Assay of peptidase activities of intestinal brush border membrane with l -amino acid oxidase

ANALYTICAL

BIOCHEMISTRY

39, 15-23

(1971)

Assay of Peptidase Border Membrane S. AURICCHIO, Institute

for Child Health,

Activities of with L-Amino M. PIERRO,

Intestinal Brush Acid Oxidase M. ORSATTI

AND

University of Naples, Naples, de& Ricerche, Rome, It& Received

March

and

Consiglio

Nazionale

4, 1970

Some of the methods currently used for the assay of peptidase activities measure the disappearance of the substrate (1) ; others are based on the measurement of the amino acids produced during the reaction, but they give high blank values for most of the substrates (2, 3). In both cases a small percentage of hydrolysis cannot be determined with sufficient accuracy and kinetic studies are difficult. These shortcomings could be avoided by development of a method which permits the measurement of the amino acids produced without any intereference from the peptides. Recently, enzyme activities splitting L-phenylalanine-containing peptides have been determined by measuring the released L-phenylalanine (4) with a procedure employing L-amino acid oxidase (5). In this report we describe a method for the assay of a variety of peptidase activities. The procedure is based on the oxidative deamination of the L-amino acids with L-amino acid oxidase coupled with the oxidation of o-dianisidine by hydrogen peroxide in the presence of peroxidase. A preliminary application of this method to the assay of peptidase activities of the brush ‘border membrane from rat small intestine is also described. METHODS Rat

Intestinal

Microvillous

Membrane

Rats of the Sprague-Dawley strain were stunned by a blow to the head and decapitated. The small bowel was excised, opened longitudinally, and washed out with a 5 mM EDTA solution adjusted to pH 8.5 with NaOH. Mucosal scrapings obtained from the proximal third of the small bowel were suspended in the same solution (74 ml/gm tissue) and homogenized in a Waring Blendor for 30 sec. From the homogenate the microvillous membrane was prepared according to Forstner et al. 15

16

AURICCHIO,

PIERRO,

AND

ORSATTI

(6) and was found to be homogeneous by electron microscope examination. In order to have a marker of the microvillous membrane, sucrase activity was determined by the method of Auricchio et al. (7) : its specific activity increased 14.6- to 28.4-fold (mean: 21.6) in the membrane fraction as compared to the original homogenate. Enzyme

Assay

The standard assay mixture contains 0.4 Fole of dipeptide and L-leucylglycylglycine or 0.2 pmole of tri- and tetra-L-alanines, 6 ,pmoles of phosphate/acetate/borate ‘buffer, pH 8.5 (8), enzyme solution, and other components when indicated, in a total volume of 100 ~1. Blanks were prepared without enzyme and without substrate. The incubation was carried out at 37°C for 5-20 min. The reaction was stopped by addition of 10 ,JJ of 60% percloric acid. Then 10 ~1 of 11 M KOH was added to neutralize the solution and the volume was brought to 0.8 ml with O.lM potassium phosphate buffer, pH 7.4. After centrifugation at 20009 for 5 mm, 0.4 ml of supcrnatant was added to 0.2 ml of dye/peroxidase/Triton (9) and to 5 ,J or 10 JJ of L-amino acid oxidase solution (35 or 70 pg of enzyme, respectively, in 0.01 M sodium phosphate buffer, pH 7.4, and 0.01 M KCl). The mixture was incubated at 37°C for 10 min or 2 hr, depending on the substrate used. Then 0.3 ml of 50% H,SO, was added. The absorbance was read with an Unicam spectrophotometcr at 530 nip on a 2 cm light pathway against a blank prepared with 0.4 ml of distilled water instead of supernatant. For the standard curves, mixtures of amino acids (1030 mpmoles) were prepared in a volume of 100 ~1, corresponding to 2.5-7.570 hydrolysis of the substrate. The mixtures mcrc deproteinized and treated as for the enzyme assay. Reagents Hippuryl-L-phenylalanine, hippuryl-L-arginine hydrate, L-lysyl-n-leutine, L-arginyl-L-leucine, L-arginyl-L-valine acetate, tri-L-phenylalanine, and the j?-naphthylamides of L-leucine, L-phenylalanine, n-histidine, n-isoleucine, L-alanine, L-valine, L-alanyl-L-alanine, L-cystine, L-seryln-tyrosine, L-tyrosine, and .a-L-glutamyl-P-naphthylamide, were obtained from Cycle Chemical Corp., Los Angeles, Calif. L-Leucylglycine, glycyl-L-leucine, L-leucine amide hydrochloride, Lleucylglycylglycine, L-glutamyl-L-valine, glycyl-L-valine, L-valyl-n-glutamic acid, L-alanyl-L-proline, tri-L-alanine, tetra-L-alanine, and pentaL-alanine were obtained from Miles Laboratory, Elkart, Ind. L-Phenylalanyl-L-alanine, n-alanyl-L-glutamic acid, Triton X-100, dimethyl sulfoxide, and the different amino acids were obtained from Sigma Chemical Co., St. Louis, MO.

L-AMINO

ACID

OXIDASE

ASSAY

OF

17

PEPTIDASES

L-Amino acid oxidase (Crotalus adamanteus venom) was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio and peroxidase (horseradish), a soluble salt-free lyophilized preparation, from Worthington Biochemical Corp., Freehold, N. J. Peptides, arylamides, and amino acids were tested for purity by highvoltage paper electrophoresis (4) and were found to be at least 99.7% pure. RESULTS

AND

DISCUSSION

Color Development for Different Amino Acids Different amino acids show different behaviors when tested for their reactivity with L-amino acid oxidase (Fig. 1). Some of them, including n-leucine, n-tyrosine, L-phenylalanine, and L-methionine, display high reactivity. As indicated in Figure 2, they develop very rapidly a color which is stable for at least 2 hr. No difference in color development was observed for these amino acids when larger amounts of n-amino acid oxidase were used. Other amino acids, including n-valine, n-arginine, n-alanine, L-lysine, and n-glutamic acid, show much lower reactivity. L-Valine and n-arginine (Fig. 3), as well as L-lysine, exhibit very low color development, which does not reach completeness in 120 min of incubation. The re-

1

FIG. 1. Comparative mpmoles amino acid mmoles amino acid mpmoles amino acid 2 hr at 37”C, 0.3 ml mean values and the

reactivity of L-amino and 35 pg L-amino acid and 70 pg L-amino acid and 70 ,ug L-amino acid HaSO, is added. Analyses ranges are reported.

acids. The test tube contains: 10 oxidase (blank rectangles), or 10 oxidase (spotted rectangles), or 30 oxidase (hatched rectangles). After were

performed

three

times:

the

18

AURICCHIO,

PIEREO,

AND

ORSATTI

i=

p 400 x 2 e t 2oq,-+-e-*-e-+-* 0 ’

.

30

60

90

120

MINUTES

FIG. 2. Color development mpmoles (-) amino acid in +35 pg enzyme. Absorbance of 0.3 ml 50% H80, ( 1 ), L-tyrosine and L-phenylalanine.

for L-leucine. The test tube contains 5 (---) or 15 0.4 ml distilled water +0.2 ml dye/peroxidase/Triton was read at 440 rnp for 2 hr and then, after addition at 530 nq.~ (I). Similar results were obtained with

activity of these amino acids is higher when larger amounts of L-amino acid oxidase are used (Fig. 3). Finally, some amino acids, including glycine and proline, do not react at all. As shown in Figure 4 for several amino acids, including both fast reacting and slow reacting ones, color development is proportional to the amino acid concentration. On the basis of these results, 35 pg of L-amino acid oxidase and an incubation time of 10 min before addition of sulfuric acid were selected for the assay of enzymic activities hydrolyzing peptides or arylamides containing fast-reacting amino acids such as leucine and phenylalanine. Under these conditions there is essentially no interference from the slowly reacting amino acids eventually released from the substrate. In contrast, the assay of enzyme activities splitting substrates containing only slowly reacting amino acids needs larger quantities of L-amino acid oxidase and longer incubation time: these conditions must be defined for each subst,rate, depending on the constituent amino acids. Pepticlase Activity

Assay

In order to use the measurement of the released amino acids with L-amino acid oxidase for determination of peptidase activities, the following preliminary points were checked out: first, all the peptides and arylamides listed under “Methods” were tested for their reactivity

LAMINO

ACID

OXIDASE

ASSAY

OF

PEPTIDASES

19

1. . . n

MINUTES

FIG. 3. Color development for L-valine (0) and L-arginine (0). The t,est tube contains 15 mpmoles amino acid in 0.4 ml distilled water +0.2 ml dye/peroxidase/ Triton -j-35 pg (---) or 70 pg (-) L-amino acid oxidase. Absorbance was read at 440 mp for 2 hr and then, after addition of 0.3 ml 50% H&30, (1 ), at 530 mp (0,

rn).

Amirnacld

quantity

( rnN moles )

FIG. 4. Color development as function of L-amino acid concentration. The test tubes contain different concentrations of amino acids in 0.4 ml distilled water f0.2 ml dye/peroxidase/Triton +35 ,gg L-amino acid oxidase for phenylalanine (O), tyrosine (@), and leucine (X) and ‘70 pg enzyme for valine (M), lysine (A), and arginine (0). After 2 hr at 37”C, 0.3 ml 50% H&J01 is added.

20

AURECHIO,

PIERRO,

AND

OBSATTI

with the L-amino acid oxidase and none of them was found to develop any color, with the single exceptions of tri-L-phenylalanine and L-seryln-tyrosine-/3-naphthylamide; second, when “fast-reacting amino acids” were added to the standard incubation mixtures at concentrations corresponding to 2.575% hydrolysis of the peptides, essentially complete recovery was obtained. Accordingly, a procedure based on L-amino acid oxidase permits small quantities of amino acids to be measured without any interference from much higher concentrations of peptides, making it possible to determine peptidase activities under conditions giving a small percentage of peptide hydrolysis. Therefore the method described under “Methods” was designed and applied to the assay of peptidase activities in the brush border membrane from the rat small bowel. The reproducibilities of the peptidase assays were +2.07%, -+2.52%, and %3.71%, respectively, for the hydrolytic activities toward n-leucine amide, n-phenylalanyl-n-alanine, and L-leucylglycylglycine. The rates of hydrolysis of L-leucine amide, n-phenylalanyl-n-alanine, n-leucylglycine, and n-leucylglycylglycine were linear with respect to both the incubation time and the amount of enzyme solution (Figs. 5 and 6). Kinetics of 0 order were also obtained for the enzyme activities splitting n-arginyl-n-valine, n-alanyl-n-glutamic acid, n-glutamyl-nvaline, n-valyl-n-glutamic acid, n-alanyl-n-proline, glycyl-n-valine, and tri- and tetra-n-alanines, when the percentage of hydrolysis range be-

5

10 MINUTES

15

20

FM. 5. F’eptide hydrolysis as function of incubation time in brush border membrane of rat intestine. Hydrolysis of the substrates is expressed in mpmoles substrate hydrolyzed in 100 pl incubation mixture: (A) L-leucine amide; (0) Lphenylalanyl+alanine ; (0) L-leucylglycylglycine ; ( X) L-leucylglycine.

L-AMINO

ACID

OXIDASE

j.lg denzymatic Fro. 6. Peptide membrane of rat tration are referred of Fig. 5. Different

Peptidase

Activities

ASSAY

OF

21

PEPTIDASES

proteins

hydrolysis as function of enzyme concentration in brush border intestine. Both hydrolysis of the substrate and enzyme concento 10 .uI incubation mixture. Symbols are identical with those animals were used for the different substrates.

of Three

TABLE 1 Different Preparations Rat Intestinal Mucosa

of Brush

Specific

Border

Membrane

of

activity”

Substrate

Glycyl-n-leucine GArginyl-rJeucine I.-Lysyl-L-leucine n-Arginyl-n-valine tiAlanyl-bglutamic LGlutamyl-L-valine LValyl-Lglutamic LAlanyl-Lproline Glycyl-r,-valine L-Leucylglycine LPhenylalanyl-n-alanine LLeucylglycylglycine Tri-L-alanine Tetra-L-alanine tiLeucine amide Sucrose a rmoles hydrolyzed drolysis of tri- and protein.

acid

acid

Mean

Range

0.034 0.017 0.024 0.056 0.019 0.032 0.003 0.007 0.051 0.496 1.080 1.213 9.897 7.731 0.634 1.482

0.027-0.041 0.015-0.019 0.020-O. 027 0.042-0.069 0.018-0.020 0.013-0.063 0.003-0.004 0.006-0.008 0.028-0.087 0.343-0.626 0.772-1.409 0.858-1.417 7.645-11.526 6.240-10.572 0.514-0.794 1.070-1.932

substrate/min/mg protein. The specific activity for the tetra-tialanines is expressed in ~moles liberated alanine/min/mg

hy-

22

AURIOOHIO,

PIERRE,

AND

ORSATTI

5 and 20%. Since these enzyme activities hydrolyze peptide containing slowly reacting amino acids, sulfuric acid was added after 2 hr of incubation, with 70 Jpg of L-amino acid oxidase. Different peptidases are variously affected by metals in such a way that the use of various metal ions provides a mean to recognize and characterize the individual enzymes in animal tissues. Therefore, it is of interest that complete recovery of L-leucine was obtained from standard incubation mixtures cont,aining one of the following reagents: 1 mM CoCl,, 1 m&I MnCl,, 1 n-&I CaCl,, 1 mM ZnCl,, 1 m&I CdCl,, 200 mM NaCl, 200 mlM KCl, 1 mM puromycin. As shown in Table 1, the brush border membrane splits some peptides to a large extent. The specific activities for the hydrolysis of L-leucylglycine, L-leucine amide, L-phenylalanyl-L-alanine, L-leucylglycylglytine, and tri- and tetra-L-alanines are of the same order of magnitude as the specific activity of an intrinsic digestive enzyme of the membrane such as sucrase. This agrees with the results of other authors (10, 11) and suggests that the brush border membrane plays a role in terminal digestion of proteins. tween

SUMMARY

A method is described for the assay of peptidase activities which depends on the measurement of the amino acids liberated as a result of hydrolytic reaction. The amino acid determination is performed calorimetrically by a procedure based on oxidative deamination coupled with oxidation of o-dianisidine by hydrogen peroxide in the presence of peroxidase. The main advantage of this procedure over other methods described is that the products of the enzymic hydrolysis are measured without any interference from the peptides. This permits the actual measurement of a small percentage of peptide hydrolysis. Preliminary studies indicate that leucine amide and some di- and tripeptides are rapidly hydrolyzed by brush border membrane prepared from rat small intestine. ACKNOWLEDGMENT The authors assistance.

are

indebeted

to

Mr.

Francesco

Vollaro

for

excellent

technical

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L., AND LINDBERQ, T., Biochim. A. T., AND TATTRIE, B. L., Can.

Biophys. Acta 165, 149 (1965). J. Biochem. 42, 95 (1964),

L-AMINO

3. BINKLEY,

mw

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ACID

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OXIDASl

AND

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ASSAY

N.,

OF

Arch.

23

PEPTIDASES

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Biophys.

128, 397

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4. HEIZER, W. D., AND LASTER, L., J. Clin. Invest. 48, 210 (1969). 5. LA Du, B. N., AND MICHAEL, P. J., J. Lab. Clin. Med. 55, 491 (1960). 6. FORSTNER, G. G., SABESIN. S. M., AND ISSELBACHER, K. J., Biochem. J. 106, 381 (196s). 7. A-URICCHIO, S., RUBINO, A., TOSI, R.. SEMENZA, G., LANDOLT, M., KISTLER, H. J., AND PRADER, A., Enzymol. Biol. Clin. 3, 193 (1963). 8. TEORELL, T., AND STENHAGEN, E., Biochem. 2. 299, 416 (1938). 9. VAN DYKJG, K., AND SZUSTKIEWICZ, C., Clin. Chem. 15, 154 (1969). 10. RHODES, J. B., EICHHOLZ, A., AND CRANE, R. K., Biochim. Biophys. Acta 135, 959 (1967). 11. PETERS, T. J., J. Physiol. 202, 13~ (1969).