ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 1, November, pp. 49-58,1978
Human
Epidermal
MARY
Transglutaminase: Stimulation by Trypsin, Solvents, and Chaotropic Salts’ FOSTER
Duke University
PLISHKER,* JUDITH M. THORPE, LOWELL A. GOLDSMITH3 Medical
Center, Durham,
North
Carolina
Organic
AND
27710
Received April 5, 1978; revised May 30, 1978 The activity of pure human epidermal transglutaminase was enhanced 3- to lo-fold by various treatments. Incubation with trypsin caused a time-dependent enhancement in activity, up to 3 times the initial activity, with no apparent change in electrophoretic mobility as detected by disc and sodium dodecyl sulfate electrophoresis, and with no apparent change in immunological properties. This enhancement was specific for trypsin among the several enzymes tested. Preincubation of transglutaminase with 0.1 to 2.0 M potassium thiocyanate or potassium iodide, and with 10 to 50% solutions of alcohols and other organic solvents caused a time-dependent enhancement of activity up to lo-fold over control. The presence of calcium was required for the observed enhancement. Kinetic studies suggest that the K,,, values of the substrates putrescine and casein determined for the native enzyme are similar to those for the stimulated forms of the enzyme. These in vitro methods of altering enzyme activity may be indications of potential in vivo controls of transglutaminase activity.
Human epidermal transglutaminase is one of the calcium requiring enzymes which catalyze the acyl transfer of a y carboxy group derived from a peptide-bound glutamine to a primary amino receptor such as the e-amino group of a peptide-bound lyThe formation of this E-(ysine. glutamyl)lysine bond can create a covalent protease-resistant crosslink between proteins. The general features of transglutaminases and E-( y-glutamyl)lysine bonds have been recently reviewed (1). Previous work by Ogawa and Goldsmith described the purification of human epidermal transglutaminase and some of its prop-
erties (2); in those studies the activity of pure epidermal transglutaminase was enhanced by treatment of the enzyme with dimethylsulfoxide or by heating to 56°C. We now describe enhancement of enzyme activity after limited trypsin treatment, and after incubation in chaotropic salts, alcohols, and other organic solvents. Examination of the activated forms of this enzyme suggests possible mechanisms for the observed stimulation, and gives some insight into possible cellular control mechanisms for transglutaminase activity.
’ Presented in part at the American Society of Biological Chemists Meeting April 1977, Chicago, Ill., and The Society for Investigative Dermatology, April 30, 1977, Washington, D.C. Supported in part by Grants AM-07093 and AM-17253 from the National Institutes of Health. Lowell A. Goldsmith is the recipient of a Research Career and Development Award, Grant AM-09008 from the National Institutes of Health. ’ Present address: Biochemistry Department, M.D. Anderson Hospital, Houston, Texas 77030. 3 To whom all correspondence should be sent.
Materials
EXPERIMENTAL
PROCEDURES
[1,4-Y!]Putrescine and aquasol were from New England Nuclear (Boston, Mass.). Putrescine, bovine serum albumin, ovalbumin, chymotrypsinogen, cytochrome c, chymotrypsin, pronase, trypsin (type XIdiphenylcarbamyl chloride treated), ribonuclease A, alkaline phosphatase, neuraminidase, phospholipase A, papain, plasmin, and soybean trypsin inhibitor were obtained from Sigma (St. Louis, MO.). Factor XIII was a gift of P. McKee. Alkyl agarose columns were obtained from Miles Laboratories, Inc. (Elkhart, Ind.). Immunodiffusion plates were obtained from Hyland 49 0003.9861/78/1911-0049$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
50
PLISHKER,
THORPE,
(Costa Mesa, Calif.). Demineralized water was used throughout, and salts and other reagents were of the highest grades available.
Methods Enzyme assay. Epidermal transglutaminase was purified from human stratum corneum as previously described (2). The radioactive assay for transglutaminase was based on the incorporation of [‘4C]putrestine into casein at pH 9.5 by the method previously described (2, 3). An enzyme unit is defined as the amount of enzyme that catalyzes the incorporation of 1 nmol of putrescine into casein in 30 min. The specific activity of purified enzyme was approximately 350 units/mg of protein, which is in good agreement with the previous report (4). All activities and velocities are the mean values of duplicate determinations using the purified enzyme preparation. Variation between duplicate samples was less than 10%. Trypsin enhancement. Transglutaminase, 10 to 50 $, in 10 mu Tris (4) (pH 7.5), containing 1 mu EDTA, was incubated with 10 mM CaCh and 5-300 pg/ml of trypsin (dissolved in 0.001 M HCl) in a fmal volume of 25-100 d. Preincubations were performed at 37°C in a shaking water bath for the time periods described in figure legends. The preincubation was followed by the addition of 0.5 ml of complete reaction mixture and the incubation was continued as described above. Appropriate controls were performed. Effect of other enzymes, chaotropic salts and organic solvents: enzymes. Samples (50 ~1) of transglutaminase in 10 mM Tris (pH 7.5), 1 mM EDTA, and 10 mM CaClz were preincubated in a final volume of 100 ~1 at 37°C for 20 min in the presence of 125 pg/ml chymotrypsin, pronase, ribonuclease, neuraminidase, or phospholipase A; 10-1500 gg/ml plasmin, 0.01 unit of thrombin; or 50 pg/ml of papain in 10 mM Tris, 1 mM EDTA, 5 mM cysteine, and 0.6 mM 2-mercaptoethanol. Similar preincubations were performed for 0.5 to 24 h in the presence of 500 pg/ml alkaline phosphatase. Control samples were treated identically without added enzyme. Each preincubation mixture was assayed for transglutaminase activity as described above. Chaotropic salts. Twenty-five microliters of transglutaminase (90 units/ml) was preincubated in 10 mM Tris (pH 7.5), 1 mM EDTA, 10 mM CaClz with concentrations from 0.1 to 2.0 M KSCN, KI, KCl, or NaCl in a fmal volume of 50 al at 37°C in a shaking water bath. After the preincubation period, 0.5 ml of complete reaction mixture was added, and the assay continued as described above. Control samples, containing no chaotropic salts, were treated identically. In the time course studies, aliquots of the preincubation mixtures were removed at specific time intervals and added to 0.5 ml of reaction mixture and assayed as described. A control sample, containing no salt, was assayed simultaneously. Organic solvents. Several alcohols and organic re-
AND
GOLDSMITH
agents were examined for their effect on transglutaminase activity. Transglutaminase (50 fi to 0.1 ml) (20-40 units/ml) was preincubated in 10 mM Tris (pH 7.5), 1 mu EDTA, 10 mu CaCb, containing from 10 to 50% solvent in a final volume of 100-200 pl for 20 min at 37°C in a shaking water bath. Controls were preincubated under identical conditions without added solvent. After the preincubation period, 0.5 ml of reaction mixture was added and the assay continued as described above. The effect of solvents on the assay mixture itself was ascertained by adding the solvent to the assay mixture without preincubation and beginning the assay with the addition of enzyme. Gel electrophoresis. Disc electrophoresis on 7.5% acrylamide gels was performed at pH 9.5 by the method of Davis (5). After electrophoresis the unstained gels containing enzyme samples were sliced into segments and assayed for transglutaminase activity (2). To determine whether electrophoresis stimulated transglutaminase, the enzyme was electrophoresed on short (15 mm x 5 mm) 7.5% disc gels with the same buffers used for longer (6 cm) gels, with the lower end of the electrophoresis tube inserted into dialysis tubing. Gels were pre-electrophoresed, dialysis tubing was attached, and electrophoresis was performed for 18 h at 4°C (0.4 mA per tube) so that the transglutaminase was eluted from the gel into the dialysis tubing. Contents of the dialysis tubing were assayed for.transglutaminase activity and for the sensitivity of this electrophoresed enzyme to stimulation by ethanol as described above. Polyacrylamide gel electrophoresis in the presence of SDS4 was performed by the method of Neville (6) on 5% acrylamide gels in order to estimate the molecular weight and purity of the enzyme. Samples were incubated in 10% SDS with 0.05 M dithiothreitol and 0.1% pyronin Y for 10 min at 100°C before application to gels. Bovine serum albumin, ovalbumin, chymotrypsinogen, and cytochrome c were used as molecular weight standards. Gels were stained with Coomassie brilliant blue. Zmmunodiffusion. Gel double diffusion precipitation was performed as described by Ouchterlony (7) using the previously described antibodies (8). Alkyl agarose chromatography. Transglutaminase samples were dialyzed against 10 mru Tris-HCl (pH 7.5) containing 10 mM CaCl2, 0.15 M NaCl, and 1 mM EDTA (Buffer B) at 4°C. Columns (1 ml) of alkylagarose complexes were equilibrated with the same buffer. Samples were applied and the column washed sequentially with 10 mu Tris-HCl (pH 7.5) containing 10 mM CaC12, 1 mu EDTA, and increasing concentrations of NaCk (B) 150 mM NaCk (C) 0.5 M NaCI; (D) 2.0 M NaCI; and (E) 2.0 M NaCl with 50% ethylene glycol (v/v) (9). Fractions were collected and moni4 Abbreviations used: SDS, sodium dodecyl sulfate; Me&SO, dimethylsulfoxide.
EPIDERMAL
tored at 280 nm and transglutaminase activity determined. Protein determination. Protein was measured by the method of Lowry et al. (10) with bovine serum albumin as a standard. RESULTS
Stimulation
51
TRANSGLUTAMINASE
by Trypsin
There was an enhancement in activity after limited treatment with trypsin. As shown in Fig. 1, this stimulation was dependent on the concentration of trypsin between 5 and 100 pg/ml. At values between 100 and 200 Erg/ml, the stimulation appears to have reached a maximum, with stimulated activity 300% of control, and at higher trypsin concentrations losses in activity were observed. In the presence of 25 pg/ml or 50 pg/ml trypsin, stimulation continued for at least 1 h with a loss in activity found after 2 h. The presence of calcium during preincubation with trypsin was required for this enhancement, as indicated by the loss in activity observed when transglutaminase and trypsin were preincubated in the presence of excess EDTA (Fig. 1). Crude epidermal enzyme was less sensitive
to stimulation by trypsin, possibly because of trypsin substrates or inhibitors in the crude extract. Stimulation was observed only if transglutaminase and trypsin were preincubated before the addition of the complete reaction mixture. Simultaneous addition of reaction mixture and trypsin, followed by the usual 30 min of incubation, caused essentiahy no increase in activity over control values. The presence of soybean trypsin inhibitor (1:l with trypsin) prevented stimulation by trypsin. Characterization of trypsin-stimulated transglutaminase was attempted by several procedures. As shown in Fig. 2, the electrophoretic mobility of the trypsin-stimulated enzyme on 7.5% polyacrylamide gels was the same as that of the control. Electrophoresis of control and stimulated enzyme in
TRYPSIN ACTIVATED
-5:
FIG. 1. Enhancement of transglutaminase activity by trypsin. Samples of transglutaminase were preincubated with varying concentrations of trypsin for 20 mm as described under Methods. Samples of purified transglutaminase (0) and tissue extract (O), and tissue extract in the presence of 1 mM EDTA (no added calcium) (*) were examined. AU of these were compared with the control which contained 10 mM calcium. The preincubation of enzyme in excess EDTA was 76% of the calcium-containing control. Tissue extract was the concentrated supernatant from the 12,000g centrifugation of tissue which had been homogenized in 10 mM Tris, 1 mM EDTA, pH 7.5. The molar ratios of trypsin to transglutaminase varied from 31 (25 pg/ml trypsin) to 3O:l (250 pg/ml trypsin).
7
CONTROL
1 A I 20
40 DISTANCE (mm)
60
FIG. 2. Electrophoresis on 7.5% polyacrylamide gels. Samples (100 fl) of control and trypsin-treated transglutaminase were electrophoresed, and the unstained gels were eluted and assayed as described previously (2). The trypsin-treated sample was produced by preipcubation in the presence of 200 pgg/ml trypsin for 20 min at 37°C. This resulted in 2.1-fold enhancement in activity. Samples of trypsin alone produced no activity (cpm) in the gel.
52
PLISHKER,
THORPE,
the presence of SDS, as shown in Fig. 3, demonstrated that the mobility of the enzyme on the 5% polyacrylamide gel was unchanged, and that no new protein bands were apparent in the activated sample. To determine whether the electrophoresis could cause stimulation, transglutaminase activity was eluted by electrophoresis from a 7.5% disc gel into dialysis tubing. The enzyme recovered by this technique was enhanced 5-fold by preincubation in ethanol, as compared with 6.6-fold enhancement by ethanol of an aliquot of control enzyme which had not been electrophoresed. Thus, it appears unlikely that stimulation occurred during electrophoresis. The total activity recovered from the acrylamide gels was the same for the trypsin-activated and control samples; similar equal recoveries of heat-activated and control samples after electrophoresis were also reported (2). Since electrophoresis does not activate it is possible that activation may be reversed by electrophoresis. Immunologic properties of the trypsintreated enzyme were examined by immu-
FIG. 3. Electrophoresis on 7.5% polyacrylamide gel in sodium dodecyl sulfate. Samples (50 al) of control and trypsin-stimulated transglutaminase, and of trypsin were electrophoresed as described under Methods. The stimulation occurred after preincubation with 100 pg/ml trypsin resulting in activity which was 160% of the control. Pattern A is trypsin (TRY) alone, B is transglutaminase (TGL) alone, C is trypsin-stimulated enzyme (which still contains trypsin). Two adjacent wells were filled in A, B, and C. The last pattern (D) is a mixture of cytochrome c (CC), chymotrypsinogen (CHY), ovalbumin (OVA), and bovine serum albumin (BSA). The band at the top of B and C represents c 5 4
53
TRANSGLUTAMINASE
*-*\*
9
1000
>
900 800 700 600 500 400 300 200 100
*/*'
t
+ ,*’
,*'
I 160 TIME (mln)
05 10 15 20 SALT CONCENTRATION (M) FIG. 5. Effects of salts on transglutaminase. Fifty microliters of transglutaminase (100 units/ml) was incubated in 10 mu Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM CaCl2, containing increasing concentrations of KSCN (O), KC1 (W) or NaCl (0) in a final volume of 100 ~1 at 37°C for 20 min. Transglutaminase (25 4) in a fmal volume of 50 ~1 was treated identically with KSCN (0). Each sample was assayed by adding 0.5 ml of complete reaction mixture as described under Methods. Data obtained with KI are essentially identical to those for KSCN (0).
these conditions. Preincubation in KC1 and NaCl caused very little stimulation. There was no stimulation with any of these salts when the preincubation was done in the absence of calcium. The time course for stimulation of transglutaminase activity in the presence of 0.3 M KSCN is shown in Fig. 6. Activity continued to increase for at least 2 h under these preincubation conditions. As previously noted (2) incubation at 37°C in calcium caused a time related increase in activity. To determine whether stimulation occurred only during preincubation with the salts, or if the enhancement occurred after the addition of reaction mixture for assay, preincubations were performed at the same chaotropic salt concentrations in different final volumes, 50 and 100 ~1..The dilution with 0.5 ml of assay mixture resulted in different final (assay) salt concentrations for identical initial (preincubation) concentrations. As shown in Fig. 5, the stimulation for the 50 and 100 d preincubation volumes is essentially identical. Thus, the observed stimulation was apparently the result of the
200
240
FIG. 6. Time course of stimulation by KSCN. Samples (25 ~1) of transglutaminase (40 units/ml) were (pH 7.5), 1 mM EDTA, incubated in 10 mM Tris-HCl 10 mu CaClz, 0.3 M KSCN (*) in a final volume of 50 4. At the indicated time intervals samples were removed and assayed for transglutaminase activity as described in Methods. Aliquots from a control sample (0) were treated identically without added KSCN. V refers to the nanomoles of substrate incorporated.
reagent concentrations during the preincubation, not the actual assay. The effect of preincubation in trypsin (180 pg/ml) alone, 0.44 M KSCN alone, and in both trypsin and KSCN simultaneously showed the effect of KSCN and trypsin together was only slightly greater than the added effects of the two treatments done separately (unpublished observations).
Stimulation
by Organic Solvents
Preincubation of transglutaminase in the presence of several different alcohols or organic solvents also resulted in stimulation of activity. As shown in Fig. 7, stimulation occurred with all the solvents tested, even at concentrations of 50% solvent. In addition to those solvents listed in Fig. 7, similar stimulation was also seen with 10-50s t-butanol, chloroform, ethylene glycol, butanediol, ethanol, and acetone. None of the solvents tested, even at 50% solvent, caused any decrease in activity below the control level. Calcium was required in the preincubation procedure for stimulation to occur, as was previously shown for the enhancement by Me&GO (2). The effect of these organic solvents also apparently occurred during preincubation, as less enhancement in activity was observed if the solvent was added with the reaction mixture for assay. Treatment of Factor XIII
54
PLISHKER.
THORPE, AND GOLDSMITH
FIG. 7. Effect of organic solvents on transglutaminase. Transglutsminase (25 ~1) (60 units/ml) was incubated with lo-508 of each of the solvents shown in a final volume of 50 ah Me&O (0), methanol (O), n-propanol (W), isopropanol (Cl), n-butanol (A), and n-octanol (A). Control samples were treated similarly without added solvent. The reaction conditions are as described under Methods. Each sample was assayed by adding 0.5 ml of complete reaction mixture to the 50 al of preincubation mixture and the assay performed as described under Methods.
and the transglutaminase in a crude liver homogenate with MezSO or ethanol, under conditions which stimulated epidermal transglutaminase, caused a loss in the activity of these two transglutaminases. The reversibility of the observed activation was examined in the case of ethanol stimulated transglutaminase. A decrease in activity was observed in the ethanol stimulated sample after extensive dialysis. Treatment of this dialyzed sample with ethanol resulted in only a slight stimulation of activity. This overall decrease in activity and stimulation could represent a reversal in activation or an irreversible change in the enzyme associated with a loss of activity.
Alkyl Agarose Chromatography Epidermal transglutaminase was chromatographed on a series of alkyl agarose columns as shown in Fig. 8. Under the conditions used, the enzyme was retained on the hexyl (C,), octyl (C,), and decyl (C,,) agaroses. The enzyme was eluted with buffer containing 2.0 M NaCl and 50% ethylene glycol. The activity recovered from the Ck, G, and Cl0 columns was 200%, 330%, and 260% respectively, of the applied activity. This level of stimulation is similar to that observed with trypsin preincuba-
ALKYL CHAIN LENGTH
FRACTION
FIG. 8. Chromatography of transglutaminase on alkyl agarose. 0.2 ml samples of transglutaminase in 10 mu Tris-HCl, 1 mM EDTA, 0.15 NaCl were applied to the series of alkyl agarose columns (12 x 7 mm) equilibrated in the same buffer at 4°C: agarose hexyl (Cs), octyl (CS), (Co) and ethyl (CZ), butyl and decyl (C,,) agarose. The columns were eluted with 6 ml starting buffer (B), and 4 ml each of starting buffer containing 0.5 NaCl (C), 2.0 NaCl (D), and 2.0 NaCl and 50% ethylene glycol. Fractions (0.8 ml) were collected and monitored for ODZW;the transglutaminase activity in 0.2 ml of eluate was assayed in the standard reaction mixture. Activity is represented as cpm incorporated in 30 min (M). The total activity recovered (*) is shown on the right as a function of alkyl chain length. Transglutaminase was stimulated by ethylene glycol at both 37°C and at 4°C. Preincubation at 37°C in 10 Tris (pH 7.5), 10 mM CaC12,2.0 NaCl containing lO%, 20%, 30% and 50% ethylene glycol resulted in activities which were 200%,440%, 500%, and 540%, respectively, of the control. Preincubations at 4°C for 20 min in 10 mM Tris (pH 7.5), 10 CaCl2, 2.0 NaCl containing 12%, 25%, and 50% ethylene glycol resulted in activities which were lOO%,150%,and 2008, respectively, of the control. The total recovered activity from the CS,CS, Cl0 columns was greater than could be accounted for by ethylene glycol stimulation alone (see Results).
(Cd, M
M
M
M
mM
M
mM M
EPIDERMAL
TRANSGLUTAMINASE
tion. Incubation of transglutaminase in buffer containing 2.0 M NaCl and 50% ethylene glycol for 20 min (approximate time of exposure of chromatographed protein to this solvent) at 4’C resulted in a 2-fold enhancement in activity (legend to Fig. 8). If this 200% stimulation by ethylene glycol was subtracted from the activity recovered by elution with ethylene glycol from the CS, Cg, and Cl0 columns, there were still recoveries of total activity which were over 100%: 160%, 220%, and 160%, respectively, for these columns. Thus, the ethylene glycol stimulation did not account for all of the enhancement seen by this chromatographic procedure; the hydrophobic interaction of the enzyme with the alkyl agarose must have contributed to the observed enhancement. Kinetic Studies Samples of transglutaminase which had been stimulated by treatment with trypsin, KSCN, ethanol (as indicated in figure legends for Figs. 9 and lo), and control enzyme were examined kinetically. The reciprocal plots shown in Figs. 9 and 10 show the patterns obtained at saturated levels of either of the two substrates, casein (A) and putrescine (B). K,,, values were obtained for the control enzyme from secondary plots (intercepts and slopes of reciprocal plots versus l/fixed substrate) of additional data obtained for both substrates. The Km value for casein calculated from secondary plots was 0.230 mg/ml; the value obtained from the intersection of the lines in Fig. 9 was 0.133 mg/ml. The V,, values (cpm) were: 26,900 (control), 87,700 (trypsin), 238,000 (KSCN) and 154,000 (ethanol). The K,,, value for putrescine calculated from secondary plots was 0.110 mu; the Km values obtained as intercept values in the reciprocal plot (Fig. 10) were 0.114, 0.106, 0.110, and 0.123 mM for trypsin-, KSCN-, and ethanol-stimulated, and control enzymes, respectively. The V,,, values (cpm) for stimulated enzymes were: trypsin, 52,000; KSCN, 347,ooO;ethanol, 332,ooO;and control, 18,400. The Km values for both substrates for the control and stimulated forms of this enzyme are similar; the difference appears primarily in the V,,,,,.
55
3.5
30
25 rr $2 20 t> 15
IO
5
_1 20
40
60
l/A(mg/ml)-' 9. Kinetic study of stimulated transglutaminase at varying casein concentrations. Samples of transglutaminase control enzyme (*), trypsin-stimulated (*), ethanol-stimulated (O), and KSCN-stimulated (0) were assayed in 100 mu glycine-NaOH (pH 9.5), containing 10 mM CaClz, 5 mM dithiothreitol, 0.250 mru [Wlputrescine (4.0 mCi/mmol), and 9.4-337.0 ,ug/ml casein. Velocity (V) represents counts/mm incorporated and A represents casein concentration (mg/ml). Lines were calculated by a linear regression. Stimulated forms of the enzyme were prepared as described in Fig. 10. FIG.
DISCUSSION
The activity of human epidermal transglutaminase is enhanced severalfold by different types of treatments: 1) preincubation with trypsin, 2) preincubation in chaotropic salts, 3) preincubation in alcohols and other organic solvents, 4) chromatography on alkyl-agarose columns, and 5) heating to 56°C (2). Stimulation by these treatments is dependent on the time of preincubation, the concentration of stimulating agent and the presence of calcium. Epidermal transglutaminase, like the liver transglutaminase, does not require activation by proteolytic enzymes, as does plasma transglutaminase (Factor XIII);
56
PLISHKER,
IU
20
30
40
THORPE,
50
l/B mM-’ FIG. 10. Kinetic study of stimulated transglutaminase at varying putrescine concentrations. Samples of transglutaminase control enzyme (O), trypsin-stimulated (*), ethanol-stimulated (0), and KSCN-stimulated (Cl), were assayed in 110 mu glycine-NaOH (pH 9.5) containing 10 mM CaCh, 5 mu dithiothreitol, 0.8 mg/ml casein, and 0.024 to 0.924 mu [i4C]putrescine (4.0 mCi/mmol). Stimulated forms of transglutaminase were prepared by preincubation of the enzyme for 20 min at 37’C in 10 mM Tris (pH 7.5) containing 10 mu CaCh and no additions (control); I20 pg/ml trypsin (trypsin-stimulated); 1.0 M KSCN (KSCN-stimulated); or 3.5 M ethanol (ethanol-stimulated). Lines were calculated by linear regression.
however, epidermal transglutaminase is stimulated up to 3-fold by preincubation with trypsin. The stimulation of epidermal transglutaminase was apparently specific for trypsin under the conditions tested. Since high trypsin concentrations and long preincubation times cause a loss of transglutaminase activity, proteolytic degradation of transglutaminase during trypsin stimulation may have prevented the 10 fold stimulation attained with other pretreatments. Using several different procedures, attempts were made to characterize the trypsin-stimulated transglutaminase. There
AND
GOLDSMITH
was no apparent change in molecular weight of the stimulated enzyme when compared with the native enzyme by SDS electrophoresis, nor were there differences seen by disc electrophoresis, immunoelectrophoresis, or double immunodiffusion. Therefore, any structural changes which may have occurred during stimulation did not drastically alter the molecular weight or the immunologic properties of this enzyme. Epidermal transglutaminase was also stimulated by preincubation in the chaotropic salts KSCN and KI. Preincubation in 1.5 M KSCN at 37°C caused an increase in transglutaminase activity of up to lo-fold over control values (Fig. 5). Chaotropic ions have been shown to affect the solubilization of membrane bound proteins and the components of multicomponent enzymes, and to increase the water solubility of biopolymers and small organic molecules (11). The order of potency of the anions is SCN->II>Cl-, at high and low concentrations. Mechanisms for enhancement might be: 1) conformational change of the enzyme, possibly due to changes in the structure and lipophilicity of the aqueous solvent, 2) removal by solubilization of an inhibitor which may have been complexed with epidermal transglutaminase, or 3) disaggregation of multimers. The effect was apparently not on the solubility of either of the substrates, or of the product of this reaction, since the preincubation concentration of the salts rather than the final (assay) concentration determined the extent of activation. A possible model for the stimulation by chaotropic salts and trypsin might be the enzyme collagenase. A stimulatory effect by KSCN and trypsin without apparent molecular weight change has also been reported for human skin collagenase (12-14). Preincubation of this enzyme in any of several alcohols or organic solvents also resulted in concentration-dependent enhancement of activity (Fig. 7); there was no obvious relationship between the structure of the solvent and the extent of stimulation. The binding of proteins to immobilized straight chain hydrocarbons is primarily due to hydrophobic interactions (15). Chromatography of transglutaminase on alkyl
EPIDERMAL
TRANSGLUTAMINASE
agarose columns (Fig. 8) demonstrated the presence of a hydrophobic region on the molecule which interacted with hydrocarbon chains 6-10 carbons in length. The interaction was reversed by the presence of ethylene glycol and the enzyme eluted. The observed enhancement of recovered activity may be a result of this hydrophobic interaction enhancement since incubation with ethylene glycol did not account for the total observed increase in activity. In. an effort to understand the stimulatory effects of trypsin, KSCN, and alcohols, the stimulated forms of transglutaminase were examined kinetically (Figs. 9 and 10). The stimulated forms of the enzyme had similar apparent K,,, values for each substrate. The affinity of the enzyme for the substrates was apparently not drastically affected by stimulation. The V,,, did change, those for the stimulated enzymes being much higher than control values. The plot of data obtained at saturated putrescine (B) concentration and varying casein (A) concentration (Fig. 10) was an intersecting plot. The apparent effect of stimulation was similar to that seen by increasing the putrescine concentration of control enzyme. A similar effect (Fig. 10) was seen on casein concentration. Thus it appears that stimulation increased the effective concentration of both substrates, possibly by increasing the number of active sites or the effectiveness of the active sites on the enzyme. The former explanation is unlikely considering the molecular weight of the enzyme. A more likely explanation is an increase in the effectiveness of existing active sites, possibly by making the sites less hindered and more accessible to substrates possibly by changing the enzyme hydration (16, 17). A proteolytic cleavage, a conformational change,, or removal of a small complexed molecule might all result in increased exposure of active sites. A charged complexed molecule, such as an antizyme (18) or a phospholipid, might be expected to become separated from transglutaminase during disc electrophoresis. Enzyme which had been electrophoresed and recovered was still sensitive to stimulation by ethanol suggesting that removal of a charged molecule was not the mechanism of stimulation.
57
The most likely explanation for the observed stimulation appears to be a conformational change and would account for previous data concerning heat-stimulated and MeaLSO-stimulated enzyme (2) and the data presented here. The observed changes in activity are generally slow processes, occurring over a period from 5 min for MezSOstimulated enzyme (2), to 1 h (trypsin), and up to at least 3 h (KSCN) suggesting a conformational change. Antibody induced activation of liver (19) and cow epidermal (20) transglutaminase is reported and may be related to antibody induced conformational changes (21). Previous studies (22) have suggested that activation of other enzymes, such as plasminogen, factor XIII, and complement factor Cl, occurs by conformational transitions induced by proteolytic enzymes rather than by peptide bond cleavage and might explain the lack of a molecular weight change with trypsin activation. The fact that the activity of this enzyme can be enhanced up to lo-fold by treatments presented here may be of importance in understanding the physiological controls of this enzyme. ACKNOWLEDGMENTS The authors appreciate preliminary experiments by Dr. Hideoki Ogawa on trypsin activation and the secretarial assistance of Ms. Susan Boos. This is publication number 34 of the dermatological research laboratories, Duke University Medical Center. REFERENCES 1. FOLK, J. E., AND
FINLAYSON, J. S. (1977) Adv. Protein Chem. 31, I-133. 2. OGAWA, H., AND GOLDSMITH, L. A. (1976) J. Biol. Chem. 251, 7281-7266. 3. GOLDSMITH, L. A., AND MARTIN, C. M. (1975) J. Invest. Dermatol. 64, 316-321. 4. OGAWA, H., AND GOLDSMITH, L. A. (1977) in Biochemistry of Cutaneous Epidermal Differentiation (Seiji, M., and Bernstein, I. M., eds.), pp. 419-432, University of Tokyo Press, Tokyo. 5. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 6. NEVILLE, D. M., JR. (1971) J. Biol. Chem. 246, 6328-6334. 7. OUCHTERLONY, 0. (1958) Prog. Allergy, 5, 1-7. 8. OGAWA, H., AND GOLDSMITH, L. A. (1977) J. Invest. Dermatol. 66.32-35. 9. DAVEY,
M. W., SULKOWKI,
E., AND CARTER,
W.
A. (1976) J. Biol. Chem. 251, 7620-7625. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
58
PLISHKER,
THORPE,
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. HATEFI, Y., AND HANSTEIN, W. G. (1969) Proc.
Nut. Acad. Sci. USA 62,1129-1136. 12. SELLERS, A., CARTWRIGHT, E., MURPHY, G., AND REYNOLDS, J. d. (1977) Biochem. J. 163, 303-307. 13. STRICKLIN, G. P., BAUER, E. A., JEFFREY, J. J., AND EISEN, A. Z. (1977) Biochemistry 16, 1607-1615. 14. ABE, S., AND NAGAI, Y. (1972) Biochim. Biophys. Acta 278, 125-132. 15. SHALTIEL, S. (1974) in Methods in Enzymology (Jakoby, W. B., and Wilchek, M., eds.), Vol. 34, pp. 126-140, Academic Press, New York.
AND
GOLDSMITH
16. KERTESY, M., KOLLER, J., AND AZMAN, A. (1977)
Nature 266,276-278. 17. Low, P. S., AND SOMERO, G. N. (1975) Proc. Nut.
Acad. Sci. USA 72, 3305-3309. 18. HELLER, J. S., FONG, W. F., AND CANELLAKIS, E.
S. (19?6) Proc. Nut. Acad. Sci. USA 73, 1858-1862. 19. FbsBs, L., AND LARI, K. (1977) Biochemistry 16, 4061-4066. 20. BUXMAN, M. M., AND WUEPPER, K. D. (1976)
Biochim. Biophys. Acta 452,356-369. 21. ARNON, R. (1974) in The Antigens (Sela, M., ed.), Vol. I, pp. 109-116, Academic Press, New York. 22. NEURATH, H., AND WALSH, K. A. (1976) Proc.
Nut. Acad. Sci. USA 73,3825-3832.
Human
Epidermal
MARY
Transglutaminase: Stimulation by Trypsin, Solvents, and Chaotropic Salts’ FOSTER
Duke University
PLISHKER,* JUDITH M. THORPE, LOWELL A. GOLDSMITH3 Medical
Center, Durham,
North
Carolina
Organic
AND
27710
Received April 5, 1978; revised May 30, 1978 The activity of pure human epidermal transglutaminase was enhanced 3- to lo-fold by various treatments. Incubation with trypsin caused a time-dependent enhancement in activity, up to 3 times the initial activity, with no apparent change in electrophoretic mobility as detected by disc and sodium dodecyl sulfate electrophoresis, and with no apparent change in immunological properties. This enhancement was specific for trypsin among the several enzymes tested. Preincubation of transglutaminase with 0.1 to 2.0 M potassium thiocyanate or potassium iodide, and with 10 to 50% solutions of alcohols and other organic solvents caused a time-dependent enhancement of activity up to lo-fold over control. The presence of calcium was required for the observed enhancement. Kinetic studies suggest that the K,,, values of the substrates putrescine and casein determined for the native enzyme are similar to those for the stimulated forms of the enzyme. These in vitro methods of altering enzyme activity may be indications of potential in vivo controls of transglutaminase activity.
Human epidermal transglutaminase is one of the calcium requiring enzymes which catalyze the acyl transfer of a y carboxy group derived from a peptide-bound glutamine to a primary amino receptor such as the e-amino group of a peptide-bound lyThe formation of this E-(ysine. glutamyl)lysine bond can create a covalent protease-resistant crosslink between proteins. The general features of transglutaminases and E-( y-glutamyl)lysine bonds have been recently reviewed (1). Previous work by Ogawa and Goldsmith described the purification of human epidermal transglutaminase and some of its prop-
erties (2); in those studies the activity of pure epidermal transglutaminase was enhanced by treatment of the enzyme with dimethylsulfoxide or by heating to 56°C. We now describe enhancement of enzyme activity after limited trypsin treatment, and after incubation in chaotropic salts, alcohols, and other organic solvents. Examination of the activated forms of this enzyme suggests possible mechanisms for the observed stimulation, and gives some insight into possible cellular control mechanisms for transglutaminase activity.
’ Presented in part at the American Society of Biological Chemists Meeting April 1977, Chicago, Ill., and The Society for Investigative Dermatology, April 30, 1977, Washington, D.C. Supported in part by Grants AM-07093 and AM-17253 from the National Institutes of Health. Lowell A. Goldsmith is the recipient of a Research Career and Development Award, Grant AM-09008 from the National Institutes of Health. ’ Present address: Biochemistry Department, M.D. Anderson Hospital, Houston, Texas 77030. 3 To whom all correspondence should be sent.
Materials
EXPERIMENTAL
PROCEDURES
[1,4-Y!]Putrescine and aquasol were from New England Nuclear (Boston, Mass.). Putrescine, bovine serum albumin, ovalbumin, chymotrypsinogen, cytochrome c, chymotrypsin, pronase, trypsin (type XIdiphenylcarbamyl chloride treated), ribonuclease A, alkaline phosphatase, neuraminidase, phospholipase A, papain, plasmin, and soybean trypsin inhibitor were obtained from Sigma (St. Louis, MO.). Factor XIII was a gift of P. McKee. Alkyl agarose columns were obtained from Miles Laboratories, Inc. (Elkhart, Ind.). Immunodiffusion plates were obtained from Hyland 49 0003.9861/78/1911-0049$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
50
PLISHKER,
THORPE,
(Costa Mesa, Calif.). Demineralized water was used throughout, and salts and other reagents were of the highest grades available.
Methods Enzyme assay. Epidermal transglutaminase was purified from human stratum corneum as previously described (2). The radioactive assay for transglutaminase was based on the incorporation of [‘4C]putrestine into casein at pH 9.5 by the method previously described (2, 3). An enzyme unit is defined as the amount of enzyme that catalyzes the incorporation of 1 nmol of putrescine into casein in 30 min. The specific activity of purified enzyme was approximately 350 units/mg of protein, which is in good agreement with the previous report (4). All activities and velocities are the mean values of duplicate determinations using the purified enzyme preparation. Variation between duplicate samples was less than 10%. Trypsin enhancement. Transglutaminase, 10 to 50 $, in 10 mu Tris (4) (pH 7.5), containing 1 mu EDTA, was incubated with 10 mM CaCh and 5-300 pg/ml of trypsin (dissolved in 0.001 M HCl) in a fmal volume of 25-100 d. Preincubations were performed at 37°C in a shaking water bath for the time periods described in figure legends. The preincubation was followed by the addition of 0.5 ml of complete reaction mixture and the incubation was continued as described above. Appropriate controls were performed. Effect of other enzymes, chaotropic salts and organic solvents: enzymes. Samples (50 ~1) of transglutaminase in 10 mM Tris (pH 7.5), 1 mM EDTA, and 10 mM CaClz were preincubated in a final volume of 100 ~1 at 37°C for 20 min in the presence of 125 pg/ml chymotrypsin, pronase, ribonuclease, neuraminidase, or phospholipase A; 10-1500 gg/ml plasmin, 0.01 unit of thrombin; or 50 pg/ml of papain in 10 mM Tris, 1 mM EDTA, 5 mM cysteine, and 0.6 mM 2-mercaptoethanol. Similar preincubations were performed for 0.5 to 24 h in the presence of 500 pg/ml alkaline phosphatase. Control samples were treated identically without added enzyme. Each preincubation mixture was assayed for transglutaminase activity as described above. Chaotropic salts. Twenty-five microliters of transglutaminase (90 units/ml) was preincubated in 10 mM Tris (pH 7.5), 1 mM EDTA, 10 mM CaClz with concentrations from 0.1 to 2.0 M KSCN, KI, KCl, or NaCl in a fmal volume of 50 al at 37°C in a shaking water bath. After the preincubation period, 0.5 ml of complete reaction mixture was added, and the assay continued as described above. Control samples, containing no chaotropic salts, were treated identically. In the time course studies, aliquots of the preincubation mixtures were removed at specific time intervals and added to 0.5 ml of reaction mixture and assayed as described. A control sample, containing no salt, was assayed simultaneously. Organic solvents. Several alcohols and organic re-
AND
GOLDSMITH
agents were examined for their effect on transglutaminase activity. Transglutaminase (50 fi to 0.1 ml) (20-40 units/ml) was preincubated in 10 mM Tris (pH 7.5), 1 mu EDTA, 10 mu CaCb, containing from 10 to 50% solvent in a final volume of 100-200 pl for 20 min at 37°C in a shaking water bath. Controls were preincubated under identical conditions without added solvent. After the preincubation period, 0.5 ml of reaction mixture was added and the assay continued as described above. The effect of solvents on the assay mixture itself was ascertained by adding the solvent to the assay mixture without preincubation and beginning the assay with the addition of enzyme. Gel electrophoresis. Disc electrophoresis on 7.5% acrylamide gels was performed at pH 9.5 by the method of Davis (5). After electrophoresis the unstained gels containing enzyme samples were sliced into segments and assayed for transglutaminase activity (2). To determine whether electrophoresis stimulated transglutaminase, the enzyme was electrophoresed on short (15 mm x 5 mm) 7.5% disc gels with the same buffers used for longer (6 cm) gels, with the lower end of the electrophoresis tube inserted into dialysis tubing. Gels were pre-electrophoresed, dialysis tubing was attached, and electrophoresis was performed for 18 h at 4°C (0.4 mA per tube) so that the transglutaminase was eluted from the gel into the dialysis tubing. Contents of the dialysis tubing were assayed for.transglutaminase activity and for the sensitivity of this electrophoresed enzyme to stimulation by ethanol as described above. Polyacrylamide gel electrophoresis in the presence of SDS4 was performed by the method of Neville (6) on 5% acrylamide gels in order to estimate the molecular weight and purity of the enzyme. Samples were incubated in 10% SDS with 0.05 M dithiothreitol and 0.1% pyronin Y for 10 min at 100°C before application to gels. Bovine serum albumin, ovalbumin, chymotrypsinogen, and cytochrome c were used as molecular weight standards. Gels were stained with Coomassie brilliant blue. Zmmunodiffusion. Gel double diffusion precipitation was performed as described by Ouchterlony (7) using the previously described antibodies (8). Alkyl agarose chromatography. Transglutaminase samples were dialyzed against 10 mru Tris-HCl (pH 7.5) containing 10 mM CaCl2, 0.15 M NaCl, and 1 mM EDTA (Buffer B) at 4°C. Columns (1 ml) of alkylagarose complexes were equilibrated with the same buffer. Samples were applied and the column washed sequentially with 10 mu Tris-HCl (pH 7.5) containing 10 mM CaC12, 1 mu EDTA, and increasing concentrations of NaCk (B) 150 mM NaCk (C) 0.5 M NaCI; (D) 2.0 M NaCI; and (E) 2.0 M NaCl with 50% ethylene glycol (v/v) (9). Fractions were collected and moni4 Abbreviations used: SDS, sodium dodecyl sulfate; Me&SO, dimethylsulfoxide.
EPIDERMAL
tored at 280 nm and transglutaminase activity determined. Protein determination. Protein was measured by the method of Lowry et al. (10) with bovine serum albumin as a standard. RESULTS
Stimulation
51
TRANSGLUTAMINASE
by Trypsin
There was an enhancement in activity after limited treatment with trypsin. As shown in Fig. 1, this stimulation was dependent on the concentration of trypsin between 5 and 100 pg/ml. At values between 100 and 200 Erg/ml, the stimulation appears to have reached a maximum, with stimulated activity 300% of control, and at higher trypsin concentrations losses in activity were observed. In the presence of 25 pg/ml or 50 pg/ml trypsin, stimulation continued for at least 1 h with a loss in activity found after 2 h. The presence of calcium during preincubation with trypsin was required for this enhancement, as indicated by the loss in activity observed when transglutaminase and trypsin were preincubated in the presence of excess EDTA (Fig. 1). Crude epidermal enzyme was less sensitive
to stimulation by trypsin, possibly because of trypsin substrates or inhibitors in the crude extract. Stimulation was observed only if transglutaminase and trypsin were preincubated before the addition of the complete reaction mixture. Simultaneous addition of reaction mixture and trypsin, followed by the usual 30 min of incubation, caused essentiahy no increase in activity over control values. The presence of soybean trypsin inhibitor (1:l with trypsin) prevented stimulation by trypsin. Characterization of trypsin-stimulated transglutaminase was attempted by several procedures. As shown in Fig. 2, the electrophoretic mobility of the trypsin-stimulated enzyme on 7.5% polyacrylamide gels was the same as that of the control. Electrophoresis of control and stimulated enzyme in
TRYPSIN ACTIVATED
-5:
FIG. 1. Enhancement of transglutaminase activity by trypsin. Samples of transglutaminase were preincubated with varying concentrations of trypsin for 20 mm as described under Methods. Samples of purified transglutaminase (0) and tissue extract (O), and tissue extract in the presence of 1 mM EDTA (no added calcium) (*) were examined. AU of these were compared with the control which contained 10 mM calcium. The preincubation of enzyme in excess EDTA was 76% of the calcium-containing control. Tissue extract was the concentrated supernatant from the 12,000g centrifugation of tissue which had been homogenized in 10 mM Tris, 1 mM EDTA, pH 7.5. The molar ratios of trypsin to transglutaminase varied from 31 (25 pg/ml trypsin) to 3O:l (250 pg/ml trypsin).
7
CONTROL
1 A I 20
40 DISTANCE (mm)
60
FIG. 2. Electrophoresis on 7.5% polyacrylamide gels. Samples (100 fl) of control and trypsin-treated transglutaminase were electrophoresed, and the unstained gels were eluted and assayed as described previously (2). The trypsin-treated sample was produced by preipcubation in the presence of 200 pgg/ml trypsin for 20 min at 37°C. This resulted in 2.1-fold enhancement in activity. Samples of trypsin alone produced no activity (cpm) in the gel.
52
PLISHKER,
THORPE,
the presence of SDS, as shown in Fig. 3, demonstrated that the mobility of the enzyme on the 5% polyacrylamide gel was unchanged, and that no new protein bands were apparent in the activated sample. To determine whether the electrophoresis could cause stimulation, transglutaminase activity was eluted by electrophoresis from a 7.5% disc gel into dialysis tubing. The enzyme recovered by this technique was enhanced 5-fold by preincubation in ethanol, as compared with 6.6-fold enhancement by ethanol of an aliquot of control enzyme which had not been electrophoresed. Thus, it appears unlikely that stimulation occurred during electrophoresis. The total activity recovered from the acrylamide gels was the same for the trypsin-activated and control samples; similar equal recoveries of heat-activated and control samples after electrophoresis were also reported (2). Since electrophoresis does not activate it is possible that activation may be reversed by electrophoresis. Immunologic properties of the trypsintreated enzyme were examined by immu-
FIG. 3. Electrophoresis on 7.5% polyacrylamide gel in sodium dodecyl sulfate. Samples (50 al) of control and trypsin-stimulated transglutaminase, and of trypsin were electrophoresed as described under Methods. The stimulation occurred after preincubation with 100 pg/ml trypsin resulting in activity which was 160% of the control. Pattern A is trypsin (TRY) alone, B is transglutaminase (TGL) alone, C is trypsin-stimulated enzyme (which still contains trypsin). Two adjacent wells were filled in A, B, and C. The last pattern (D) is a mixture of cytochrome c (CC), chymotrypsinogen (CHY), ovalbumin (OVA), and bovine serum albumin (BSA). The band at the top of B and C represents c 5 4
53
TRANSGLUTAMINASE
*-*\*
9
1000
>
900 800 700 600 500 400 300 200 100
*/*'
t
+ ,*’
,*'
I 160 TIME (mln)
05 10 15 20 SALT CONCENTRATION (M) FIG. 5. Effects of salts on transglutaminase. Fifty microliters of transglutaminase (100 units/ml) was incubated in 10 mu Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM CaCl2, containing increasing concentrations of KSCN (O), KC1 (W) or NaCl (0) in a final volume of 100 ~1 at 37°C for 20 min. Transglutaminase (25 4) in a fmal volume of 50 ~1 was treated identically with KSCN (0). Each sample was assayed by adding 0.5 ml of complete reaction mixture as described under Methods. Data obtained with KI are essentially identical to those for KSCN (0).
these conditions. Preincubation in KC1 and NaCl caused very little stimulation. There was no stimulation with any of these salts when the preincubation was done in the absence of calcium. The time course for stimulation of transglutaminase activity in the presence of 0.3 M KSCN is shown in Fig. 6. Activity continued to increase for at least 2 h under these preincubation conditions. As previously noted (2) incubation at 37°C in calcium caused a time related increase in activity. To determine whether stimulation occurred only during preincubation with the salts, or if the enhancement occurred after the addition of reaction mixture for assay, preincubations were performed at the same chaotropic salt concentrations in different final volumes, 50 and 100 ~1..The dilution with 0.5 ml of assay mixture resulted in different final (assay) salt concentrations for identical initial (preincubation) concentrations. As shown in Fig. 5, the stimulation for the 50 and 100 d preincubation volumes is essentially identical. Thus, the observed stimulation was apparently the result of the
200
240
FIG. 6. Time course of stimulation by KSCN. Samples (25 ~1) of transglutaminase (40 units/ml) were (pH 7.5), 1 mM EDTA, incubated in 10 mM Tris-HCl 10 mu CaClz, 0.3 M KSCN (*) in a final volume of 50 4. At the indicated time intervals samples were removed and assayed for transglutaminase activity as described in Methods. Aliquots from a control sample (0) were treated identically without added KSCN. V refers to the nanomoles of substrate incorporated.
reagent concentrations during the preincubation, not the actual assay. The effect of preincubation in trypsin (180 pg/ml) alone, 0.44 M KSCN alone, and in both trypsin and KSCN simultaneously showed the effect of KSCN and trypsin together was only slightly greater than the added effects of the two treatments done separately (unpublished observations).
Stimulation
by Organic Solvents
Preincubation of transglutaminase in the presence of several different alcohols or organic solvents also resulted in stimulation of activity. As shown in Fig. 7, stimulation occurred with all the solvents tested, even at concentrations of 50% solvent. In addition to those solvents listed in Fig. 7, similar stimulation was also seen with 10-50s t-butanol, chloroform, ethylene glycol, butanediol, ethanol, and acetone. None of the solvents tested, even at 50% solvent, caused any decrease in activity below the control level. Calcium was required in the preincubation procedure for stimulation to occur, as was previously shown for the enhancement by Me&GO (2). The effect of these organic solvents also apparently occurred during preincubation, as less enhancement in activity was observed if the solvent was added with the reaction mixture for assay. Treatment of Factor XIII
54
PLISHKER.
THORPE, AND GOLDSMITH
FIG. 7. Effect of organic solvents on transglutaminase. Transglutsminase (25 ~1) (60 units/ml) was incubated with lo-508 of each of the solvents shown in a final volume of 50 ah Me&O (0), methanol (O), n-propanol (W), isopropanol (Cl), n-butanol (A), and n-octanol (A). Control samples were treated similarly without added solvent. The reaction conditions are as described under Methods. Each sample was assayed by adding 0.5 ml of complete reaction mixture to the 50 al of preincubation mixture and the assay performed as described under Methods.
and the transglutaminase in a crude liver homogenate with MezSO or ethanol, under conditions which stimulated epidermal transglutaminase, caused a loss in the activity of these two transglutaminases. The reversibility of the observed activation was examined in the case of ethanol stimulated transglutaminase. A decrease in activity was observed in the ethanol stimulated sample after extensive dialysis. Treatment of this dialyzed sample with ethanol resulted in only a slight stimulation of activity. This overall decrease in activity and stimulation could represent a reversal in activation or an irreversible change in the enzyme associated with a loss of activity.
Alkyl Agarose Chromatography Epidermal transglutaminase was chromatographed on a series of alkyl agarose columns as shown in Fig. 8. Under the conditions used, the enzyme was retained on the hexyl (C,), octyl (C,), and decyl (C,,) agaroses. The enzyme was eluted with buffer containing 2.0 M NaCl and 50% ethylene glycol. The activity recovered from the Ck, G, and Cl0 columns was 200%, 330%, and 260% respectively, of the applied activity. This level of stimulation is similar to that observed with trypsin preincuba-
ALKYL CHAIN LENGTH
FRACTION
FIG. 8. Chromatography of transglutaminase on alkyl agarose. 0.2 ml samples of transglutaminase in 10 mu Tris-HCl, 1 mM EDTA, 0.15 NaCl were applied to the series of alkyl agarose columns (12 x 7 mm) equilibrated in the same buffer at 4°C: agarose hexyl (Cs), octyl (CS), (Co) and ethyl (CZ), butyl and decyl (C,,) agarose. The columns were eluted with 6 ml starting buffer (B), and 4 ml each of starting buffer containing 0.5 NaCl (C), 2.0 NaCl (D), and 2.0 NaCl and 50% ethylene glycol. Fractions (0.8 ml) were collected and monitored for ODZW;the transglutaminase activity in 0.2 ml of eluate was assayed in the standard reaction mixture. Activity is represented as cpm incorporated in 30 min (M). The total activity recovered (*) is shown on the right as a function of alkyl chain length. Transglutaminase was stimulated by ethylene glycol at both 37°C and at 4°C. Preincubation at 37°C in 10 Tris (pH 7.5), 10 mM CaC12,2.0 NaCl containing lO%, 20%, 30% and 50% ethylene glycol resulted in activities which were 200%,440%, 500%, and 540%, respectively, of the control. Preincubations at 4°C for 20 min in 10 mM Tris (pH 7.5), 10 CaCl2, 2.0 NaCl containing 12%, 25%, and 50% ethylene glycol resulted in activities which were lOO%,150%,and 2008, respectively, of the control. The total recovered activity from the CS,CS, Cl0 columns was greater than could be accounted for by ethylene glycol stimulation alone (see Results).
(Cd, M
M
M
M
mM
M
mM M
EPIDERMAL
TRANSGLUTAMINASE
tion. Incubation of transglutaminase in buffer containing 2.0 M NaCl and 50% ethylene glycol for 20 min (approximate time of exposure of chromatographed protein to this solvent) at 4’C resulted in a 2-fold enhancement in activity (legend to Fig. 8). If this 200% stimulation by ethylene glycol was subtracted from the activity recovered by elution with ethylene glycol from the CS, Cg, and Cl0 columns, there were still recoveries of total activity which were over 100%: 160%, 220%, and 160%, respectively, for these columns. Thus, the ethylene glycol stimulation did not account for all of the enhancement seen by this chromatographic procedure; the hydrophobic interaction of the enzyme with the alkyl agarose must have contributed to the observed enhancement. Kinetic Studies Samples of transglutaminase which had been stimulated by treatment with trypsin, KSCN, ethanol (as indicated in figure legends for Figs. 9 and lo), and control enzyme were examined kinetically. The reciprocal plots shown in Figs. 9 and 10 show the patterns obtained at saturated levels of either of the two substrates, casein (A) and putrescine (B). K,,, values were obtained for the control enzyme from secondary plots (intercepts and slopes of reciprocal plots versus l/fixed substrate) of additional data obtained for both substrates. The Km value for casein calculated from secondary plots was 0.230 mg/ml; the value obtained from the intersection of the lines in Fig. 9 was 0.133 mg/ml. The V,, values (cpm) were: 26,900 (control), 87,700 (trypsin), 238,000 (KSCN) and 154,000 (ethanol). The K,,, value for putrescine calculated from secondary plots was 0.110 mu; the Km values obtained as intercept values in the reciprocal plot (Fig. 10) were 0.114, 0.106, 0.110, and 0.123 mM for trypsin-, KSCN-, and ethanol-stimulated, and control enzymes, respectively. The V,,, values (cpm) for stimulated enzymes were: trypsin, 52,000; KSCN, 347,ooO;ethanol, 332,ooO;and control, 18,400. The Km values for both substrates for the control and stimulated forms of this enzyme are similar; the difference appears primarily in the V,,,,,.
55
3.5
30
25 rr $2 20 t> 15
IO
5
_1 20
40
60
l/A(mg/ml)-' 9. Kinetic study of stimulated transglutaminase at varying casein concentrations. Samples of transglutaminase control enzyme (*), trypsin-stimulated (*), ethanol-stimulated (O), and KSCN-stimulated (0) were assayed in 100 mu glycine-NaOH (pH 9.5), containing 10 mM CaClz, 5 mM dithiothreitol, 0.250 mru [Wlputrescine (4.0 mCi/mmol), and 9.4-337.0 ,ug/ml casein. Velocity (V) represents counts/mm incorporated and A represents casein concentration (mg/ml). Lines were calculated by a linear regression. Stimulated forms of the enzyme were prepared as described in Fig. 10. FIG.
DISCUSSION
The activity of human epidermal transglutaminase is enhanced severalfold by different types of treatments: 1) preincubation with trypsin, 2) preincubation in chaotropic salts, 3) preincubation in alcohols and other organic solvents, 4) chromatography on alkyl-agarose columns, and 5) heating to 56°C (2). Stimulation by these treatments is dependent on the time of preincubation, the concentration of stimulating agent and the presence of calcium. Epidermal transglutaminase, like the liver transglutaminase, does not require activation by proteolytic enzymes, as does plasma transglutaminase (Factor XIII);
56
PLISHKER,
IU
20
30
40
THORPE,
50
l/B mM-’ FIG. 10. Kinetic study of stimulated transglutaminase at varying putrescine concentrations. Samples of transglutaminase control enzyme (O), trypsin-stimulated (*), ethanol-stimulated (0), and KSCN-stimulated (Cl), were assayed in 110 mu glycine-NaOH (pH 9.5) containing 10 mM CaCh, 5 mu dithiothreitol, 0.8 mg/ml casein, and 0.024 to 0.924 mu [i4C]putrescine (4.0 mCi/mmol). Stimulated forms of transglutaminase were prepared by preincubation of the enzyme for 20 min at 37’C in 10 mM Tris (pH 7.5) containing 10 mu CaCh and no additions (control); I20 pg/ml trypsin (trypsin-stimulated); 1.0 M KSCN (KSCN-stimulated); or 3.5 M ethanol (ethanol-stimulated). Lines were calculated by linear regression.
however, epidermal transglutaminase is stimulated up to 3-fold by preincubation with trypsin. The stimulation of epidermal transglutaminase was apparently specific for trypsin under the conditions tested. Since high trypsin concentrations and long preincubation times cause a loss of transglutaminase activity, proteolytic degradation of transglutaminase during trypsin stimulation may have prevented the 10 fold stimulation attained with other pretreatments. Using several different procedures, attempts were made to characterize the trypsin-stimulated transglutaminase. There
AND
GOLDSMITH
was no apparent change in molecular weight of the stimulated enzyme when compared with the native enzyme by SDS electrophoresis, nor were there differences seen by disc electrophoresis, immunoelectrophoresis, or double immunodiffusion. Therefore, any structural changes which may have occurred during stimulation did not drastically alter the molecular weight or the immunologic properties of this enzyme. Epidermal transglutaminase was also stimulated by preincubation in the chaotropic salts KSCN and KI. Preincubation in 1.5 M KSCN at 37°C caused an increase in transglutaminase activity of up to lo-fold over control values (Fig. 5). Chaotropic ions have been shown to affect the solubilization of membrane bound proteins and the components of multicomponent enzymes, and to increase the water solubility of biopolymers and small organic molecules (11). The order of potency of the anions is SCN->II>Cl-, at high and low concentrations. Mechanisms for enhancement might be: 1) conformational change of the enzyme, possibly due to changes in the structure and lipophilicity of the aqueous solvent, 2) removal by solubilization of an inhibitor which may have been complexed with epidermal transglutaminase, or 3) disaggregation of multimers. The effect was apparently not on the solubility of either of the substrates, or of the product of this reaction, since the preincubation concentration of the salts rather than the final (assay) concentration determined the extent of activation. A possible model for the stimulation by chaotropic salts and trypsin might be the enzyme collagenase. A stimulatory effect by KSCN and trypsin without apparent molecular weight change has also been reported for human skin collagenase (12-14). Preincubation of this enzyme in any of several alcohols or organic solvents also resulted in concentration-dependent enhancement of activity (Fig. 7); there was no obvious relationship between the structure of the solvent and the extent of stimulation. The binding of proteins to immobilized straight chain hydrocarbons is primarily due to hydrophobic interactions (15). Chromatography of transglutaminase on alkyl
EPIDERMAL
TRANSGLUTAMINASE
agarose columns (Fig. 8) demonstrated the presence of a hydrophobic region on the molecule which interacted with hydrocarbon chains 6-10 carbons in length. The interaction was reversed by the presence of ethylene glycol and the enzyme eluted. The observed enhancement of recovered activity may be a result of this hydrophobic interaction enhancement since incubation with ethylene glycol did not account for the total observed increase in activity. In. an effort to understand the stimulatory effects of trypsin, KSCN, and alcohols, the stimulated forms of transglutaminase were examined kinetically (Figs. 9 and 10). The stimulated forms of the enzyme had similar apparent K,,, values for each substrate. The affinity of the enzyme for the substrates was apparently not drastically affected by stimulation. The V,,, did change, those for the stimulated enzymes being much higher than control values. The plot of data obtained at saturated putrescine (B) concentration and varying casein (A) concentration (Fig. 10) was an intersecting plot. The apparent effect of stimulation was similar to that seen by increasing the putrescine concentration of control enzyme. A similar effect (Fig. 10) was seen on casein concentration. Thus it appears that stimulation increased the effective concentration of both substrates, possibly by increasing the number of active sites or the effectiveness of the active sites on the enzyme. The former explanation is unlikely considering the molecular weight of the enzyme. A more likely explanation is an increase in the effectiveness of existing active sites, possibly by making the sites less hindered and more accessible to substrates possibly by changing the enzyme hydration (16, 17). A proteolytic cleavage, a conformational change,, or removal of a small complexed molecule might all result in increased exposure of active sites. A charged complexed molecule, such as an antizyme (18) or a phospholipid, might be expected to become separated from transglutaminase during disc electrophoresis. Enzyme which had been electrophoresed and recovered was still sensitive to stimulation by ethanol suggesting that removal of a charged molecule was not the mechanism of stimulation.
57
The most likely explanation for the observed stimulation appears to be a conformational change and would account for previous data concerning heat-stimulated and MeaLSO-stimulated enzyme (2) and the data presented here. The observed changes in activity are generally slow processes, occurring over a period from 5 min for MezSOstimulated enzyme (2), to 1 h (trypsin), and up to at least 3 h (KSCN) suggesting a conformational change. Antibody induced activation of liver (19) and cow epidermal (20) transglutaminase is reported and may be related to antibody induced conformational changes (21). Previous studies (22) have suggested that activation of other enzymes, such as plasminogen, factor XIII, and complement factor Cl, occurs by conformational transitions induced by proteolytic enzymes rather than by peptide bond cleavage and might explain the lack of a molecular weight change with trypsin activation. The fact that the activity of this enzyme can be enhanced up to lo-fold by treatments presented here may be of importance in understanding the physiological controls of this enzyme. ACKNOWLEDGMENTS The authors appreciate preliminary experiments by Dr. Hideoki Ogawa on trypsin activation and the secretarial assistance of Ms. Susan Boos. This is publication number 34 of the dermatological research laboratories, Duke University Medical Center. REFERENCES 1. FOLK, J. E., AND
FINLAYSON, J. S. (1977) Adv. Protein Chem. 31, I-133. 2. OGAWA, H., AND GOLDSMITH, L. A. (1976) J. Biol. Chem. 251, 7281-7266. 3. GOLDSMITH, L. A., AND MARTIN, C. M. (1975) J. Invest. Dermatol. 64, 316-321. 4. OGAWA, H., AND GOLDSMITH, L. A. (1977) in Biochemistry of Cutaneous Epidermal Differentiation (Seiji, M., and Bernstein, I. M., eds.), pp. 419-432, University of Tokyo Press, Tokyo. 5. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 6. NEVILLE, D. M., JR. (1971) J. Biol. Chem. 246, 6328-6334. 7. OUCHTERLONY, 0. (1958) Prog. Allergy, 5, 1-7. 8. OGAWA, H., AND GOLDSMITH, L. A. (1977) J. Invest. Dermatol. 66.32-35. 9. DAVEY,
M. W., SULKOWKI,
E., AND CARTER,
W.
A. (1976) J. Biol. Chem. 251, 7620-7625. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
58
PLISHKER,
THORPE,
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. HATEFI, Y., AND HANSTEIN, W. G. (1969) Proc.
Nut. Acad. Sci. USA 62,1129-1136. 12. SELLERS, A., CARTWRIGHT, E., MURPHY, G., AND REYNOLDS, J. d. (1977) Biochem. J. 163, 303-307. 13. STRICKLIN, G. P., BAUER, E. A., JEFFREY, J. J., AND EISEN, A. Z. (1977) Biochemistry 16, 1607-1615. 14. ABE, S., AND NAGAI, Y. (1972) Biochim. Biophys. Acta 278, 125-132. 15. SHALTIEL, S. (1974) in Methods in Enzymology (Jakoby, W. B., and Wilchek, M., eds.), Vol. 34, pp. 126-140, Academic Press, New York.
AND
GOLDSMITH
16. KERTESY, M., KOLLER, J., AND AZMAN, A. (1977)
Nature 266,276-278. 17. Low, P. S., AND SOMERO, G. N. (1975) Proc. Nut.
Acad. Sci. USA 72, 3305-3309. 18. HELLER, J. S., FONG, W. F., AND CANELLAKIS, E.
S. (19?6) Proc. Nut. Acad. Sci. USA 73, 1858-1862. 19. FbsBs, L., AND LARI, K. (1977) Biochemistry 16, 4061-4066. 20. BUXMAN, M. M., AND WUEPPER, K. D. (1976)
Biochim. Biophys. Acta 452,356-369. 21. ARNON, R. (1974) in The Antigens (Sela, M., ed.), Vol. I, pp. 109-116, Academic Press, New York. 22. NEURATH, H., AND WALSH, K. A. (1976) Proc.
Nut. Acad. Sci. USA 73,3825-3832.
