THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 23, Issx. of December 10, PP. 5518-5525, 1966

Printed in U.S.A.

Mechanism of Action of Guinea Pig Liver Transglutaminase

I. PURIFICATION AND PROPERTIES OF THE ENZYME: IDENTIFICATION OF A FUNCTIONAL CYS- TEINE ESSENTIAL FOR ACTIVITY

(Received for publication, June 13, 1966)

J. E. FOLK AND P. W. COLE

From the National Institute of Dental Research, National Institutes of Health, Bethesda, Adaryland 20014

SUMMARY

Guinea pig liver transglutaminase has been purified 230- fold in high yield by means of diethylaminoethyl cellulose chromatography of liver homogenate supernatant fluid, pre- cipitation of the enzyme with protamine, selective extraction with ammonium sulfate solution, and rechromatography on the cellulose absorbent.

Estimates of molecular weight made by several procedures, 76,900 f 5,000 (sedimentation and diffusion), 90,000 f 4,000 (sedimentation equilibrium), and 90,000 f 10,000 (14C- iodoacetamide incorporation), show some variation. The s&+ and Dzs,w values for transglutaminase were determined as 5.40 x 10-u and 6.44 x lo* cm* set-l, respectively. The -SH content, 16 to 17 residues/90,000 g, was determined on native and denatured enzyme by two procedures. The amino acid composition of transglutaminase is reported.

The effects of several -SH reagents on enzymatic activity have been investigated. Irreversible inhibition by 14C-iodo- acetamide at pH 6.8 occurs only in the presence of the essen- tial cation, Ca++. One mole of i4C-carbamidomethyl is in- corporated per mole of enzyme with the complete loss of enzymatic activity. Substrate affords efficient protection against inactivation and 14C-carbamidomethyl incorporation. Analyses of the 14C-iodoacetamide-inactivated enzyme show that inactivation results from alkylation of 1 cysteine residue.

The &*-dependent transglutaminase activity of guinea pig liver was first described by Waelsch and co-workers (l-3). This enzyme catalyzes two reactions: (a) the replacement by primary amines and (b) the hydrolysis of amide groups of protein- and peptide-bound glutamine residues (2-4). A recent report from this laboratory has presented a simplified scheme of transgluta- minase action as derived from kinetic and inhibitor studies (5). This postulated mechanism involves a three-point attachment of substrate to enzyme and the intermediate formation of an acyl enzyme through a thiol ester bond. The present report describes a purification of transglutaminase, enumerates certain of the physical and chemical properties of the purified enzyme, and identifies, as cysteine, a residue essential for enzyme activity. The sequence of amino acids around this functional cysteine is the subject of a separate report (6).

EXPERIMENTAL PROCEDURE

Material-The carbobenzoxy-L-glutaminylglycine was pre- pared as outlined previously (5) or purchased from Cycle Chemi- cal Corporation. Sephadex G-25, G-100, and G-200 were pur- chased from Pharmacia; DEAE-cellulose was from Carl Schleicher and Schtil Company; protamine sulfate was from Eli Lilly and Company; p-chloromercuribenzoate and iodoacetamide were from Calbiochem.; 5,5’-dithiobis(2-nitrobenzoic acid) was from Aldrich; l-14C-iodoacetamide was from Tracerlab; N-ethyl- maleimide was from Mann; l-14C-NEM1 was from Schwarz Bio- Research.

The purity of the l-C14-iodoacetamide was evidenced by single radioactive areas in three chromatographic systems before and after reaction with excess cysteine at pH 9. The concentration of iodoacetamide was determined by reaction with excess GSH at pH 9.0 followed by spectrophotometric CMB titration (7) of unreacted GSH. A measure of specific radioactivity was ob- tained by quantitative determination of S-(14C-carboxymethyl)- cysteine in acid hydrolysates of the 14C-iodoacetamide-GSH reac- tion product with the use of an amino acid analyzer equipped with a scintillation-counting device as described under “Methods.”

The purification and determination of radiopurity and specific radioactivity of l-14C-NEM were carried out as outlined (8). The concentration of this reagent was determined spectrophoto- metrically at 302 rnp (9).

The CMB was purified by the procedure of Boyer (7). iMethods-Transglutaminase activity was measured by the

calorimetric hydroxamate procedure (4, 5). Each test was made in a final volume of 0.5 ml of 0.2 M Tris-acetate buffer, pH 6.0, containing 0.1 M hydroxylamine, 5 rnrvr CaC&, 10 mM GSH, and 30 mM CBZ-n-glutaminylglycine. After a 5- or lo-min incuba- tion with enzyme at 37”, 0.5 ml of ferric chloride-trichloracetic acid reagent (10) was added, and the resulting red color was meas- ured at 525 mp. An enzyme unit is defined as the amount of en- zyme which catalyzes the formation of 0.5 pmole of hydroxa- mate per min in the test. Specific activity is expressed as enzyme units per mg of protein.

Radioactivity was measured in naphthalene-dioxane-counting fluid (11) with the use of a Packard model 314 liquid scintillation spectrometer.

1 The abbreviations used are: NEM, N-ethylmaleimide; CMB, p-chloromercuribenzoate; CBZ-, carbobenzoxy-; DTNB, 5,5’- dithiobis(2-nitrobenzoic acid).

5518

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Amino acid analyses were performed on acid-hydrolyzed sam- ples by an ion exchange procedure (12, 13). Tryptophan was determined on separate unhydrolyzed samples by an ultraviolet spectrophotometric method (14). Samples (1 to 1.5 mg) of lyophilized transglutaminase were hydrolyzed with 3 ml of con- stant boiling HCl under nitrogen in sealed Pyrex glass tubes at 106” for 24, 48, and 90 hours. Corrections for destruction of serine, threonine, and methionine and for rate of release of valine and isoleucine were calculated from the timed hydrolysates. Performic acid oxidization of the enzyme and determination of cysteic acid in oxidized samples were carried out according to a published procedure (15). Radioactive X-carboxymethylcys- teine was estimated with an amino acid analyzer equipped with a scintillation-counting device (16), and specific radioactivity of this amino acid was calculated by comparison with uniformly labeled 14C-phenylalanine which had been standardized in the Packard spectrometer.

Spectrophotometric titrations of protein thiol groups with CMB (7) were carried out in 0.1 M Tris-acetate buffer, pH 7.0, and in 8 M urea in the same buffer at 25”. Buffered urea solution was prepared immediately before use and enzyme was equili- brated with buffer or urea solution by gel filtration through columns of Sephadex G-25 (coarse) which were equilibrated with the appropriate solution. CMB was added stepwise and absorp- tion was measured at 250 rnp, allowing 20 to 45 min for complete reaction between additions.

Calorimetric estimation of protein thiol groups was performed by the procedure of Ellman (17) at pH 8 in 0.1 M Tris-chloride buffer at 25“. The yellow color resulting upon the addition of DTNB was measured at 412 rnp, and total -SH groups were calculated by the use of the molar extinction coefficient of 13,600.

Protein thiol groups were also estimated by their capacity to react with r4C-NEM. Reactions were carried out in 0.1 M Tris- acetate buffer, pH 6.8, and in 8 M urea in the same buffer at 25”. After incubations with 14C-NEM, the reagents were removed from the enzyme by gel filtration of a 0.5-ml portion through a column, 0.7 x 39 cm, of Sephadex G-25 (coarse) which was equilibrated with 5 mM Tris-chloride buffer, pH 7.5, containing 2 mM EDTA and maintained at 4”. Aliquots of the resulting enzyme solutions were tested for protein concentration, enzyme activity, and radioactivity.

RESULTS

Purijication of Transglutaminase

Ten Hartley strain guinea pigs (250 to 500 g) were killed by decapitation. The livers were removed and homogenized in 75-g portions, each with 150 ml of cold 0.25 M sucrose, in a stainless steel Lourdes homogenizer. All further operations were carried out at temperatures below 5”. The combined homogenates were centrifuged for 2 hours at 30,000 rpm in a Spinco preparative ultracentrifuge with the No. 30 rotor. The resulting clear super- natant fluid was pumped directly into a column, 3.5 x 20 cm, of DEAE-cellulose which was equilibrated with 5 mM Tris-chloride buffer, pH 7.5, containing 2 mM EDTA. After applicationof the sample, the column was washed with 200 ml of the pH 7.5 Tris- EDTA buffer, and the chromatogram was developed with a 1.5- liter linear gradient of 0 to 1.0 M NaCl in the same buffer. Five- milliliter fractions were collected at a flow rate of 5 ml per min. A typical chromatogram of the guinea pig liver homogenate supernatant fluid is shown in Fig. 1A.

FRACTION NUMBER

3.0 t

2= 2.5 2 s t

1 25

FRACTION NUMBER

FIG. 1. Chromatographic separation of transglutaminase on DEAE-cellulose at pH 7.5. A, chromatography of supernatant fluid from guinea pig liver homogenate; B, chromatography of (NH&SO4 extracts of first protamine precipitate. See text for details.

The fractions rich in transglutaminase activity, collected be- tween about 0.3 and 0.5 M NaCl, and indicated by the horizontal

arrow of Fig. IA, were pooled. To this enzyme eluent (approxi- mately 200 ml) were added gradually with stirring 20 ml of a freshly prepared 1% (w/v) solution of protamine sulfate. The precipitate, which contained essentially all of the enzyme ac- tivity, was collected by centrifugation (15 min at 14,600 X g). This precipitate was washed by homogenizing it in an all glass homogenizer with 20 ml of 0.2 M Tris-acetate buffer, pH 6, fol- lowed by centrifugation (5 min at 15,000 x g). The enzyme has been stored as long as 3 days as the protamine precipitate under the pH 6 Tris buffer without noticeable loss in activity. Transglutaminase was extracted from the protamine precipitate with 0.1 M (NH&S04 in 5 mM Tris-chloride buffer containing 2 mM EDTA by the use of the above homogenizing and centri- fuging procedure. Extractions with one 20-ml and three lo- ml portions of the (NH&SO4 solution were usually sufficient to remove most of the enzyme activity. The efficiency of extrac- tion, however, was routinely evaluated by assay, and extrac- tion was continued if necessary.

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5520 Mechanism of Transgbtaminase Action. I Vol. 241, No. 23

The combined clear extracts were chromatographed on DEAE- cellulose by a procedure similar to that outlined above. The extracts were pumped into a column, 2 x 12 cm, of adsorbent and the column was washed with 50 ml of the equilibrating buffer, 5 mM Tris-chloride-2 lll~ EDTA, pH 7.5. The protein was eluted with a 300-ml linear gradient from 0 to 1.0 M NaCl in the same buffer. Three-milliliter fractions were collected at a flow rate of 3 ml per min. A typical chromatogram of the extracts of the first protamine precipitate is shown in Fig. 1B. It may be noted that transglutaminase specific activity is low in the first few fractions of the peak and that in this portion of the peak the ratio of absorbance at 260 and 280 rnp is higher than that over the remainder of the peak. It was found useful to measure the absorbance of the fractions at both 260 rnp and 280 rnp and to discard all fractions having ratios at 260 rnB to 280 rnp higher than 0.64.

The fractions indicated by the horizontal arrow of Fig. 1B were combined and 15 ml of freshly prepared 1% protamine sulfate solution were added gradually with stirring. The precipitate was collected by centrifugation, washed with 0.2 M Tris-acetate buffer, pH 6, and extracted with 0.1 M (NH&SO4 in 5 mM Tris- chloride buffer, pH 7.5, containing 2 mM EDTA in a manner similar to that used for the first protamine extraction. Four 5-ml portions of the (NH&S04 solution were generally sufficient to extract all of the activity. The combined extracts were chromatographed on a column, 2 x 7 cm, of DEAE-cellulose with the use of the Tris-chloride-EDTA buffer, pH 7.5, and an 80-ml

TABLE I Purijication of transglutaminase

1 t 1 1 Total

~--- w %

Liver homogenate. 24,200” 2555 0.11 100 Spinco supernatant fluid. 9,450” 2463 0.26 96 First DEAE chromatography. 464” 2310 5.0 90 (NH&S04 extract of first prota-

mine precipitate. . 89b 2040 23.0 80 Second DEAE chromatography.. 70b 1680 24.0 66 (NH&S04 extract of second pro-

tamine precipitate. 55b 1360 24.7 53 Third DEAE chromatography.. 52b 1300 25.0 51

a Protein was estimated by the method of Lowry et al. (18). b Protein was estimated from the E!&, as 15.8.

linear gradient, 0 to 1.25 M in NaCl. Two-milliliter fractions were collected at a flow rate of about 3 ml per min. The com- bined fractions of transglutaminase, containing between 1.5 and 3 mg of enzyme per ml, were stored frozen at -10”. In Table I are recorded the total recoveries of activity and enrichment of the enzyme. Transglutaminase is purified approximately 230- fold and has been obtained in greater than 50% yield from origi- nal liver homogenate.

Properties of Purijied Transglutaminase

Transglutaminase prepared by the described procedure has been stored in the dilute Tris-EDTA buffer containing NaCl for periods up to 3 months with no detectable loss in activity. Freezing and thawing of these solutions several times also caused no loss in activity. The enzyme could be salted out by 70% saturation of its solutions with (NH&S04 which had been crys- tallized twice from 0.01 M EDTA. The use of commercial crys- talline (NH&SO1 for this purpose resulted in considerable losses in activity. Solutions of transglutaminase were generally main- tained 1 to 2 mM in EDTA as a precaution against loss in enzyme activity.

The enzyme showed a specific activity of 25 & 0.5. The K, for CBZ-n-glutaminylglycine in the hydroxamate procedure was found to be 66 & 2 x low3 M with V,, of 37 f 1 pmoles min-l mg+ .

Transglutaminase displayed a typical protein absorption spec- trum in the ultraviolet range with a maximal extinction at 280 rnp and a ratio of absorbances at 260 and 280 rnp of 0.605. The Eiz calculated from the nitrogen content of the dry ash-free protein (16.1% nitrogen) was 15.8 i 0.6. That determined by comparison in the biuret procedure with ribonuclease and rabbit muscle aldolase was 15.5 f 0.4. The value of 15.8 was used to determine protein concentration in the studies reported here.

A sedimentation pattern of purified transglutaminase is illus- trated in Fig. 2. A small amount of a rapidly sedimentating component was noted in the early stages of each run, as may be observed in this figure. Four concentrations of enzyme, ranging from 1.65 to 6.6 mg of protein per ml (buffer conditions of Fig. 2), showed gradually increasing sedimentation coefficients with decreasing protein concentration. Linear extrapolation to zero protein concentration yielded an s$,~ of 5.40 x 10-13. Diffusion measurements made by the procedure of Koike, Reed, and Car- roll (19) in the Spinco model E ultracentrifuge at enzyme concen- trations of 0.66 and 0.435% (buffer conditions of Fig. 2) and at 3.8” and 5”, respectively, showed Dz~,~ values of 6.73 and 6.78 X l(r cm2 set+, respectively; those made by the procedure of

FIG. 2. Schlieren diagrams of an ultracentrifuge run of purified transglutaminase in a Kel-F analytical cell at 5.9”. The protein concentration was 1% in 5 mM Tris-chloride buffer with 2 mM EDTA and 0.2 M NaCl, pH 7.5. The photographs were taken at 17, 49, 81, 113, 145, and 177 min after attaining 59,780 rpm. Sedimentation proceded from left to right.

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Issue of December 10, 1966 J. E. Folk and P. W. Cole 5521

Longsworth (20) at 3.7” in the Aminco model B electrophoresis apparatus at an enzyme concentration of 0.435% (buffer condi- tions of Fig. 2) showed a D2s,w of 6.44 X lo-’ cm2 see-I. A small amount of heterogeneity was evident from the fringe distribution in the diffusion runs. This may correspond to and can be ac- counted for by the small amount of heavy component observed during sedimentation. A molecular weight value of 76,900, with an estimated maximum error of 5,000, was caluclated from the Dissw obtained by the procedure of Longsworth and the above s&+ and by the use of a partial specific volume of 0.734 cm3 per g estimated from the amino acid data reported in Table II.

Transglutaminase was examined by sedimentation equilibrium with the use of the meniscus depletion method of Yphantis (21). Essentially the same results were obtained with the enzyme puri- fied as described above, and with an enzyme fraction of highest specific activity from a Sephadex G-200 gel filtration (peak frac- tion of Sephadex G-200 run, Fig. 3). Runs were performed in the Spinco model E ultracentrifuge at 17,250 and 24,630 rpm in the buffered salt solution described in Fig. 2 and at protein con- centrations between 0.01 and 0.03%. Some upward curvature was observed in the In C with respect to x2 plots. The weight average molecular weights for the whole cell, determined by the use of the partial specific volume of 0.734 cm3 per g, ranged from 86,000 to 94,000.

The molecular weight values calculated from the data obtained in the sedimentation-diffusion runs and that obtained by sedi- mentation equilibrium are a’ some variance. 14C-Iodoacetamide incorporation studies detailed later in this paper define the equivalent weight of trans;lutaminase as 90,000 f 10,000, on the assumption that the enzyme is fully active. On the basis of these results, a molecular weight of 90,000 is accepted as a work-

TABLE II

Amino acid composition of transglutaminase The values are those corrected for destruction of serine, threo-

nine, and methionine and for rate of release of valine and isoleu- tine from 24., 48-, and go-hour hydrolysates. The value for amide nitrogen is that obtained by extrapolation to zero time; that for half-cystine is the go-hour value. Tryptophan was determined spectrophotometrically on unhydrolyxed samples. The data have been normalized to the molecular weight of 90,000.

Amino acid Amino acid residues per 90,000

Aspartic acid. ..................... Threonine ....................... Serine .... ......................... Glutamic acid. ..................... Proline ............................ Glycine ............................ Alanine ........................... Half-cystine .................... Valine ............................. Methionine ......................... Isoleucine ..................... Leucine ........................ Tyrosine. ......................... Phenylalanine ...................... Lysine ............................ Histidine ........................ Arginine .......................... Tryptophan ...................... Amide nitrogen. ...................

83.2 39.8 53.9 93.2 40.0 60.1 52.4 17.4 63.3 15.7 37.4 79.1 29.2 27.0 34.4 15.0 46.4 17.1

(81.8)

0.8

0.1

O- i2

-J 20 40

FRACTION NUMBER

60

FIG. 3. Gel filtration of transglutaminase on Sephadex G-100 (---) and Sephadex G-200 containing 10% cellulose (- - -) at 4”. Two-milliliter samples containing 4 mg of enzyme were applied to columns, 1 X 50 cm, of the gels which had been equilibrated with 5 mM Tris-chloride buffer containing 2 mM EDTA and 0.2 M NaCl, pH 7.5. Protein was eluted with the same buffer. Fractions of 0.65 ml were collected at flow rates of 20 and 12 ml per hour for Sephadex G-100 and G-200, respectively.

ing value. Further studies are underway to define this parame- ter more closely.

A single peak of enzymatic activity, which corresponded within limits of the method to the single protein peak, was observed when transglutaminase was subjected to sucrose density gradient centrifugation (22). The enzyme did not move out of the sample gel on disc electrophoresis in polyacrylamide gel at pH 8.6 (23). When large samples (0.1 to 0.2 mg) of enzyme were subjected to this procedure, two or three faint bands were observed on the running gel, indicating minor protein impurities.

In Fig. 3 are illustrated the results of gel filtration of samples of the enzyme on Sephadex G-100 and G-200. The shape of the protein peaks observed in these two Sephadex runs and the spe- cific activities in the fractions across these peaks is indicative of no significant heterogeneity. Minor ultraviolet-absorbing im- purity in the first few fractions of the peak in each run is, how- ever, suggested by the lower specific enzymatic activities of these fractions.

The results of the amino acid analyses of transglutaminase are summarized in Table II. The data have been normalized to the molecular weight of 90,000. Cystine was found in acid hydroly- sates of transglutaminase. The value for this amino acid in- creased 15% between 24 and 90 hours of hydrolysis with a value of 17.4 half-cystines per mole at 90 hours. Two separate samples of enzyme were oxidized and analyzed for cysteic acid. Values of 17.9 and 18.2 moles per mole of enzyme were obtained. Spec- trophotometric titration of transglutaminase thiol groups with

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5522 Mechanism of Transglutaminase Action. I Vol. 241, No. 33

1 OS9 4 I s 0.8 --I \ f 0.7 - \ cu \ s 0.6 - \

2 \ zo.5- \ =:

b z 0.4 - \

\ 0.3 - !

- 100

- 90 it =:

-80 2 +

-70;

5 -60 r

z

-50 z

-40"

-30 .!

0,2 \

- .I

20 \

0.1 ‘L 1

IO

0 0

‘Y+- k-0 0.05 0.1 0.15

ml PMB

FIG. 4. Spectrophotometric titration of transglutaminase with CMB and effect of CMB on enzyme activity. The CMB solution, 1.136 X 1OV M, was added stepwise to 1 ml of 9.06 PM enzyme in 0.1 Tris-chloride buffer, pH 7.0. Appropriate corrections were made for the separate absorptions of the enzyme and the reagent. Samples of enzyme used for activity measurements were removed from separate reaction mixtures which were prepared in the same manner and at the same time as that titrated spectrophoto- metrically. 0, assayed by the procedure described under “Methods.” A, assayed by the procedure described under “Methods,” except that the 10 rnM GSH was replaced by 1 mM EDTA in assay solutions.

TABLE III Reaction of NEM with transglutaminase

Enzyme was incubated with i4C-NEM (0.6 mC per mmole) in 0.2 M Tris-acetate buffer, 5 mM CaClt at pH 6.8 and 25”. The enzyme and 14C-NEM concentrations (micromolar), respectively, were: Experiment I, 5.82 and 510; in Experiment II, 4.54 and 780; and in Experiment 111, 4.54 and 3060.

Experiment ‘“C-NEM

bound per 90,000 g of

enzyme I

moles

I. 3-min incubation A. B. Without CaCL. C. With 33 mM CBZ-L-glu.

taminylglycine Il. 2-min incubation

A. B. Without CaCh. C. With 33 mM CBZ-L-glu

taminylglycine III. 30-min incubation,

3.6 2.8

3.0

2.3 1.9

2.0 6.3

nhibition of enzymatic

activity

%

91 0

25

50 0

0 100

CMB in two separate experiments with native enzyme and in four separate experiments with enzyme in 8 M urea showed the following values for moles of -SH per 90,000 g of transglutamin- ase: native, 17.0, 15.9; enzyme in urea, 16.1, 15.9, 16.6, 16.2. A CMB titration curve for the native enzyme is shown in Fig. 4.

Estimation of enzyme thiol groups by means of the colori- metric DTNB procedure showed the following values for moles of -SH per 90,000 g of enzyme: native, 17.4, 17.4, 17.1; enzyme in 8 M urea, 16.3, 16.4; enzyme in 5 M guanidine, 16.4, 17.2.

Estimation of enzyme -SH groups by their capacity to bind i4C-NEM gave consistently lower and more variable values. Thus, four separate experiments with enzyme in 8 M urea gave the following equivalents of i4C-NEM bound per mole of enzyme: 10.8 (5.8 PM enzyme and 1.6 mM NEM for 30 min), 9.5 (5.8 PM

enzyme and 1.5 mM NEM for 60 min), 9.1 (4.5 PM enzyme and 1.5 mM NEM for 60 min), 8.9 (4.5 PM enzyme and 1.6 mM NEM for 30 min). With the native enzyme, the highest level of NEM bound was 6.3 eq (Table III, Experiment III). This level was not increased by longer incubation of enzyme with NEM.

Effects of Thiol Reagents on Transglutaminase Activity

CMB-A CMB titration curve for native transglutaminase is presented in Fig. 4. The degree of reversible and irreversible inhibition of enzyme activity by CMB is also illustrated in this figure. After reaction of approximately five enzyme -SH groups with CMB (0.04 ml of CMB) only 5 to 10% of the enzyme activity remained. Essentially full activity was restored by including GSH in the assay mixtures. Reaction of the first 7.5 to 8 moles of -SH with CMB (0.06 ml) was practically instan- taneous; i.e. maximum absorbance was reached by the time that the solutions were mixed and absorbance was measured. Upon further addition of CMB, however, the length of time required for maximum absorbance to be reached increased pronouncedly, suggesting that the enzyme was undergoing conformational changes which need to precede reaction with CMB (7). At the same time, significant irreversible loss in enzyme activity was noted. Incubation of the enzyme, which had been allowed to react with 0.13 ml of CMB, with 0.01 M GSH for 30 or 60 min preceding the assay resulted in no further increase in activity over that recorded in Fig. 4. It would appear that these slowly occurring changes in the enzyme are not reversed by GSH, al- though this reducing agent readily restores activity at the early stages of CMB inhibition.

DTNB-The rate of reaction of DTNB with native trans- glutaminase was similar to that observed with CMB. The first four to five -SH groups reacted with reagent within 1 min after addition of reagent, and the rate of reaction became progressively slower requiring, after about 10 groups had reacted, over3 min for color formation equivalent to one -SH group. Between 70 and 85 min were required for the full reaction to occur. Addi- tion of DTNB to enzyme solution at the level of 1 mole per mole of enzyme resulted in loss of 85% of the initial transglutaminase activity. This inhibition was not reversed by GSH (0.1 M GSH, 30 min at pH 8).

NEM-The reactivity of 14C-NEM with native transglutamin- ase and the effects on enzymatic activity are summarized in Table III. Under the conditions used, 2 or 3 moles of r4C-NEM were incorporated per mole of enzyme in the absence of Ca++ without loss in enzymatic activity (Experiments IB and IIB). In the presence of Ca++, which is essential for enzyme activity, the increase in r4C-NEM incorporated (Table III: A - B, Ex-

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periments I and II) was roughly proportional to the loss in activity. Substrate protected against this loss in activity in agreement with previous findings (5). Substrate also appeared to cause a concomitant proportional reduction in the amount of i4C-NEM bound by the Ca++-activated enzyme over that bound in the absence of the metal ion.

Iodoacetamide-In Table IV are summarized the results of two separate experiments on the inactivation of transglutaminase by low levels of W-iodoacetamide. At pH 6.8, the loss in enzymatic activity was accompanied by a concomitant pro- portional incorporation of r4C-carbamidomethyl up to the level of approximately 1 male/90,000 g of enzyme (Experiments IA and IIA). The values for equivalent weight of enzyme calcu- lated on the basis of inhibition of activity with respect to 14C- carbamidomethyl bound are recorded in this table. In calcu- lating these values, it was assumed that iodoacetamide reacts with a single enzyme group and that the enzyme is fully active.

The very reactive nature of the enzyme group involved is evident from the rapid, essentially complete reaction with iodo- acetamide at concentration ratios of iodoacetamide to enzyme of less than 1 (Experiments IA and IIA). Effective protection against enzyme inactivation and against carbamidomethyl incorporation by substrate is evident (Experiments IB and IIB). In the absence of the activating cation, Ca++, and at the low pH, the enzyme was not labeled and no activity was lost (Ex- periments IC and IIC). At pH values of 9.3 and 9.5, some carbamidomethyl was bound in the absence of Ca++. Activity, however, was lost only in the presence of this metal and with the uptake of 1 additional eq of the labeled group (Experiments I, D and E, and II, D and E).

Two samples of transglutaminase which had been labeled with 1 eq of carbamidomethyl were reacted with DTNB. In this case, 15.9 and 16.0 -SH groups per mole of enzyme were found, as compared with 17.1 to 17.4 determined per mole of the native enzyme.

Identi$cation of r4C-Carbamidomethyl Amino Acid Residue in i4C-Iodoacetamide-inactivated Transglutaminase

r4C-Carbamidomethyl-labeled transglutaminase was prepared essentially as outlined in Table IV (13 MM enzyme, 103 NM 14C- iodoacetamide (1 mC per mmole), 5 mM CaC12, 2 min in 0.1 M Tris-acetate buffer at pH 6.8). The reaction mixture (30 ml) was passed rapidly through a column, 1.8 x 90 cm, of Sephadex G-25 (coarse) which was equilibrated with 5 mM Tris-acetate buffer, pH 7.5, containing 2 mu EDTA. The reagent-free pro- tein solution was brought to 70% saturation with solid (NHS2- SOc (47 g/100 ml); the protein was collected by centrifugation, dissolved in a small volume of distilled water, dialyzed free of salts with respect to distilled water, and lyophilized. The yield of protein on a dry ash-free basis was 30 mg (86% recovery). It contained 1 eq of 14C-carbamidomethyl and displayed no enzyme activity.

Approximately 1-mg samples of this labeled protein were hydrolyzed with HCl for 18 hours. The HCI was removed and a hydrolysate was applied as two equal portions to paper for chromatography or electrophoresis. One-half of the hydrolysate was oxidized in situ on the paper by the use of 30% Hz02 and 0.02% ammonium molybdate. A marker of S-carboxymethyl- cysteine was oxidized in the same manner. The chromato- graphic and electrophoretic locations of S-carboxymethylcysteine

TABLE IV

Reaction of iodoacetamide with transglutaminase

In Experiment I, 5.28 PM enzyme, and in Experiment II, 5.45 PM enzyme were incubated at 25” with the recorded concentra- tions of l-14C-iodoacetamide (Experiment I, 0.8 mC per mmole; Experiment II, 1 mC per mmole) in 0.1 M Tris-acetate buffer, 5 mM CaC12. Following incubation, the protein was freed of reagents by gel filtration as described in “Methods” for NEM and aliquots of the resulting enzyme solutions were assayed for enzymatic activity, protein, and radioactivity.

Experiment ‘GIodo- nhibition

acetamidr of concentra uymatic

tion activity

I. A. 2 min at pH 6.8. 103.0 10.3

3.4 2.6 1.7

B. 2 min at pH 6.8 with 20 mM CBZ-L-gluta- minylglycine 3.4

C. 2 min at pH 6.8 without CaC12 10.3

D. 3 min at pH 9.5. . 103.0 E. 3 min at pH 9.5 with-

out CaCL.. 103.0 II. A. 2 min at pH 6.8. 125.0

12.5 4.2 3.1 2.1

B. 2 minat pH6.8 with68 mM CBZ-L-gluta- minylglycine 4.2

C. 2min at pH 6.8 with- out CaCh. 12.5

D. 3min at pH 9.3. 125.0 E. 3 min at pH 9.3 with-

out CaClz. 125.0

-

1‘

n c 1

IC-Carba. mido-

methyl in- orporatec ?er 90,000

g of tXlZYIlW

% moles

100 1.05 92 0.83 61 0.57 43 0.44 34 0.32

>l

0 100

0 100

95 75 53 34

2

0 100

0

0

0 2.70

1.85 1.00 0.87 0.73 0.52 0.39

0.03

0 2.50

1.49 -

:alculated quivalent weight of enzyme

85,700 99,800 96,300 89,000 95,600

90,000 98,300 92,500 91,700 78,500

TABLE V

Chromatographic and electrophoretic location of S-carboxymethyl- cgsteine and oxidization product

system

1-butanol-acetic acid-Hz0 (4: 1:5) Ethanol-HSO-NHdOH (0.88 M) (90:5:5). Phenol-NaCN. 4y0 formic acid, pH 1.8, with 23.5 X 104 cm

sheets of Whatman No. 3MM for 135 min at 4800 volts...........................

RF values

SCarboxy- methyl- cystenle

Oxidation Droduct of

0.24 0.12 0.04 0.01 0.16 0.05

23” 120

and its oxidization product are given in Table V. In each sys- ~1 Distance traveled in centimeters from origin.

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5524 Mechctnim of Transglutaminase Action. I Vol. 241, No. 23

tern, the hydrolysate and the oxidized hydrolysate showed a single radioactive area corresponding to X-carboxymethylcysteine and its oxidization product, respectively.

A portion of hydrolysate of the labeled enzyme equivalent to 0.016 pmole of protein (as determined from the observed values for glutamic acid, glycine, and alanine), and containing approxi- mately 75% of the radioactivity originally present in an equiva- lent amount of unhydrolyzed protein, was subjected to analysis in an amino acid analyzer equipped with a scintillation-counting device (16). A single ninhydrin-positive, radioactive peak was observed immediately preceding aspartic acid and corresponding in position to S-carboxymethylcysteine. This amino acid (approximately 0.01 pmole on ninhydrin basis, 0.01 pmole on basis of radioactivity) contained 89 % of the radioactivity applied to the analyzer. A small amount of radioactivity (11%) was unretarded by the ion exchange column.

DISCUSSlON

A simplified schematic mechanism of transglutaminase action has been postulated on the basis of the findings of kinetic and inhibitor studies (5). In order to obtain direct chemical evidence for the enzyme groups involved in transglutaminase catalysis and to determine some characteristics of their reactivity, chemical and physical environment, and spatial and functional relation- ships, it was first necessary to obtain the enzyme in an essentially pure form. Thus, the present report is devoted in part to de- tailing a purification of transglutaminase. It has been possible to obtain the enzyme in high yield from a rich source and to accomplish an approximately 230-fold purification.

The molecular weight of 90,000, accepted at present as a working value, is based on three measurements which agree within about 15%. It is possible, because of the very reactive nature of the essential enzyme group and the lability of the enzyme to heavy metals (2), that small portions of the enzyme protein are inactivated during purification and, thus, high values for equivalent weight were obtained. Approximately 15 prep- arations of enzyme, however, have shown essentially the same specific activity, 25 + 0.5. The reasonable agreement of the molecular weight values determined by physical procedures and the equivalent weight obtained from 14C-carbamidomethyl incorporation suggests that transglutaminase has one active site per molecule. This suggestion is strengthened by the observed efficient protection by substrate against inactivation and incorporation with 14C-iodoacetamide and 14C-NEM, and from the fact that inactivation by these reagents under the present experimental conditions occurs only in the presence of Ca++ which is essential for enzyme activity.

Identification of cysteine as the amino acid carbamidomethyl- ated by iodoacetamide is in agreement with a postulated thiol ester intermediate in transglutaminase catalysis (5). The kinetic data and earlier inhibitor studies have suggested the participation of an enzyme -SH of apparent low pK. The rapid and efficient reaction of iodoacetamide with a single enzyme cysteine -SH group at pH 6.8 resulting in complete loss in activity would seem to implicate this particular -SH per se in the catalytic mechanism. The fact that, in the absence of Ca++, even at pH 9.5, the essential -SH group does not react with iodoacetamide would show that Ca+f functions in some manner to expose or condition this group, rather than simply to affect a proper orientation of the already reactive and exposed -SH group with other essential groups in the enzyme active center.

Significant irreversible losses in transglutaminase activity occur as the result of reaction with CMB only after the rapidly re- acting -SH groups have been bound (Fig. 4). These losses are probably the result of irreversible conformational changes in the molecule induced by the reagent. Whether the reversible losses in activity which occur at low levels of CMB (Fig. 4) and the irreversible loss in activity caused by low levels of DTNB re- sult from reactions of these reagents with the “active center” -SH is not known. Evidence concerning the nature of these inhibitions should contribute further to our knowledge of the availability and structural environment of this unique enzyme -SH group.

Transglutaminase shows no unusual features in its amino acid composition. Estimation of -SH groups by CMB titration and by reaction with DTNB shows that essentially all of the cysteic acid formed by oxidization of transglutaminase (18 moles per mole) can be accounted for as cysteine in the native protein (16 to 17 moles per mole). It seems extraordinary that cysteine is oxidized quantitatively to cystine (17.4 moles of 3 cystine per mole) during acid hydrolysis. In this regard, significant quanti- ties of cystine have been found in acid hydrolysates of rabbit muscle aldolase which contains cysteine but is believed to con- tain no cystine (24). The lower estimate of transglutaminase -SH groups derived from binding of r4C-NEM may be the re- sult of the unaviailability of certain -SH groups for reaction with this reagent, even in 8 M urea. Indeed, NEM is less reactive than CMB or DTNB with the native enzyme. These data leave some doubts as to the actual cystine-cysteine composition of transglutaminase. It seems likely, however, that the enzyme contains 16 to 18 cysteine residues and few, if any, disulfide bonds. Further studies are underway to establish this important structural feature of the molecule.

Acknourledgments-Our thanks are due Mr. E. R. Mitchell for the ultracentrifugal analyses, Dr. K. A. Piez for the sedimenta- tion equilibrium analyses, Mr. H. L. Wolff and Dr. W. R. Carroll for the diffusion analyses, and Dr. E. J. Miller and Miss Re- becca Butler for the amino acid analyses.

1.

2.

3.

SARKAR, N. K., CLARKE, D. D., AND WAELSCH, H., &o&em. Biophys. Acta, 261, 451 (1957).

CLARKE, D. D., MYCEK, M. J., NEIDLE, A., AND WAELSCH, H., Arch. Biochem. Biophys., 79, 338 (1959).

MYCEK, M. J., AND WAELSCH, H., J. Biol. Chem., 236, 3513 (1960).

4. FOLK, J. E., AND COLE, P. W., J. Biol. Chem., 240,295l (1965). 5. FOLK, J. E., AND COLE, P. W., Biochem. Biophys. Acta, 122, 244

(1966). 6. FOLK, J. E., AND COLE, P. W., J. Biol. Chem., 241, 3228 (1966). 7. BOYER, P. D., J. Am. Chem. Xoc., 76, 4331 (1954). 8. COLMAN. R. F.. AND BLACK, S., J. Biol. Chem., 240, 1796

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(1965): GREGORY, J. D., J. Am. Chem. Sot., 77, 3922 (1955). LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem., 169, 21

(1944). BRAY, G. A., Anal. Biochem., 1, 279 (1960).

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17. ELLMAN, G. L., Arch. Biochem. Biophys., 82, 70 (1959). 22. MARTIN, R. C., AND AMES, B. N., J. Biol. Chem., 236, 1372 18. LOWRY, 0. H., ROSEBROUGH, M. J., FARR, A. L., AND RAND- (1961).

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Biopiys. kes. Gokmun.,“l, 16 (1962). 20. LONGSWORTH, L. G., J. Am. Chem. Sot., 74, 4155 (1952). 21. YPHANTIS, D. A., Biochemistry, 3, 297 (1964).

24. RUTTER, W: J., in P. D. BOYER, H. LARDY, AND K. MYRBHCK (Editors), The enzymes, VoZ. V., Ed. 2, Academic Press, Inc., New York, 1961, p. 341.

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J. E. Folk and P. W. ColeACTIVITY

IDENTIFICATION OF A FUNCTIONAL CYSTEINE ESSENTIAL FORPURIFICATION AND PROPERTIES OF THE ENZYME:

Mechanism of Action of Guinea Pig Liver Transglutaminase: I.

1966, 241:5518-5525.J. Biol. Chem. 

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