosal cell scrapings. Membrane vesicles were suspended (15 mg protein/ml) in buffer containing 300 mM D-mannit and 10 mM Hepes/Tris,’ pH 7.5. Aliquots of 300 ~1 were dispensed into 2ml glass vials (Kimble 120 12-LAB) which were then frozen in liquid nitrogen, selaed with an oxygen-methane flame, and stored in dry ice. These membranes were irradiated at - 135°C with a beam of 13 MeV electrons as described elsewhere (9). After irradiation the sample vials were held at -80°C until assay. The frozen vials were opened and the gas phase purged with air. The samples were then thawed, placed on ice, and aliquots removed for assay of each enzymatic activity at 23°C. For each unirradiated enzyme, >90% of the original “fresh” activity was retained. The y-glutamyltranspeptidase activity was measured in a mixture containing 5 mM L-y-glutamyl-p-nitroanilide, 10 mM MgC12, and 5 mM glycylglycine in 100 mM Tris-HCI, pH 9.0. The reaction was stopped with 1 N acetic acid, and the reaction product, p-nitroaniline, was measured calorimetrically

’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid, Tris, tris(hydroxymethylt aminomethane; GGT, y-glutamyltranspeptidase; AP-N, aminopeptidase N.

ENZYMES

279

at 4 10 nm ( 10). Aminopeptidase N activity was measured using an assay mixture of 3 10 PM L-leucyl-fl-naphthylamide in 100 mM sodium phosphate, pH 7.0. Enzymatic activity was based on liberation of the product, ,& naphthylamine, which was diazotized and measured calorimetrically at 560 nm (11). Suerase activity was measured in 100 mM sodium maleate buffer, pH 6.0, containing 28 mM sucrose. Liberated glucose was determined by the method of Dahlqvist ( 12). All reaction activities were measured during the initial linearrate time period. Enzymatic reaction activities (A) were measured relative to the control (unirradiated) activity (AO). The surviving biochemical activity at each radiation dose was expressed as A/Ao. The data in each experiment were fitted by a leastsquares analysis to the relation (6) ln(A/AO) = -kD where D is the radiation dose in megarads. The fit was constrained to A/A,, = 1 when D = 0. The molecular weight of the functional unit, or target size was determined from MW=

17.9X 1O’k.

This equation (13) includes the correction factor for irradiations performed at - 135°C. RESULTS

Each experiment consisted of a separate membrane preparation irradiated at seven different doses. Each enzyme was assayed in triplicate at each dose. Results from a typical experiment are shown in Fig. 1. Loss in enzymatic activity was measured to at least 92% that of the control. In all cases the loss was a simple exponential function of radiation exposure as required by the single target analysis formula above, and implies that the measured activity is due to a single-sized enzyme. The loss in activity of the three enzymes was comparable at every dose, suggesting similar-sized functional units for all three. The target sizes, calculated form the radiation inactivation as described in Materials and Methods, are in-

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AND WRIGHT

(6). Attached lipids do not affect the polypeptide target size determination ( 14- 16), nor do covalently linked carbohydrates ( 17- 19). Three Plasma Membrane

DOSE IMradsl

FIG. 1. Loss of activity of sucrase, AP-N and GGT after exposure at - 135°C to ionizing radiation from a linear electron accelerator. Data shown are from a single experiment in which a frozen membrane preparation was irradiated and subsequently assayed for all three enzymes. For each activity the data was fit by a single exponential function by a constrained least-squares analysis. For this experiment, target sizes obtained in this manner were: suerase 63 kDa, AP-N 58 kDa, and GGT 57 kDa.

dicated for each enzyme in the figure legend. The results were reproducible and the summary from several independent experiments is given in Table 1. Irradiation of lyophilized membranes irradiated at low temperature gave essentially the same results (data not shown). DISCUSSION

Radiation

Inactivation

Interpretation

Exposing frozen proteins to ionizing radiation leads to loss of biological function which is independent of the surrounding microenvironment. This loss can be analyzed by target theory to yield the size of the functional unit

Enzymes

The plasma membrane-bound enzymes GGT, AP-N, and sucrase each play a role in the final stages of digestion. These enzymes share an analogous synthesis route, similar type of membrane attachment via a short Nterminal anchor, and a similar mature topology. Because their enzymatic properties are unrelated, they will be discussed individually. y-Glutamyltranspeptidase (EC 2.3.2.2). Recently the biosynthesis and structure of GGT has been investigated using cell-free translation systems and several tissue preparations (2022). These systems produced nonglycosylated, glycosylated, and membrane-bound and unbound forms. The translation product of the mRNA is a 60 to 63-kDa polypeptide which is glycosylated by the microsomal membranes to increase the chain size to about 78 kDa. This structure is inserted into the apical plasma membrane by a short segment at the NH2terminus. The chain is subsequently cleaved into a larger fragment anchored to the membrane, and a smaller subunit which remains noncovalently attached to the larger unit but is not in contact with the membrane (3,22). The small unit contains the y-glutamyl binding site. This mature form of the enzyme has

TABLE I RABBIT

Enzyme y-Glutamyltranspeptidase Aminopeptidase N Sucrase

INTESTINAL

BRUSH

BORDER

MEMBRANE

ENZYMES

Membrane-bound enzyme target size &Da)

Isolated enzyme (carbohydrate-free form) &Da)

59?3(n=5) 59* 1 (n=4) 63 + 1 (n = 4)

60” 105b 122’

Note. The in situ target sizes were determined by individual experiments as explained under Results; the mean f SE is shown for ail experiments (number of experiments in parentheses). a Ref. (2 I). ’ Ref. (25). ‘Ref. (1).

RADIATION

INACTIVATION

PROBE

been extracted and treated with endo+-Nacetylglucosaminidase H to remove core glycosylation; this revealed that the original 60to 63-kDa polypeptide chain had been cleaved into the larger 40- to 42-kDa fragment plus the smaller one of 23-25 kDa (20-22). The interpretation of the present radiation results is straightforward in light of previous biochemical studies, and the virtually identical fundamental structure of GGT in kidney and intestine (3). The 59-kDa target size (Table 1) corresponds accurately to the sum of the protein components of the larger subunit (including the anchor) and the small unit containing the substrate binding site. Since there is no covalent bond between these fragments, the radiation results strongly suggest that both units are required for enzymatic activity. This confirms the biochemical work of Tate and colleagues (23,24). Using isolated and reconstituted heavy and light subunits, these investigators found that both subunits together are required for transpeptidase activity. Further biochemical studies (24) suggested that the active center is located in the intersubunit contact region; both subunits must be in close approximation. The agreement between the biochemical and radiation inactivation data make GGT a good internal control for the analysis of functional units of other membrane-bound enzymes, sucrase and aminopeptidase N. Sucrase (sucrose a-D-glucohydrolase; EC 3.2.1.48). Sucrase is a distinct domain within the sucrase-isomaltase complex (5,25,26). The pro-sucrase-isomaltase precursor is translated and glycosylated as a single polypeptide chain containing both sucrase and isomaltase activities. The single chain is inserted into the apical plasma membrane where pancreatic proteases in the intestinal lumen cleave the chain to form two discrete enzymatic domains. In the final mature form the whole sucrase-isomaltase complex (about 280 kDa) is anchored to the membrane via the isomaltase subunit (about 150 kDa), with the sucrase domain noncovalently attached to the isomaltase unit. It is not known whether the in vivo membrane bound sucrase undergoes further cleavage to

OF

MEMBRANE-BOUND

ENZYMES

281

smaller peptides. The present data (Table 1) show a target size of 63 kDa which suggests that further cleavage does occur, thus creating additional independent noncovalently attached functional subunits within the sucrase domain. Aminopeptidase N (leucine aminopeptidase; EC 3.4.11.2). As with the above enzymes, rabbit intestinal aminopeptidase N is translated, glycosylated, and inserted into the apical membrane as a single polypeptide chain. An N-terminal hydrophobic anchor of about 4 kDa holds the enzyme to the membrane. From studies of detergent- and/or protease-extracted enzyme, denaturing and native gel electrophoresis, and amino acid analyses, it is currently speculated that the glycosylated enzyme is a monomer of about 130 kDa (the carbohydrate-free form is 105 kDa) and is not cleaved to a dimer in the rabbit intestinal apical membrane (1,2,27). However, this monomeric interpretation is unique to the rabbit intestine; in other species and membranes AP-N is composed of at least 2 subunits which are noncovalently aggregated (1). The in situ situation is uncertain due to conflicting results arising from differences in solubilization methods and subsequent proteolytic attack which gave multiple fragment bands on SDS gels. In view of the present finding (Table 1) that the mature functional unit in situ (59 kDa) is smaller than the carbohydrate-free extracted form previously reported, our interpretation is that (in common with the other tissues and species) the rabbit intestinal AP-N exists as a dimer of noncovalently aggregated subunits. Because only one anchor and one zinc atom is detected per enzyme (25) we suggest that one subunit is noncovalently attached to a second which is anchored in the membrane in the same fashion as in the GGT topology. With this arrangement, only one subunit need actually participate in catalytic activity, while the other merely tethers the enzyme to the membrane. Concluding, we find that these three membrane-bound enzymes assume a similar in situ mature topology. All are glycosylated poly-

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peptides anchored to the membrane by a short hydrophobic amino-terminal region which is attached to a larger cleaved fragment containing the active site and extending into the external environment. The similarity is even more intriguing because of the structural correspondence with unrelated surface enzymes such as HMG CoA reductase (28). These properties may reflect a general principle widely applicable in cell physiology. The radiation method can reveal the size of biologically active proteins in the membrane. Structurally associated polypeptides which are not required for the measured activity, and which are not covalently linked, will not be detected in the target size determination. The methodology can be applied to any function which withstands the freezing and thawing procedure. Although the technique will give no clues about the character of the surrounding membrane, it will reveal accurately the mass of the active polypeptides in the native state. No other method presently available can yield such information. REFERENCES 1. Kenny, A. J., and Maroux, S. (1982) Physiol. Rev. 62,91-128. 2. Maroux, S., and Feracci, H. (1983) in Methods in Enzymology (Fleischer, S., and Fleischer, B., eds.), Vol. 96, pp. 406-423, Academic Press,New York. 3. Tate, S., and Meister, A. (198 1) Mol. Cell. Biochem. 39,357-368. 4. Ciba Foundation Symp. (1983) Vol. 95. 5. Bnmner, J., Wacker, H., and Semenza, G. (1983) in Methods in Enzymology (Fleischer, S., and Fieischer, B., eds.), Vol. 96, pp. 386-406, Academic Press, New York. 6. Pollard, E. C., Guild, W. R., Hutchinson, F., and Setlow, R. B. (1955) Progr. Biophys. 5, 72-108. 7. Suarez, M. D., Revzin, A., Narlock, R., Kempner, E. S., Thompson, D. A., and Ferguson-Miller, S. (1984) J. Biol. Chem. 259, 13,791-13,799. 8. Stevens, B. R., Ross, H. R., and Wright, E. M. (1982) J. Membrane Biol. 66, 2 13-225.

AND WRIGHT

9. Harmon, J. T., Nielson, T. B., and Kempner, E. S. (1985) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 117, pp. 65, Academic Press, New York. IO. Orlowsky, M., and Meister, A. (1963) Biochitn. Eiophys. Acta 73,679-68 I. 11. Martinek, R. G., Berger, L., and Broida, D. (1964) Clin. Chem. 10, 1087. 12. Dahlqvist, A. (1984) Stand. J. Clin. Lab. Invest. 44, 169-172. 13. Pestka, S., Kelder, B., Familletti, P. C., Maschera, J. A., Crowl, R., and Kempner, E. S. (1983) J. Biol. Chem. 258,9706-9709. 14. Innerarity, T. L., Kempner, E. S., Hui, D. Y., and Mahley, R. W. (1981) Proc. Natl. Acad. Sci. USA 78,4378-4382. 15. Beauregard, G., Potier, M., and Roufogalis, B. D. (1980) Biochem. Biophys. Res. Commun. %,12901295. 16. Edwards, P. A., Kempner, E. S., Lan, S.-F., and Erickson, S. K. (1985) J. Biol. Chem. 260, 10,27810,282. 17. Lowe, M. E., and Kempner, E. S. (1982) J. Biol. Chem. 257, 12,478- 12,480. 18. Kempner, E. S., and Schlegel, W. (1979) Anal. Biochem. 92,2-10. 19. Kempner, E. S., Miller, J. H., and McCreery, M. J. (1986) Anal. Biochem. 156, 140-146. 20. Finidori, J., Laperche, Y., Haguenauer-Tsapsi, R., Barouki, R., Guellaen, G., and Hanoune, J. (1984) J. Biol. Chem. 259,4687-4690. 21. Capraro, M. A., and Hughey, R. P. (1983) FEBS Lett. 157, 139-143. 22. Nash, B., and Tate, S. S. (1984) J. Bioi. Chem. 259, 678-685. 23. Gardell, S. J., and Tate, S. S. (1981) .I. Biol. Chem. 256,4799-4804. 24. Gardell, S. J., and Tate, S. S. (1982) Arch. Biochem. Biophys. 216, 719-726. 25. Hauri. H-P., Wacker, H., Ricki, E. E., Bigler-Meier, B., Quaroni. A., and Semenza, G. (1982) J. Biol. Chem. 257,4522-4528. 26. Bruner, J., Hauser, H., Braun, H., Wilson, K. J., Wacker, H., O’Neill, B., and Semenza, G. (1979) J. Biol. Chem. 254, 1821-1828. 27. Feracci, H., and Maroux, S. (1980) B&him. Biophys. Acta 599, 448-463. 28. Liscum, L., Finer-Moore, J., Stroud, R. M., Luskey, K. L., Brown, M. S., and Goldstein, J. L. (1985) J. Biol. Chem. 260, 522-530.