The purification and properties of a β-aspartyl N-acetylglucosylamine amidohydrolase from hen oviduct

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

The Purification

130,

and

Properties

N-Acetyiglucosyiamine from ANTHONY Division

(1969)

295-303

of a P-Aspartyl

Amidohydrolase

Hen Oviduct’

I,. TARENTINO

AND

FRANK

MALEY

of Laboratories and Research, New York State Department Health, Albany, New York 22101 Received

October

14, 1968; accepted

November

of

27, 1968

An enzyme capable of hydrolyzing 1-r-fi-aspartamido[-2-acetamido].1,2-dideoxy&u-glucose (Asn-GlcNAc) and related glycopeptides, such as those from ovalbumin, ribonuclease B, and transferrin, was purified about 1500-fold from hen oviduct. Substitution of the amino or carboxyl groups of the aspartate moiety prevented the enzyme from hydrolyzing the substrate. The products of the reaction were aspartate, ammonia, and N-acetylglucosamine, with I-amino-N-acetylglucosamine an apparent intermediate. The molecular weight of the protein, determined by sucrose density gradient centrifugation and gel filtration, fell in the range of 101,00&110,000. The enzyme was irreversibly inhibited by 5-diazo-4-oxo-n-norvaline (DONV), an asparaginase inhibitor, with a Ki of 9.5 x 10-” M.

The enzyme hydrolyzing Asn-GlcNa@, a linkage group in many glycoproteins, has been reported to be associated with epididyma1 tissue (l), guinea pig serum (a), and the lysosomes of rat liver and kidney (3). In the course of purifying ,&N-acetylglucosaminidase from hen oviduct, we also found Asn-GlcNAc (or glycopeptide) amidohydrolase present in this tissue to a relatively high degree. Since this enzyme was important to us in characterizing the structure of a glycopeptide from ribonuclease B (4), an extensive purification of the amidohydrolase was undertaken. In addition, the properties were studied and compared with similar enzymes described previously (2, 3). The 1 Supported in part by U. S. Public Health Service Grant CA 06406 from the National Cancer Institute and Grant GB 4589 from the National Science Foundation. l-L-pused : Asn-GlcNAc, 2 Abbreviations aspartamido[ -2.acetamido] -1,2-dideoxy-p-o-glucase; DONV, 5-diazo-4-oxo-n-norvaline; CONV, 5-chloro-4-oxo-n-norvaline; dansyl chloride, ldimethylaminonapthalene-5-sulfonylchloride. 295

results of these efforts form the basis of this report. EXPERIMENTAL

PROCEDURE

MATERIALS

Synthetic Asn-GlcNAc was prepared by the procedure of Marks et al. (5), modified by Yamamoto et al. (6), for the synthesis of 1-azido-2acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-~-D-glucase. The preparation and analysis of ovalbumin and ribonuclease B glycopeptides were described previously (4). The molar ratio of the constituents of these glycopeptides was as follows: Ovalbumin ylycopeptide. Aspartic acid, 1.00; leucine, 0.19; threonine, 0.12; glucosamine, 2.99; mannose, 5.89. Ribonuclease B glycopeptide. Aspartic acid, 1.00; leucine, 1.02; threonine, 0.31; glucosamine, 1.87; mannose, 6.44. Pure ribonuclease B glycopeptide. Aspartic acid, 1.00; leucine, 0.00; threonine, 0.00; glucosamine, 1.84; mannose, 6.05. The sialo-glycopeptide from transferrin was approximately 70y0 pure and was a gift of Dr. G. A. Jamieson, American National Red Cross, Washington, D. C. The aspartate analogs, DONV and CONV, were

296

TARENTINO

kindly provided by Dr. R. E. Handschumacher, Department of Pharmacology, Yale University. These compounds were dissolved in 0.05 M potassium phosphate buffer (pH 7.5) and stored at -20”. The glutamate analogs, 6-diazo-5-oxo-Lnorleucine and azaserine, were gifts from ParkeDavis and Company, Ann Arbor, Michigan. The p-nitrophenyl-glycosides of P-N-acetylglucosamine, a-mannose, CY- and fl-glucose, ,+galactose, and @-glucuronic acid were purchased from Pierce Chemical Co., Rockford, Illinois, as was dansyl chloride. Phosphocellulose and DEAE-cellulose were prepared as described by Lorenson et al. (7). Amberlite IRC-50 (Mallinckrodt) was sized by the procedure of Crestfield el aZ. (8). These absorbents were suspended in the appropriate buffer (30% slurry) and packed into the columns under a pressure of 10 psi. Glutamic-oxalacetate transaminase and malate dehydrogenase were purchased from the Sigma Chemical Co., and had specific activities (&moles per min per mg protein) of 290 and 125, respectively, at pH 7.5. METHODS

Assays for glycopeptide amidohydrolase. Assay I. The enzymic release of N-acetylglucosamine from Asn-GlcNAc was measured calorimetrically (4). One unit represents the amount of enzyme that will release 1 mrmole of N-acetylglucosamine from Asn-GlcNAc/hr at pH 5.1 and 37”. Assay II. A continuous spectrophotometric assay was developed, based on the enzymic release of aspartic acid from Asn-GlcNAc or from glycopeptides containing this linkage. In the presence of an excess of glutamic-oxalacetate transaminase and malate dehydrogenase, the decrease in absorbancy at 340 rnp, due to the liberation of aspartic acid, was strictly proportional to the amount of glycopeptide amidohydrolase added. A stock solution containing the following components was prepared: 0.2 M potassium phosphate (pH 7.5), 2.0 ml; 0.012 M reduced nicotinamide adenine dinucleotide (prepared in 1% bicarbonate), 0.1 ml; 0.1 M cu-ketoglutarate (neutralized to pH 7), 0.1 ml; glutamic-oxalacetate transaminase (29 units), 0.5 ml; and malate dehydrogenase (6 units), 0.5 ml. An aliquot (0.32 ml) of this solution was added to cuvettes with a 0.63-ml total capacity (10 mm light path, 2 mm width), followed by Asn-GlcNAc (0.02 ml of a 0.05 M solution), water, and enzyme to give a final volume of 0.5 ml. The rate of decrease of absorbance at 340 rnp was followed continuously at 30” in a Gilford Model 2400 recording spectrophotometer. The enzyme fractions obtained prior to tne Amberlite IRC-50 step were not pure enough to be assayed accurately by the spectrophotometric procedure. For a comparison of As-

AND

MALEY

says I and II (at pH 7.5), the specific activity of the Step VII enzyme, described in the purification scheme, was found to be 7920 and 9620, respectively. Protein concentration was determined by the 280/260 method, by use of the data of Warburg and Christian (9). The assays for P-N-acetylglucosaminidase, a-mannosidase, 01- and /3-glucosidase, P-galactosidase, and B-glucuronidase were performed by a modification of the method of Conchie et al. (10). Sucrose density gradient centrifugation. Sucrose density centrifugation and calculation of the sedimentation coefficient were performed essentially as described by Martin and Ames (11). A 4% ml linear gradient of b200j, sucrose in 0.05 M TrisHCl (pH 8.0) was equilibrated in the cold several hours before use. Six hundred units of the Step VII enzyme in a O.l-ml vol were layered on the top of the gradient. After centrifugation overnight (12 hr) at 40,000 rpm in a Spinco Model L ultracentrifuge equipped with an SW-39 swinging bucket rotor, the tubes were punctured and 12.drop fractions were collected. The peak enzyme activity was determined by Assay I. The recovery of enzyme units in the peak area was approximately 86%. Catalase and alcohol dehydrogenase were used as standards. The linearity of the gradient was determined with an Abbe refractometer. Sephadex G-200 chromatography. Proteins of known molecular weight were used to calibrate a column of Sephadex G-200 (1.5X 85.5 cm; 63%88-M mesh size), previously equilibrated with 0.01 M potassium phosphate (pH 7.5). The samples were placed on the column in a total volume of 0.50 ml, and were eluted at a flow rate of 5.5 ml/hr. Fractions of 1.5-1.8 ml were collected. The void volume of the column (48.5 ml) was determined with T2 bacteriophage and the elution volume was approximated from the midpoint of the eluted protein and enzyme peaks. The recovery of glycopeptide amidohydrolase was approximately 80%. Identijxation of products resulting from the reaction of substrate with the amidohydrolase. I. Synthetic Bsn-GlcNAc. Asn-GlcNAc (2.0 pmoles) and purified glycopeptide amidohydrolase (2400 units) were incubated at 37” in either 0.1 M potassium phosphate (pH 7.5), or sodium citrate (pH 5.1), in a final volume of 0.1 ml, for 0,3, 6, 9, and 24 hr. The reactions were terminated by heating at loO* for 3 min. The products of the reaction were dansylated and chromatographed on Dowex 1-formate columns as described by Plummer et al. (4). The water eluents from the columns were evaporated to dryness, and the residues were subjected to borate electrophoresis (12), to identify the IVacetylhexosamine formed. The dansy!ated prod-

GLYCOPEPTIDE

AMIDOHYDROLASE

ucts were eluted from the columns with 2 N formic acid and subjected to electrophoresis and thinlayer chromatography (4). Another series of reactions (O.lO-ml total volume) were incubated at pH 7.5 and 5.1 for 3 hr at 37” and then were stopped by adding 0.90 ml of 0.2 M sodium citrate (pH 2.2). An aliquot of each was examined on the amino acid analyzer to confirm the presence of aspartic acid. ZZ. Glycopeptides. Ovalbumin, ribonuclease B, and transferrin glycopeptides (0.045 pmoles each) were incubated with purified glycopeptide amidophoshydrolase (2400 units) in 0.1 M potassium phate (pH 7.5) for 1 hr (final volume 0.85 ml). The reactions were stopped with 0.85 ml of 0.2 M sodium citrate (pH 2.2) and an aliquot (0.75 ml) was chromatographed on the amino acid analyzer for the identification of aspartic acid. Stoichiomelry. Duplicate tubes containing AsnGlcNAc (0.5 pmoles) and enzyme (160 units, specific activity 7920) in 0.1 M potassium phosphate (pH 7.5) were incubated at 37” for 30 min. (total volume 0.1 ml). Analyses were then conducted for N-acetylglucosamine release (Assay I), aspartic acid, and ammonia. The latter two compounds were quantitated on the short and long columns of the amino acid analyzer. Polyacrylamide gel electrophoresis. Samples of the most purified enzyme fraction were subjected to polyacrylamide gel electrophoresis in the system described by Davis (13), for 1 hr at 5”. Gels were removed and stained with Buffalo black for protein. Duplicate samples were cut into 2-mm sections and examined for N-acetylhexosamine by Assay I. Enzyme

Purijkation

All operations were carried out from 0 to 4”. Step I. Extract. Hen oviducts (750 g) were homogenized in 600 ml of 0.01 M potassium phosphate (pH 7.5). The crude extract was centrifuged at 27,000g for 20 min and the supernatant fraction (740 ml) was filtered through glass wool. Step ZZ. Ammonium sulfate fractionation. The oviduct extract was diluted with 0.01 M potassium phosphate (pH 7.5) to give a final protein concentration of 34 mg/ml (volume 2.0 liters). Ammonium sulfate was added to 0.40 saturation (22.5 g/100 ml of extract). After being stirred for 30 min, the suspension was centrifuged at 10,000g for 20 min, and the precipitate was discarded. The supernatant fraction was raised to 0.65 saturation by adding 15.3 g per 100 ml, and stirred for 30 min, followed by centrifugation at 10,ooOg for 20 min. The precipitate was dissolved in a minimum volume of 0.01 M potassium phosphate (pH 7.5), and the resulting solution was dialyzed overnight against two B-liter changes of the same buffer.

PURIFICATION

297

Step III. Calcium phosphate gel. The extract from Step II (545 ml, 78.2 mg protein per ml) was diluted with 0.01 M potassium phosphate (pH 7.5) to a protein concentration of 43 mg/ml. A calcium phosphate gel suspension (700 ml, containing 31 mg/ml) was added with stirring over a 15-min period. After being stirred for an additional 20 min, the extract was centrifuged at 10,OOOgfor 20 min. The gel supernatant fraction (1630 ml) was then adjusted to 0.70 ammonium sulfate saturation by adding 43.6 g of salt per 100 ml. The extract was stirred for 30 min and centrifuged at 10,OOOgfor 20 min. The precipitate was dissolved in a minimum volume of 0.01 M Tris-HCl (pH 8.1) and dialyzed overnight against two g-liter changes of the same buffer. Step IV. DEAE-cellulose chromatography. The enzyme from Step III (475 ml, 56.4 mg protein per ml) was absorbed to a DEAE-cellulose column (7.6X16 cm), that was equilibrated previously with 0.01 M Tris-HCl (pH 8.1). The column was washed with 0.01 M Tris-HCl (pH 8.1) until the 280rnp absorbance of the column effluent decreased to near zero, followed by elution with 0.01 M Tris-HCl (pH 8.1) containing 0.03 M NaCl, until the absorbance at 280 rnp was negligible. The enzyme was eluted from the column by increasing the NaCl concentration in the buffer greater to 0.05 M. Fractions with a specific activity than 30 were pooled (520 ml) and brought to 0.70 saturation with ammonium sulfate (43.6 g/100 ml). The precipitate was centrifuged, dissolved in a minimum volume of 0.01 M potassium phosphate (pH 6.5), and dialyzed overnight against two 4liter changes of the same buffer. Step V. P-cellulose chromatography. Enzyme from Step IV was added to a P-cellulose column (2x28 cm), that was equilibrated previously with 0.01 M potassium phosphate (pH 6.5). Fractions of 11 ml were collected at a flow rate of 2 ml/min/cm%. About 60% of the protein passed through the column in the first 20 tubes (Fig. 1) on elution with 0.01 M potassium phosphate (pH 6.5). At tube 32, the column was developed with a linear gradient consisting of 350 ml of 0.01 M potassium phosphate (pH 6.5) in the mixing chamber and 350 ml of 0.20 M phosphate (PH 6.5) in the reservoir. The enzyme contained in tubes 44-63 was pooled (213 ml) and concentrated to 12 ml with an Amicon ultrafilter, followed by dialysis overnight against two 4.liter changes of 0.01 M potassium citrate (pH 6.5). Step VI. Amberlite ZRC-60 chromatography. The enzyme from Step V (15 ml) was applied to a column of IRC-50H+ (2X15 cm) that was equilibrated previously with 0.01 M potassium citrate (pH 6.5). The column was eluted at a flow rate of 1 ml/min/cm2 and fractions of 4.5 ml were col-

298

TARENTINO

AND

MALEY

lected at a flow rate of tubes 47-61 was pooled Amicon ultrafilter to 7.0 The various steps in are summarized in Table RESULTS

TUBE

NUMBER

FIG. 1. Chromatography of oviduct glycopeptide amidohydrolase on phosphocellulose. More specific details on the purification are given in Methods. The solid line represents the absorbancy at 280 rnb, and the broken line, the enzyme activity. The column was washed with 0.01 M potassium phosphate up to tube 32, at which point a gradient elution (diagonal solid line) was initiated, as described in Step V of the Methods.

FIG. 2. Chromatography of oviduct glycopeptide amidohydrolase on Amberlite IRC-50. More specific details on the purification are given in Methods. The solid line represents the absorbancy at 280 rnb, and the broken line, the enzyme activity. After elution of the column with 0.01 M potassium citrate (pH 6.5), the column was eluted with 0.05 M and 0.20 MI potassium citrate (pH 6.5) at points A and B, respectively.

lected. As seen in Fig. 2, most of the protein was strongly absorbed to the resin except for the enzyme, which passed through in the breakthrough volume of the column. The contents of tubes 611 were pooled (33 ml, 0.5 mg protein per ml) and concentrated in the Amicon ultrafiltration apparatus to 8 ml. Step VII. Sephadex G-200 chromatography. The enzyme from Step VI was chromatographed on a column of Sephadex G-200 (2x90 cm) that was equilibrated previously with 0.01 M potassium phosphate (pH 7.5). Fractions of 2.5 ml were col-

9 ml/hr. The enzyme in and concentrated in the ml. the purification scheme I.

AND

DISCUSSION

Enzyme purity. Polyacrylamide gel electrophoresis of the most purified enzyme fraction revealed the presence of one major protein band with which the enzyme migrated, and at least five minor bands. The following enzyme activities could not be detected in the purified enzyme preparation: L-asparagine amidohydrolase, P-Nacetylglucosaminidase, cy- and ,&glucosidase, and P-galactosidase. Traces of ar-mannosidase and ,B-glucuronidase activity were still present. Characteristics of the enzyme assay. The enzymic release of N-acetylglucosamine from Asn-GlcKAc was linear for at least 2 hr at 37” and the reaction rate was strictly proportional to enzyme concentration (Fig. 3). The hen oviduct glycopeptide amidohydrolase appeared to be relatively stable in that it could be stored at -20” for 3 months without loss of activity, and retained full activity after several weeks of repeated freezing and thawing. pH Optimum and stoichiometry of the reaction. Figure 4 shows the activity of the purified amidohydrolase as a function of pH. As indicated, the amount of N-acetylhexosamine released was maximal at pH 5.1 with only a gradual decrease in activity from pH 6.5 to 8.5. As yet there is no explanation for this apparent plateau in activity, which is similar to that reported by Makino et al. (15) for the guinea pig serum amidohydrolase. These results are in contrast to those obtained by Mahadevan and Tappel (3) for the lysosomal amidohydrolase where an optimal pH of 7.5 was described. In agreement with the findings of Makino et al. (15), the hydrolysis at pH 5.1 was found to yield aspartate, N-acetylglucosamine, and ammonia in a ratio of 1: 1: 1, which is in contrast to the results at pH 7.5 where a ratio of 1: 1: 0.3 was obtained. The latter

effect

is

believed

due

to

the

slow

GLYCOPEPTIDE

AMIDOHYDROLASE TABLE

PURIFICATION Purification step Extract Ammonium sulfate Calcium phosphate DEAE-cellulose P-cellulose Amberlite IKC-50 Sephadex G-200

I

OF HEN OVIDUCT GLYCOPEPTIDE AMIDOHYDROLSSE Volume (ml)

Total units

Total protein bg)

Specificaactivity

Recovery (%)

740 545 475 100 213 33 7

549,860 539,550 373) 000 198 ) 000 143) 750 111,075 68,600

67,050 42,730 27,840 2480 142 16.5 5.3

8.2 12.G 13.4 79.8 1012 6732 12,943

100 98.1

.. (0.40-0.65) gel

a Based on the hydrolysis

299

PURIFICATION

of 1 m,umole of Asn-GlcNAc

per hour per milligram

protein

07.8 3G.0 20.4 20.2 12.5 at pH 5.1 and

37”

MINUTES

,,‘I

ENZYME

FIG. 3. The effect of incubation time and enzyme concentration on glycopeptide amidohydrolase activity. N-acetylglucosamine release at pH 5.1, with Step VII enzyme, was measured by Assay I. of ammonia from Lamino-N-acetylan intermediate produced glucosamine, under mildly alkaline conditions (3, 15). As shown previously (15), 1-amino-l\i-acetylglucosamine rapidly releases ammonia nonenzymically at acid pH and more slowly at pH 7.5. These results led them to propose that the hydrolysis could be divided into two stages : (1) Asn-GlcNAc + Hz0 + l-aminoN-acetylglucosamine + aspartate; (2) I-amino-N-acetylglucosamine + Hz0 -+ NHJ + N-acetylglucosamine. This sequence appears to be irreversible, as radioactivity could not be detected in AsnGlcNAc when we conducted the hydrolysis in the presence of aspartateJ4C or asparagine-14C. Similarly, the presence of an excess of NH, + N-acetylglucosamine or l-aminoN-acetylglucosamine alone, did not promote release

FIG. 4. The effect of pH on the glycopeptide amidohydrolase activity. Assay I was used to measure the activity of Step VII enzyme. A wide range buffer similar to that described by Gerwin et al. (14) was used, but with 0.1 M glycylglycine instead of borate (14). The pH for each reaction tube was determined at room temperature with a microelectrode.

the incorporation of labeled aspartate into Asn-GlcNAc. When the enzyme reaction II-as conducted at pH 7.5, we were able to detect a major reducing sugar that migrated exactly with I-amino-N-acetylglucosamine (15). Sucrose density

The glycocentrifugation. peptide amidohydrolase activity migrated in duplicate experiments as a single sharp peak of s!$F = 6.01 (Fig. 5), when compared with catalase (11.3s) and alcohol dehydrogenase (7.4s). The recovery of enzyme units was about 86 70. Sephadex G’-200 chromatography. Figure 6 presents a plot of the elution volume (Ve) of several well characterized proteins as a

300

TARENTINO

AND MALEY

Hemoglobin \

Amido Hydrotase

65L lo

FIG. 5. Sucrose density centrifugation of glycopeptide amidohydrolase. The enzyme (0.1 ml containing 600 units, Step VII) was layered on a 5 to 20% linear gradient and after centrifugation for 12 hr, fractions were collected as described in Methods. The amidohydrolase was located by measuring 0.05-ml aliquots from each fraction for enzyme activity (Assay I). Catalase and alcohol dehydrogenase were centrifuged at the same time, and their respective peak enzyme activities are indicated by the arrows.

function of the logarithm of their molecular weights (16) and, as seen, the elution volume of the amidohydrolase (77.8 ml and 78.3 ml in duplicate studies) corresponds to a molecular weight of about 110,000. The molecular weight of the amidohydrolase was also calculated by combining the data obtained from the sucrose density gradient study with that from the Sephadex G-200 chromatography. The following relationship was used (17) : Molecular

weight

67rNq as = (1 _ ep) ,

where a = Stokes radius (40.5 A), calculated according to Ackers (18) from the Sephadex data; s = sedimentation coeEicient,, obtained from the centrifugation experiments ; 7] = viscosity at 25” of the medium; p = density at, 25” of the medium; fi = partial specific volume (0.725 cm/g; N = Avogadro’s number. The combined data yielded a value for the molecular weight of about 101,000, which

I II 20)

30 4ojo’ MOLECULAR

loo WElGHTx

200 ’ IO-’

I 300

II 500

FIG. 6. Chromatography of glycopeptide amidohydrolase on Sephadex G-206. The column was calibrated with the indicated proteins as described in Methods. The enzyme activity was located by Assay I.

agrees reasonably well with the 110,000 obtained from Sephadex chromatography alone and thus indicates that the enzyme is a fairly symmetrical protein (17). Mahadevan and Tappel (3) reported a molecular weight of 31,000 for a purified kidney lysosomal glycopeptide amidohydrolase, a value obtained by Sephadex G-200 chromatography. In addition to size, the hen oviduct. and kidney amidohydrolases differ in their pH optimum. Comparison of Assay I and II. To obtain kinetic data with the purified amidohydrolase, a continuous spectrophotometric assay was developed, based on the liberation of aspartic acid from Asn-GlcNAc. The K, for Asn-GlcNAc was determined to be 1.0 X lo+ M by the calorimetric assay, and 4.5 X lo-’ M by the spectrophotometric assay (Fig. 7). The K, for the rat liver lysosomal enzyme (determined by the calorimetric assay) was reported as 5.9 X 1O-4 M (3). The rate of hydrolysis of the complex glycopeptides was linear with time, and proportional to the amount, of enzyme added. Because of limitations in the amounts

of these glycopeptides, parison

with

the

an accurate

hydrolysis

com-

of synthetic

Asn-GlcNAc could not be made, although the latter compound was hydrolyzed about three times faster than the ribonuclease B glycopeptide at comparable substrate concentrations (Assay II).

GLYCOPEPTIDE

AMIDOHYDROLASE

301

PURIFICATION

1 F

FIG. 7. Estimation of the Michaelis constant for Asn-GlcNAc using Assay I (0) and Assay II (0). The substrate concentration is expressed as millimolar, and the velocity as millimicromoles per hour per ml enzyme (pH 7.5).

Substrate speci.city. A comparison of the rates of hydrolysis of several glycopeptides at comparable concentrations containing the Asn-GlcNAc linkage is shown in Pig. 8. The ribonuclease B glycopeptide (line 1) containing the amino acids threonine and leucine (see Methods) attached to the carboxyl group of the aspartyl-polysaccharide, was a poor substrate for the amidohydrolase as compared to the pure ribonuclease B glycopeptide (line 4). The cleavage of the Asn-GlcNAc bond of the impure ribonuclease B glycopeptide to yield a tripeptide of aspartyl-leucyl-threonine would not be measurable in Assay II unless contaminating peptidases released aspartate. However, the absence of any of the above hydrolytic reactions was verified by the complete recovery of the impure ribonuclease B glycopeptide after treatment with Step VII amidohydrolase for 24 hr. These results indicate that a free carboxyl group on the aspartic acid residue of the glycopeptide linkage is necessary for amidohydrolase activity. Sialo-transferrin (line 2) and ovalbumin (line 3) glycopeptides were hydrolyzed at intermediate rates. Previously we reported (4) that a free amino group was also required for glycopeptide amidohydrolase activity, since the presence of a

3t 0

4 0 n

4

,L I’)

MINUTES FIG. 8. The hydrolysis of complex glycopeptides by glycopeptide amidohydrolase. The enzymic release of aspartic acid from ribonuclease B glycopeptide (curve l), sialo-transferrin glycopeptide (curve 2), ovalbumin glycopeptide (curve 3), and pure ribonuclease B glycopeptide (curve 4) was measured by Assay II. The glycopeptides (0.025 rmole of each) were incubated with 0.05 ml of Step VII enzyme and assayed, as described in Methods, for synthetic Asn-GlcNAc.

dimethylaminonapthylsulfonyl group on this functional group prevented hydrolysis of the compound. Similar results have recently been reported by Makino et al. (a), with chemically prepared substituted glycopeptides. As suggested by Makino et al. (a), the glycopeptide amidohydrolase probably functions in a degradative role, with respect to the normal turnover of glycoproteins. Thus, after the removal of the protein core by specific peptidases, the amidohydrolase could facilitate the further degradation of the polysaccharide moiety from the reducing end, as well as from the nonreducing end, by removal of aspartic acid. The existence of many types of specific carbohydrases, which can operate on complex polysaccharides, suggests this view to be reasonable. In addition, the predominant intracellular location of the amidohydrolase appears to be the lysosomes (3, 19), an organelle composed almost exclusively of degradative enzymes (20). Inhibition studies. Handschumacher et al.

302

TARENTINO

DONV

(M x IO”,

FIG. 9. Dixon plot of the inhibition of glycopeptide amidohydrolase by DONV. The enzyme (Step VII, 0.02 ml) was preincubated with the indicated concentrations of DONV in a final volume of 0.13 ml for 20 min. The stock solution was then added (0.32 ml), followed by 0.10 (0) or 0.50 (0) pmole of Asn-GlcNAc to give a final volume of 0.50 ml. The relative veiocity is expressed as the change in absorbancy at 340 mF/4 min.

(21) have recently shown that DONV specifically inactivates the enzyme L-asparagine amidohydrolase. Since the hydrolysis of L-asparagine and Asn-GlcNAc is somewhat analogous, it was suspected that DONV might, also inhibit the glycopeptide amidohydrolase. As revealed in Fig. 9, DONV did indeed prove to be an effective noncompetitive inhibitor of the enzyme, but only when preincubated with the amidohydrolase. The hydrolysis of the complex glycopeptides, in addition to Asn-GlcNAc, was inhibited by the presence of DONV. The Ki for DONV with Asn-GlcNAc as substrate was 9.5 X lo+ M, as compared with a value of 6 X lo-4 M obtained for L-asparagine amidohydrolase (21). Aspartic acid, Nacetylglucosamine, and asparagine were not effective in preventing the inhibition of the enzyme by DONV when added to the preincubation mixture at, a final concentration of 0.5 mu, a 50-fold excess with respect to DONV. Asn-GlcNAc, however, does afford the enzyme some protection against inhibition by DONV, as evidenced by the results in Fig. 10, where DONV was added to already initiated reactions containing different

levels

AND MALEY

of substrate.

It is apparent

that, the rate of inactivation of the enzyme depends on the amount of substrate in the

FIG. 10. The effect of Asn-GlcNAc concentration on the inhibition of glycopeptide amidohydrolase by DONV. The standard enzyme reaction (see Methods) was started by the addition of 0.25 (curve l), 0.50 (curve 2), 1.0 (curve 3), or 2.5 (curve 4) pmoles of Asn-GlcNAc. After 5 min, (zero time on the abscissa), 0.2 pmole of DONV was added and the rates shown by curves 1 through 4 were obtained. The reaction rate, at the indicated substrate levels, in the absence of inhibitor is represented by curve 5.

system. At, very high levels of Asn-GlcNAc, the initial reaction rate in the presence of DONV was very close to that obtained in its absence. To emphasize further the high degree of specificity of the inhibition by DONV, the related analogs, 6-diazo-5-oxo-L-norleucine and azaserine, were not inhibitory even at, a final concentration of approximately 100 times greater than the Ki for DONV. CONV, a compound which does not inhibit L-asparagine amidohydrolase (21), was found not to inhibit the glycopeptide amidohydrolase. ACKNOWLEDGMENT The authors express their appreciation to Dr. Thomas H. Plummer, Jr., for his aid with the amino acid analyses and helpful discussions. REFERENCES 1. EYLAR,E.

H., ANDMURAKAMI, M.,in“Methods in Enzymology” (E. F. Neufeld and V. Ginsburg, eds.), Vol. VIII, Press, New York (1966).

p. 597. Academic

GLYCOPEPTIDE 2. MAKINO, M., YAMASHINS,

KOJIMA, T., OHGUSHI, I., J. &o&em. (!fokyo)

AMIDOHYDROLASE T., AND 63, 186

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