ANALYTICAL
BIOCHEMISTRY
201,270-276
(1992)
A Fluorometric, High-Performance Liquid Chromatographic Assay for Transglutaminase
Activity’
Mary Lynn Fink,2 Yvonne Y. Shao, and Gilbert J. Kersh3 Department of Chemistry, Baylor University, Waco, Texas 76798
Received
July
8, 1991
A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent K,,, of 34.7 + 2.4 mru was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, a-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutamin0 1992 Academic Press, Inc. 888s.
Transglutaminases ((R)-glutaminyl-peptide: amine y-glutamyl-yltransferase, EC 2.3.2.13) are Ca2+-dependent enzymes that catalyze acyl-transfer reactions in
’ Supported by NIH Grant GM-46123 and grants from the American Heart Association, Texas Affiliate, and the Baylor University Research Committee. 2 To whom correspondence should be addressed. 3 Present address: Department of Biology, University of California at San Diego, San Diego, CA 92117.
which y-carboxamide groups of peptide-bound glutamine residues serve as acyl donors. A variety of compounds that have one or more primary alkylamines such as methylamine, putrescine, spermidine, spermine, and histamine may function in vitro as acceptor substrates with the formation of different monosubstituted yamides of peptide-bound glutamic acid. When the Eamino group of peptide-bound lysine acts as the acceptor substrate, covalent crosslinking of peptides through c-(y-glutamyl)lysine linkages results (l-5). Examples of transglutaminase-catalyzed crosslinking include stabilization of fibrin clots during hemostasis (6-8), formation of the cornified envelope by epidermal cells undergoing terminal differentiation (9), and production of a vaginal plug from seminal plasma in rodents (10). These enzymes, present in various animal tissues and body fluids and in many different cell types, have also been implicated in wound healing (1 l), crosslinking of membrane proteins (12), and cellular growth regulation (13). Many of the assays for transglutaminase activity are based on the incorporation of a radiolabeled amine into a protein substrate (1). Separation of labeled protein from unreacted amine is conveniently accomplished by precipitation of the protein. In other methods, a fluorescent amine substrate such as dansylcadaverine (7) is separated from the fluorescent product by electrophoresis, ion-exchange chromatography, thin-layer chromatography (14), or by using low- (14) or high-pressure (15) chromatography with gel filtration columns. In one variation of the fluorescent assay, the shift in wavelength of maximum emission intensity and increase in fluorescence upon incorporation of monodansylcadaverine into a protein is monitored without separation of reaction components (16). More recently, a calorimetric assay based on the incorporation of a biotinylated amine into N,N-dimethylcasein has been developed (17). Although casein is often used as a substrate for transglutaminase assays, the preparations vary widely and cannot be used to calculate a standard specific activity. Currently, the determination of specific activity re0003-2697192
270 Copyright All
rights
0 1992 of reproduction
$3.00
by Academic Press, Inc. in any form reserved.
FLUOROMETRIC
CHROMATOGRAPHIC
quires a synthetic substrate such as Z-Gln-Gly. A colorimetric assay measures the reaction of ferric chloride with peptide hydroxamates upon the incorporation of hydroxylamine into substrate at pH 6.0 (5). Although fast and reproducible, this technique is not very sensitive. Consequently, it requires relatively large amounts of substrate and enzyme. Another method relies on the separation of fluorescent substrates from products on thin layers of polyamide (14). In this method, however, quantitation is difficult to achieve and separations for substrates other than Z-Gln-Gly are not always obtained. For studies using small peptides as substrates, additional methods are required. In our studies on potential peptide inhibitors of guinea pig liver transglutaminase, we employ the use of a model system in which the protected dipeptide, benzyloxycarbonylL- glutaminylglycine (Z - Gln - Gly), is used as substrate. For these studies, we required a convenient, quantitative assay for catalytic activity and its inhibition. In this paper, we describe a sensitive, fluorometric technique that uses Z-Gln-Gly and monodansylcadaverine as substrates and makes use of the excellent resolving power of reverse-phase, high-performance liquid chromatography (HPLC) with methanol/ water as the mobile phase. This method requires only very small amounts of substrates and enzyme and is particularly well suited for kinetic studies. We have demonstrated the application of this method to the evaluation of a series of compounds as inhibitors of transglutaminase. The assay is suitable for the measurement of transglutaminase activity with purified enzyme or with crude preparations. Minor modification of the described method using isocratic or gradient elution with acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) afforded assays of transglutaminase for a number of small- and medium-molecular-weight peptide substrates. Apparent K, values for substance P, cu-endorphin, and two synthetic peptides, Arg-Leu-Leu-GlnGly-Leu-Val-NH, and His-Ser-Gln-Gly-Thr-Phe, were determined. In this assay, [Gln4]-neurotensin and [Gin’]-luteinizing hormone-releasing hormone gave no detectible, soluble products with the reaction conditions used. MATERIALS Materials Equipment.
chromatograph
AND METHODS
A Beckman Model 322 gradient liquid equipped with two Model 1lOA pumps,
4 Abbreviations used: EGTA, ethylene glycol bis(2-aminoethyl ether) N,N’-tetraacetic acid, Z-Gln-Gly, benzyloxycarbonyl-Lglutaminylglycine; Z-Glu(MDC)Gly, N-[IV-[5-[[[5-{ dimethylamino}1 - naphthalenyl] sulfonyl ] amino] pentyl] -h@ - [ { phenylmethoxy } car bonyll-L-glutaminyl]glycine; MDC or monodansylcadaverine, [N-(5aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide]; DDC or didansylcadaverine, N,N’-1,5-pentanediylbis[5-(dimethylamino)1-naphthalenesulfonamide]; TFA, trifluoroacetic acid; DTT, dithiothreitol.
ASSAY
FOR TRANSGLUTAMINASE
271
a Model 210A sample injection valve fitted with a 20-~1 sample loop, and a Model 420 microprocessor system controller was used in this study. Separations were performed on a reverse-phase, C-18 column (Beckman Ultrasphere-ODS; 150 X 4.6 mm; particle size, 5 pm). For detection, the liquid chromatograph was connected in series to a Beckman Model 164 variable wavelength detector and a Gilson Model 121 filter fluorometer (excitation, 352-360 nm; emission, 480-520 nm). Signals from the detectors were recorded on a Hewlett-Packard Model 3396A reporting integrator and/or a Houston Instrument Omniscribe D-5000 strip chart recorder. Biochemical and chemicals. The following chemicals were purchased from Fisher Scientific and were A.C.S. certified grade: Tris, calcium chloride, DTT, EDTA, EGTA, NaCl. HPLC-grade methanol, acetonitrile, and water were also purchased from Fisher Scientific. Trifluoroacetic acid was from Aldrich and was spectrophotometric grade. Propanal, butanal, pentanal, hexanal, 2-methylpropanal, 2-methylbutanal, 2methylpentanal, 2-ethylbutanal, and 2,2dimethylpropanal were also from Aldrich. 3-Methylbutanal and 3,3-dimethylbutanal were from Fluka, AG. Monodansylcadaverine, didansylcadaverine, substance P, a-endorphin, [Gln4]-neurotensin, and [Gin’]-luteinizing hormone-releasing hormone were from Sigma. Benzyloxycarbonyl-L-serylglycine was a gift of Dr. J. E. Folk and benzyloxycarbonyl-L-glutaminylglycine was synthesized as outlined (5). Polyamide sheets were from Schleicher and Schuell. Enzyme. Guinea pig liver transglutaminase was prepared as described (5). Crude preparations of transglutaminase were obtained from chicken livers. Tissue was homogenized in 1 vol of 0.25 M sucrose for approximately 1 min with a Brinkmann Polytron. The homogenate was centrifuged for 3 min in an Eppendorf microfuge and the supernatant was used as the source of the crude enzyme. Methods Enzymatic reactions. Enzymatic reactions were typically carried out in 0.1 M Trislchloride buffer containing 1 mM EDTA, 10 InM CaCl,, 1 or 2 IIIM monodansylcadaverine, varying amounts of peptide substrate, and enzyme (0.4-5 pg) in a total volume of 50 ~1 at pH 7.45, 37°C. Reactions were terminated by the addition of EGTA, pH 7.5, or organic solvent. When crude preparations were used as the source of enzyme, DTT (10 mM, final concentration) was included in the reaction mixture, and the substrate, Z-Gln-Gly, was set at a final concentration of 40 mM. For reactions with Z-Gln-Gly as substrate, samples were diluted with HPLC-grade methanol for assay. For those samples with internal standard, HPLC-grade methanol containing 0.1 mM didansylcadaverine was used for dilution. Precipitated
272
FINK,
SHAO,
proteins were removed by centrifugation or filtration. For peptide substrates other than Z-Gln-Gly, acetonitrile replaced methanol as the diluent.
Chromatographic procedures. All HPLC runs were performed at room temperature at a flow rate of 1.0 ml/min. Samples for injection were either filtered through 0.22-pm Millipore HPLC filters or centrifuged for 1 min in an Eppendorf microfuge at maximum speed. For the reaction with Z-Gln-Gly, substrates and products were eluted from the reverse-phase column with methanol/water @O/50; v/v). For other peptide substrates, products were eluted with various isocratic or gradient concentrations of acetonitrile with 0.1% TFA/water with 0.1% TFA. To establish optimal column conditions in preliminary runs, a binary gradient with organic solvent and water was used. To identify HPLC peaks, reaction samples were spiked with individual standards. Didansylcadaverine was used as an internal or external standard to calibrate the fluorescence response for quantitation. In addition, samples and standards were chromatographed on thin layers of polyamide with 1% aqueous pyridine adjusted to pH 5.6 with acetic acid as the eluting solvent. Calibration curve. An enzymatic reaction with purified guinea pig liver transglutaminase that contained 1 mM monodansylcadaverine and 20 mM Z-Gln-Gly in a total volume of 50 ~1 was allowed to go to completion. Disappearance of monodansylcadaverine was verified by HPLC. The reaction solution was diluted to 500 ~1 with methanol and was used as the standard for the reaction product. The concentration of the product was confirmed by HPLC measurement of monodansylcadaverine released upon acid hydrolysis (6 N HCl, 18 h, 115’C). A solution of 0.1 mM didansylcadaverine in methanol was employed for the internal standard. Evaluation of aldehydes as inhibitors of transglutaminuse. Aldehydes were dissolved in acetone and preincubated with purified guinea pig liver enzyme for 5 min. The maximum amount of aldehyde solution was 5% of the total reaction mixture. Z-L-Gln-Gly (1 mM, final concentration) and monodansylcadaverine (1 mM, final concentration) were added to initiate the reaction. Controls with acetone, but no aldehyde, were run for each set of experiments. Controls with aldehyde and monodansylcadaverine were also carried out. Evaluation of Z-L-Ser-Gly and sodium borate as an inhibitor of transglutaminase. Reactions were run as described for Z-L-Gln-Gly above with a 5-min preincubation of the purified guinea pig liver enzyme with Z-L-Ser-Gly and sodium borate. Final concentrations of Z-L-Ser-Gly and sodium borate were 10 mM each, that of Z-r,-Gln-Gly was 5 mM, and that of monodansylcadaverine was 1.7 mM. Individual controls with sodium borate but no Z-Ser-Gly and with Z-Ser-Gly but no sodium borate were run.
AND
KERSH
Enzyme kinetics. Rate studies were carried out as described above with 2 mM monodansylcadaverine and varying amounts of peptide substrate. The data were fitted to the equation v = (VA/K + A) using the interactive curve fitting program, MLAB, developed at the National Institutes of Health or by using the programs of Cleland (18). RESULTS
AND
DISCUSSION
Reaction of the substrate, monodansylcadaverine, with the protected dipeptide substrate, Z-Gln-Gly, yields a fluorescent product, Z-Glu(MDC)Gly (14) (Fig. 1). Separation of product from fluorescent substrate was achieved by HPLC (Fig. 2) on a reverse-phase, C-18 column using an isocratic elution solvent composed of 50% methanol and 50% unbuffered water. Under these conditions, monodansylcadaverine is retained on the column and is removed with 100% methanol at the end of the chromatographic runs. The product has a greater fluorescence in methanol than in water, and therefore, we used the minimum amount of water that would allow efficient separation of product from fluorescent substrate and internal standard. Transglutaminase-catalyzed reactions require calcium and are inhibited by organic solvents. Therefore, they were stopped either by the addition of excess EGTA or by the addition of methanol or acetonitrile. In some cases, samples were diluted with HPLC-grade methanol containing the internal standard, didansylcadaverine. Otherwise, didansylcadaverine was used as an external standard. In general, the agreement for samples run on a single day was excellent and internal standard was not required. Proteins, precipitated by the addition of methanol, were removed by centrifugation or filtration while the reaction product remained in solution. A complete recovery of the product was verified both by HPLC and by chromatography on thin layers of polyamide. The amount of product fluorescence measured was linear with respect to time (Fig. 3A) and with respect to amount of enzyme (Fig. 3B) until the concentration of the limiting substrate, monodansylcadaverine, decreased significantly. These findings indicate that the fluorescence response is proportional to the concentration of the product. That the calibration curve established with the reaction product, Z-Glu(MDC)Gly, is linear is in accordance with these observations and confirms the linearity of the detector response. The standard, didansylcadaverine, also displays a linear response of fluorescence (total peak area) with concentration and is useful for quantitation and for determining the amount of sample injected. The molar fluorescence ratio of didansylcadaverine standard to reaction product, calculated from these curves to be 1.4, also provides a means for obtaining quantitative data and for the calculation of specific activity. For the determination of sensitivity, an aliquot of the standard solution of product was diluted and ana-
FLUOROMETRIC
CHROMATOGRAPHIC
fy-NH2
ASSAY
FOR
f3 0
H2N(CH&NH-S=
a YH2?
273
TRANSGLUTAMINASE
F H2
N(W)2
CH20-C-NH-CH-C-NHCH2C02H
MDC
Z-Gin-Gly
CH20-C-NH-CH-C-NHCH2C02H
+
NH3
Z-GIu(MDC)-Gly FIG.
1.
Reaction
scheme
for the incorporation
lyzed by HPLC. By operating at the edge of sensitivity of the fluorometer, a signal-to-noise ratio for the solution studied was determined. The signal-to-noise ratio at different concentrations of product was obtained and plotted. By taking the detection limit to be the concentration of product that produced a signal-to-noise ratio of 3, the detection limit was found to be 31 pmol of product per injection. This assay is also applicable to the measurement of transglutaminase activity in biological samples. When
of MDC
into
Z-Gln-Gly
by transglutaminase.
the supernatant from crude homogenates of chicken liver was used as the source of enzyme, the product fluorescence for Z-Glu(MDC)Gly as a function of time was linear for 30 min with the reaction conditions used (Fig. 3A). Kinetic experiments in which the concentration of ZGln-Gly was varied from 1 to 80 mM gave a value of 34.7 f 2.4 InM (mean + SE) for the apparent Km for purified guinea pig liver transglutaminase under the conditions described under Materials and Methods. Although other transglutaminases such as factor XIIIa do not act on Z-Gln-Gly, somewhat larger, synthetic peptides are
DDC
J 1 J
$ 5
65-
:: 2! 0 3
45
-
ii .-:
25-
ii 5 a
5-
Product
0123456
Time
(min)
FIG. 2. HPLC elution profile of reaction product (Z-Glu(MDC)Gly), and DDC (internal standard). Methanol/water (X)/50) as the mobile phase. MDC is retained on the column. For further details, see Materials and Methods.
0
20 Time
40 (min)
60
0.01
0.02
Enzyme
0.03
0.04
(mg/ml)
FIG. 3. (A) A plot of relative fluorescence versus time for the formation of Z-Glu(MDC)Gly by purified guinea pig liver enzyme (closed symbols) and by supernatant from crude homogenates of chicken liver (open symbols). (B) Relative fluorescence versus enzyme concentration for purified liver transglutaminase. Incubations were performed at 37°C for 10 min with 8 mM Z-Gln-Gly and 2 mM MDC as described under Materials and Methods. The activity corresponds to the production of 3.5 pmol Z-Glu(MDC)Gly/min/mg enzyme.
274
FINK,
SHAO,
AND
TABLE Glutamine-Containing
Glutamine-containing
Peptides
as Potential
peptide
Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuMet-NH, cY-Endorphin: Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-SerGln-Thr-Pro-Leu-Val-Thr Arg-Leu-Leu-Gln-Gly-Leu-Val-NH, His-Ser-Gln-Gly-Thr-Phe Z-Gln-Gly [Glne]-LH-RH: p-Glu-His-TrpSer-Tyr-Gly-Leu-Gln-ProGly-NH, [Gln4]-Neurotensin: p-Glu-Leu-Tyr-Gln-Asn-Lys-Pro-ArgArg-Pro-Tyr-Ile-Leu
KERSH
1 Substrates
Mobile
Transglutaminase” Retention time (min)’
phaseb
40-100% B, (15 min) 35% B 50% 35% 50%
B B c
lo-loo% B (20 min) lo-100% B (20 min)
’ Reaction conditions described under Materials and Methods. b The mobile phase consists of a pair of solvents: A/B or C/D. A, water containing methanol; D, unbuffered water. ’ Retention time given for product formed from peptide and monodansylcadaverine. d No product detected with these assay conditions. Peptide concentration was 0.66
available as substrates (19-21). The assay described in this report is applicable to the evaluation of other transglutaminase enzymes through the use of suitable synthetic substrates. By changing the elution solvent from methanol/water to acetonitrile containing 0.1% TFA/water with 0.1% TFA, the assay described with Z-Gln-Gly as substrate can be applied to evaluate various peptides as substrates for transglutaminase. Substance P, reported as a transglutaminase substrate (22,23), was determined to have an apparent K,,, of 0.17 f 0.02 mM (Table 1). Gradient elution is used for the separation of the product with substance P and monodansylcadaverine as substrates. With these conditions, monodansylcadaverine is not retained on the column, but elutes at 2.7 min. Didansylcadaverine, the external standard, has a retention time of 8.7 min. The amount of product fluorescence measured was linear with respect to time until the concentration of substrate, substance P, decreased significantly. An apparent K,,, of 0.48 f 0.02 mM was determined for cY-endorphin and separation of the product from the transglutaminasecatalyzed incorporation of monodansylcadaverine into cw-endorphin is carried out with isocratic conditions as shown in Fig. 4. In these reactions, no correction was made for intramolecular or intermolecular crosslinking reactions, and in fact, with longer reaction times, insoluble, fluorescent material formed. At a concentration of substrate (0.62 InM) and enzyme (50 pg/ml) that results in the formation of large amounts of product as determined by relative fluorescence with substance P and a-endorphin as substrates, no product could be determined with either [Gin*]-neurotensin or [Gin’]-luteinizing hormone-releasing hormone as substrate. With conditions in which all of the substrate, substance P, was
for
0.1%
8.7
0.17 f 0.02
5.7
0.48 + 0.02
4.5 4.5 3.8
6.7
9.1 34.7
No product
detectedd
No product
detectedd
TFA,
B, acetonitrile
containing
0.1%
f 0.6 k 2.2 + 2.4
TFA,
C,
mM.
used (0.1 mg enzyme, 12 h reaction time), no product could be detected with [Gln8]-luteinizing hormone-releasing hormone as substrate. Under these conditions, with [Gln4]-neurotensin as substrate, insoluble fluorescent material formed, but no soluble fluorescent product was detected. Two synthetic peptides, ArgLeu-Leu-Gln-Gly-Leu-Val-NH, and His-Ser-GlnGly-Thr-Phe, were found to be substrates (Table 1). We have developed a model system for the evaluation of potential inhibitors of transglutaminase based on the assay with Z-Gln-Gly and monodansylcadaverine as
a3 .? z 5 K
25
5
I
I
I
1
I
I
0
2
4
6
8
1012
Time
I
(min)
FIG. 4. HPLC elution profile of reaction product with a-endorphin and MDC as substrates and DDC as external standard. Mobile phase consists of 35% acetonitrile (0.1% TFA) in water (0.1% TFA).
FLUOROMETRIC
TABLE
CHROMATOGRAPHIC
2
Aldehydes as Inhibitors of the Transglutaminase Reaction” % Inhibition Compound
10
Propanal Butanal Pentanal Hexanal 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal 2-Ethylbutanal 2-Methylpentanal 2,2-Dimethylpropanal 3,3-Dimethylbutanal a Conditions
as described
mM
5mM
100 100 100 100 100 100 100 100 100 100 80 under
Materials
50 19 24 24 40 42 90 95 95 65
16 and Methods.
ASSAY
FOR
275
TRANSGLUTAMINASE
sylcadaverine as substrates (Table 2). At 5 InM aldehyde concentrations, 2-ethylbutanal and 2-methylpentanal were most effective. While the aldehydes tested clearly inhibited transglutaminase, the results do not parallel those of the specificity of alkyl amide substrates or the inactivation pattern observed with alkyl isocyanates (3,27). Branched-chain amides in which branching occurs at the (Y or fl position to the carboxamide do not function as substrates, and structurally related, branched, alkyl isocyanates are not effective active sitedirected inhibitors while the straight-chain compounds are. Further studies are required to determine the mechanism of enzyme inhibition by aldehydes. ACKNOWLEDGMENTS The authors thank Dr. J. E. Folk for his helpful discussions. preliminary report of this work has been presented (2829).
A
REFERENCES
substrates. Serine in the presence of borate is an effective inhibitor of y-glutamyltranspeptidase (not a transglutaminase), an enzyme that acts on y-glutamyl compounds as substrates and catalyzes the utilization of glutathione (24). This inhibition is thought to act through the formation of a borate bridge complex between the serine hydroxyl and the enzyme active site nucleophile. In an effort to form an analogous complex with transglutaminase, serine was used to replace glutamine in the dipeptide substrate Z-Gln-Gly (Z-Ser-Gly). However, no inhibition of guinea pig liver transglutaminase activity was observed with a substrate concentration of Z-Gln-Gly of 5 mM and 10 mM Z-L-Ser-Gly in the presence of 10 mM sodium borate. In other, preliminary studies using this assay, we have demonstrated that alkyl aldehydes can inhibit the guinea pig liver enzyme. A variety of aldehyde compounds, structurally related to the acyl portion of substrates, are extremely effective inhibitors of serine and thiol proteases. Such inhibitors often result in the formation of hemiacetal or thiohemiacetal species with the enzyme (25,26). Although not a protease, transglutaminase has an active site thiol that participates in the formation of an acyl-enzyme thioester intermediate during the course of the reaction in a manner analogous to that of a thiol protease such as papain. Therefore, a series of aldehydes was tested for inhibition of transglutaminase. Multiple sets of reactions were carried out and the results from a typical set of data are shown in Table 2. Control reactions include those with no inhibitor, those with no enzyme, and controls with no substrate. From the controls, it was apparent that no significant amount of Schiff base formed from aldehyde and monodansylcadaverine under the conditions used. At concentrations of 10 InM aldehyde, both straight-chain aldehydes, such as butanal, pentanal, and hexanal, and compounds branched at the a-carbon inhibited the action of transglutaminase with Z-Gln-Gly and monodan-
1. Clarke, D. D., Mycek, M. J., Neidle, Arch. Biochem. Biophys. 79, 338-354.
A., and Waelsch,
H. (1959)
2. Folk,
J. E., and Finlayson, J. S. (1977) in Advances in Protein Chemistry (Anfinsen, C. B., Edsall, J. T., and Richards, F. M., Eds.), Vol. 31, pp. 1-133, Academic Press, New York.
3. Folk, J. E. (1983) Adu. Enzymol. Relat. Areas Mol. Biol. 54, l-56. 4. Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9-35.
5. Folk,
J. E., and Chung, S. I. (1985) in Methods (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. Academic Press, Orlando, FL.
6. Matacic, mun.
S., and Loewy, 30,356-362.
A. G. (1968)
Biochem.
in Enzymology 113, pp. 358-375, Biophys.
Res. Com-
7. Lorand,
L., Rule, N. G., Ong, H. H., Furlanetto, R., Jacobsen, A.. Downey, J., Oner, N., and Bnmer-Lorand, J. (1968) Biochemistry 7, 1214-1223.
8. Pisano, J. J., Finlayson, 160,892-893. 9. Rice, R. H., and Green,
J. S., and Peyton, H. (1977)
Cell
M. P. (1968)
Science
11, 417-422.
10. Williams-Ashman, H. G. (1984) Mol. Cell. Biochem. 58, 51-61. 11. Mosher, D. F., and Schad, P. E. (1979) J. Clin. Znuest. 64, 781-
787. 12. Siefring, G. E., Jr., Apostol, A. B., Velasco, (1978) Biochemistry 17, 2598-2604. 13. Birckbichler, and Carter,
P. T., and Lorand,
L.
P. J., Orr, G. R., Patterson, M. K., Jr., Conway, E., H. A. (1981) Proc. Natl. Acad. Sci. USA 78, 5005-
5008. 14. Lorand,
L., and Campbell,
L. K. (1971)
Anal.
Biochem.
44,207-
220. 15. Ando, Y., Imamura, S., Yamagata, Y., Kikuchi, T., Murachi, and Kannagi, R. (1987) J. Biochem. 101, 1331-1337. 16. Takagi, J., Saito, Y., Kikuchi, T., and Inada, Y. (1986) Anal. them. 153,295-298.
T., Bio-
17. Jeon,
W. M., Lee, K. N., Birckbichler, P. J., Conway, E., and Patterson, M. K., Jr. (1989) Anal. Biochem. 182,170-175.
18. Cleland, W. W. (1979) in Methods in Enzymology (Purich, D. L., Ed.), Vol. 63, pp. 103-138, Academic Press, New York. 19. Gorman, J. J., and Folk, J. E. (1980) J. Biol. Chem. 255,419-427. 20. Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-
2715. 21. Ichinose, 369-371.
A., Tamaki,
T., and Aoki,
N. (1983)
FEBS
Lett.
153,
276
FINK,
SHAO, AND KERSH
22. Gorman, J. J., and Folk, J. E. (1980) J. Biol. Chem. 255, 11751180. 23. Porta, R., Esposito, C., Metafora, S., Pucci, P., Malorni, A., and Marino, G. (1988) Anal. Biochem. 172,499-503. 24. Tate, S. S., and Meister, A. (1978) Proc. Natl. Acad. Sci. USA 75, 4806-4809.
25. Westerik, J. OX., and Wolfenden, 8195-8197.
R. (1972) J. Biol. Chem.
247,
26. Lewis, C. A., Jr., and Wolfenden, R. (19’77) Biochemistry 16, 4890-4895. 27. Gross, M., Whetzel, N. K., and Folk, J. E. (1975) J. Bid. Chem. 250, 7693-7699. 28. Fink, M. L., and Kersh, G. J. (1988) J. Cell Biol. 107,839a. [Abstract] 29. Fink, M. L., Shao, Y. Y., and Shey, J. (1990) J. Cell Biol. 111, 320a. [Abstract]
BIOCHEMISTRY
201,270-276
(1992)
A Fluorometric, High-Performance Liquid Chromatographic Assay for Transglutaminase
Activity’
Mary Lynn Fink,2 Yvonne Y. Shao, and Gilbert J. Kersh3 Department of Chemistry, Baylor University, Waco, Texas 76798
Received
July
8, 1991
A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent K,,, of 34.7 + 2.4 mru was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, a-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutamin0 1992 Academic Press, Inc. 888s.
Transglutaminases ((R)-glutaminyl-peptide: amine y-glutamyl-yltransferase, EC 2.3.2.13) are Ca2+-dependent enzymes that catalyze acyl-transfer reactions in
’ Supported by NIH Grant GM-46123 and grants from the American Heart Association, Texas Affiliate, and the Baylor University Research Committee. 2 To whom correspondence should be addressed. 3 Present address: Department of Biology, University of California at San Diego, San Diego, CA 92117.
which y-carboxamide groups of peptide-bound glutamine residues serve as acyl donors. A variety of compounds that have one or more primary alkylamines such as methylamine, putrescine, spermidine, spermine, and histamine may function in vitro as acceptor substrates with the formation of different monosubstituted yamides of peptide-bound glutamic acid. When the Eamino group of peptide-bound lysine acts as the acceptor substrate, covalent crosslinking of peptides through c-(y-glutamyl)lysine linkages results (l-5). Examples of transglutaminase-catalyzed crosslinking include stabilization of fibrin clots during hemostasis (6-8), formation of the cornified envelope by epidermal cells undergoing terminal differentiation (9), and production of a vaginal plug from seminal plasma in rodents (10). These enzymes, present in various animal tissues and body fluids and in many different cell types, have also been implicated in wound healing (1 l), crosslinking of membrane proteins (12), and cellular growth regulation (13). Many of the assays for transglutaminase activity are based on the incorporation of a radiolabeled amine into a protein substrate (1). Separation of labeled protein from unreacted amine is conveniently accomplished by precipitation of the protein. In other methods, a fluorescent amine substrate such as dansylcadaverine (7) is separated from the fluorescent product by electrophoresis, ion-exchange chromatography, thin-layer chromatography (14), or by using low- (14) or high-pressure (15) chromatography with gel filtration columns. In one variation of the fluorescent assay, the shift in wavelength of maximum emission intensity and increase in fluorescence upon incorporation of monodansylcadaverine into a protein is monitored without separation of reaction components (16). More recently, a calorimetric assay based on the incorporation of a biotinylated amine into N,N-dimethylcasein has been developed (17). Although casein is often used as a substrate for transglutaminase assays, the preparations vary widely and cannot be used to calculate a standard specific activity. Currently, the determination of specific activity re0003-2697192
270 Copyright All
rights
0 1992 of reproduction
$3.00
by Academic Press, Inc. in any form reserved.
FLUOROMETRIC
CHROMATOGRAPHIC
quires a synthetic substrate such as Z-Gln-Gly. A colorimetric assay measures the reaction of ferric chloride with peptide hydroxamates upon the incorporation of hydroxylamine into substrate at pH 6.0 (5). Although fast and reproducible, this technique is not very sensitive. Consequently, it requires relatively large amounts of substrate and enzyme. Another method relies on the separation of fluorescent substrates from products on thin layers of polyamide (14). In this method, however, quantitation is difficult to achieve and separations for substrates other than Z-Gln-Gly are not always obtained. For studies using small peptides as substrates, additional methods are required. In our studies on potential peptide inhibitors of guinea pig liver transglutaminase, we employ the use of a model system in which the protected dipeptide, benzyloxycarbonylL- glutaminylglycine (Z - Gln - Gly), is used as substrate. For these studies, we required a convenient, quantitative assay for catalytic activity and its inhibition. In this paper, we describe a sensitive, fluorometric technique that uses Z-Gln-Gly and monodansylcadaverine as substrates and makes use of the excellent resolving power of reverse-phase, high-performance liquid chromatography (HPLC) with methanol/ water as the mobile phase. This method requires only very small amounts of substrates and enzyme and is particularly well suited for kinetic studies. We have demonstrated the application of this method to the evaluation of a series of compounds as inhibitors of transglutaminase. The assay is suitable for the measurement of transglutaminase activity with purified enzyme or with crude preparations. Minor modification of the described method using isocratic or gradient elution with acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) afforded assays of transglutaminase for a number of small- and medium-molecular-weight peptide substrates. Apparent K, values for substance P, cu-endorphin, and two synthetic peptides, Arg-Leu-Leu-GlnGly-Leu-Val-NH, and His-Ser-Gln-Gly-Thr-Phe, were determined. In this assay, [Gln4]-neurotensin and [Gin’]-luteinizing hormone-releasing hormone gave no detectible, soluble products with the reaction conditions used. MATERIALS Materials Equipment.
chromatograph
AND METHODS
A Beckman Model 322 gradient liquid equipped with two Model 1lOA pumps,
4 Abbreviations used: EGTA, ethylene glycol bis(2-aminoethyl ether) N,N’-tetraacetic acid, Z-Gln-Gly, benzyloxycarbonyl-Lglutaminylglycine; Z-Glu(MDC)Gly, N-[IV-[5-[[[5-{ dimethylamino}1 - naphthalenyl] sulfonyl ] amino] pentyl] -h@ - [ { phenylmethoxy } car bonyll-L-glutaminyl]glycine; MDC or monodansylcadaverine, [N-(5aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide]; DDC or didansylcadaverine, N,N’-1,5-pentanediylbis[5-(dimethylamino)1-naphthalenesulfonamide]; TFA, trifluoroacetic acid; DTT, dithiothreitol.
ASSAY
FOR TRANSGLUTAMINASE
271
a Model 210A sample injection valve fitted with a 20-~1 sample loop, and a Model 420 microprocessor system controller was used in this study. Separations were performed on a reverse-phase, C-18 column (Beckman Ultrasphere-ODS; 150 X 4.6 mm; particle size, 5 pm). For detection, the liquid chromatograph was connected in series to a Beckman Model 164 variable wavelength detector and a Gilson Model 121 filter fluorometer (excitation, 352-360 nm; emission, 480-520 nm). Signals from the detectors were recorded on a Hewlett-Packard Model 3396A reporting integrator and/or a Houston Instrument Omniscribe D-5000 strip chart recorder. Biochemical and chemicals. The following chemicals were purchased from Fisher Scientific and were A.C.S. certified grade: Tris, calcium chloride, DTT, EDTA, EGTA, NaCl. HPLC-grade methanol, acetonitrile, and water were also purchased from Fisher Scientific. Trifluoroacetic acid was from Aldrich and was spectrophotometric grade. Propanal, butanal, pentanal, hexanal, 2-methylpropanal, 2-methylbutanal, 2methylpentanal, 2-ethylbutanal, and 2,2dimethylpropanal were also from Aldrich. 3-Methylbutanal and 3,3-dimethylbutanal were from Fluka, AG. Monodansylcadaverine, didansylcadaverine, substance P, a-endorphin, [Gln4]-neurotensin, and [Gin’]-luteinizing hormone-releasing hormone were from Sigma. Benzyloxycarbonyl-L-serylglycine was a gift of Dr. J. E. Folk and benzyloxycarbonyl-L-glutaminylglycine was synthesized as outlined (5). Polyamide sheets were from Schleicher and Schuell. Enzyme. Guinea pig liver transglutaminase was prepared as described (5). Crude preparations of transglutaminase were obtained from chicken livers. Tissue was homogenized in 1 vol of 0.25 M sucrose for approximately 1 min with a Brinkmann Polytron. The homogenate was centrifuged for 3 min in an Eppendorf microfuge and the supernatant was used as the source of the crude enzyme. Methods Enzymatic reactions. Enzymatic reactions were typically carried out in 0.1 M Trislchloride buffer containing 1 mM EDTA, 10 InM CaCl,, 1 or 2 IIIM monodansylcadaverine, varying amounts of peptide substrate, and enzyme (0.4-5 pg) in a total volume of 50 ~1 at pH 7.45, 37°C. Reactions were terminated by the addition of EGTA, pH 7.5, or organic solvent. When crude preparations were used as the source of enzyme, DTT (10 mM, final concentration) was included in the reaction mixture, and the substrate, Z-Gln-Gly, was set at a final concentration of 40 mM. For reactions with Z-Gln-Gly as substrate, samples were diluted with HPLC-grade methanol for assay. For those samples with internal standard, HPLC-grade methanol containing 0.1 mM didansylcadaverine was used for dilution. Precipitated
272
FINK,
SHAO,
proteins were removed by centrifugation or filtration. For peptide substrates other than Z-Gln-Gly, acetonitrile replaced methanol as the diluent.
Chromatographic procedures. All HPLC runs were performed at room temperature at a flow rate of 1.0 ml/min. Samples for injection were either filtered through 0.22-pm Millipore HPLC filters or centrifuged for 1 min in an Eppendorf microfuge at maximum speed. For the reaction with Z-Gln-Gly, substrates and products were eluted from the reverse-phase column with methanol/water @O/50; v/v). For other peptide substrates, products were eluted with various isocratic or gradient concentrations of acetonitrile with 0.1% TFA/water with 0.1% TFA. To establish optimal column conditions in preliminary runs, a binary gradient with organic solvent and water was used. To identify HPLC peaks, reaction samples were spiked with individual standards. Didansylcadaverine was used as an internal or external standard to calibrate the fluorescence response for quantitation. In addition, samples and standards were chromatographed on thin layers of polyamide with 1% aqueous pyridine adjusted to pH 5.6 with acetic acid as the eluting solvent. Calibration curve. An enzymatic reaction with purified guinea pig liver transglutaminase that contained 1 mM monodansylcadaverine and 20 mM Z-Gln-Gly in a total volume of 50 ~1 was allowed to go to completion. Disappearance of monodansylcadaverine was verified by HPLC. The reaction solution was diluted to 500 ~1 with methanol and was used as the standard for the reaction product. The concentration of the product was confirmed by HPLC measurement of monodansylcadaverine released upon acid hydrolysis (6 N HCl, 18 h, 115’C). A solution of 0.1 mM didansylcadaverine in methanol was employed for the internal standard. Evaluation of aldehydes as inhibitors of transglutaminuse. Aldehydes were dissolved in acetone and preincubated with purified guinea pig liver enzyme for 5 min. The maximum amount of aldehyde solution was 5% of the total reaction mixture. Z-L-Gln-Gly (1 mM, final concentration) and monodansylcadaverine (1 mM, final concentration) were added to initiate the reaction. Controls with acetone, but no aldehyde, were run for each set of experiments. Controls with aldehyde and monodansylcadaverine were also carried out. Evaluation of Z-L-Ser-Gly and sodium borate as an inhibitor of transglutaminase. Reactions were run as described for Z-L-Gln-Gly above with a 5-min preincubation of the purified guinea pig liver enzyme with Z-L-Ser-Gly and sodium borate. Final concentrations of Z-L-Ser-Gly and sodium borate were 10 mM each, that of Z-r,-Gln-Gly was 5 mM, and that of monodansylcadaverine was 1.7 mM. Individual controls with sodium borate but no Z-Ser-Gly and with Z-Ser-Gly but no sodium borate were run.
AND
KERSH
Enzyme kinetics. Rate studies were carried out as described above with 2 mM monodansylcadaverine and varying amounts of peptide substrate. The data were fitted to the equation v = (VA/K + A) using the interactive curve fitting program, MLAB, developed at the National Institutes of Health or by using the programs of Cleland (18). RESULTS
AND
DISCUSSION
Reaction of the substrate, monodansylcadaverine, with the protected dipeptide substrate, Z-Gln-Gly, yields a fluorescent product, Z-Glu(MDC)Gly (14) (Fig. 1). Separation of product from fluorescent substrate was achieved by HPLC (Fig. 2) on a reverse-phase, C-18 column using an isocratic elution solvent composed of 50% methanol and 50% unbuffered water. Under these conditions, monodansylcadaverine is retained on the column and is removed with 100% methanol at the end of the chromatographic runs. The product has a greater fluorescence in methanol than in water, and therefore, we used the minimum amount of water that would allow efficient separation of product from fluorescent substrate and internal standard. Transglutaminase-catalyzed reactions require calcium and are inhibited by organic solvents. Therefore, they were stopped either by the addition of excess EGTA or by the addition of methanol or acetonitrile. In some cases, samples were diluted with HPLC-grade methanol containing the internal standard, didansylcadaverine. Otherwise, didansylcadaverine was used as an external standard. In general, the agreement for samples run on a single day was excellent and internal standard was not required. Proteins, precipitated by the addition of methanol, were removed by centrifugation or filtration while the reaction product remained in solution. A complete recovery of the product was verified both by HPLC and by chromatography on thin layers of polyamide. The amount of product fluorescence measured was linear with respect to time (Fig. 3A) and with respect to amount of enzyme (Fig. 3B) until the concentration of the limiting substrate, monodansylcadaverine, decreased significantly. These findings indicate that the fluorescence response is proportional to the concentration of the product. That the calibration curve established with the reaction product, Z-Glu(MDC)Gly, is linear is in accordance with these observations and confirms the linearity of the detector response. The standard, didansylcadaverine, also displays a linear response of fluorescence (total peak area) with concentration and is useful for quantitation and for determining the amount of sample injected. The molar fluorescence ratio of didansylcadaverine standard to reaction product, calculated from these curves to be 1.4, also provides a means for obtaining quantitative data and for the calculation of specific activity. For the determination of sensitivity, an aliquot of the standard solution of product was diluted and ana-
FLUOROMETRIC
CHROMATOGRAPHIC
fy-NH2
ASSAY
FOR
f3 0
H2N(CH&NH-S=
a YH2?
273
TRANSGLUTAMINASE
F H2
N(W)2
CH20-C-NH-CH-C-NHCH2C02H
MDC
Z-Gin-Gly
CH20-C-NH-CH-C-NHCH2C02H
+
NH3
Z-GIu(MDC)-Gly FIG.
1.
Reaction
scheme
for the incorporation
lyzed by HPLC. By operating at the edge of sensitivity of the fluorometer, a signal-to-noise ratio for the solution studied was determined. The signal-to-noise ratio at different concentrations of product was obtained and plotted. By taking the detection limit to be the concentration of product that produced a signal-to-noise ratio of 3, the detection limit was found to be 31 pmol of product per injection. This assay is also applicable to the measurement of transglutaminase activity in biological samples. When
of MDC
into
Z-Gln-Gly
by transglutaminase.
the supernatant from crude homogenates of chicken liver was used as the source of enzyme, the product fluorescence for Z-Glu(MDC)Gly as a function of time was linear for 30 min with the reaction conditions used (Fig. 3A). Kinetic experiments in which the concentration of ZGln-Gly was varied from 1 to 80 mM gave a value of 34.7 f 2.4 InM (mean + SE) for the apparent Km for purified guinea pig liver transglutaminase under the conditions described under Materials and Methods. Although other transglutaminases such as factor XIIIa do not act on Z-Gln-Gly, somewhat larger, synthetic peptides are
DDC
J 1 J
$ 5
65-
:: 2! 0 3
45
-
ii .-:
25-
ii 5 a
5-
Product
0123456
Time
(min)
FIG. 2. HPLC elution profile of reaction product (Z-Glu(MDC)Gly), and DDC (internal standard). Methanol/water (X)/50) as the mobile phase. MDC is retained on the column. For further details, see Materials and Methods.
0
20 Time
40 (min)
60
0.01
0.02
Enzyme
0.03
0.04
(mg/ml)
FIG. 3. (A) A plot of relative fluorescence versus time for the formation of Z-Glu(MDC)Gly by purified guinea pig liver enzyme (closed symbols) and by supernatant from crude homogenates of chicken liver (open symbols). (B) Relative fluorescence versus enzyme concentration for purified liver transglutaminase. Incubations were performed at 37°C for 10 min with 8 mM Z-Gln-Gly and 2 mM MDC as described under Materials and Methods. The activity corresponds to the production of 3.5 pmol Z-Glu(MDC)Gly/min/mg enzyme.
274
FINK,
SHAO,
AND
TABLE Glutamine-Containing
Glutamine-containing
Peptides
as Potential
peptide
Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuMet-NH, cY-Endorphin: Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-SerGln-Thr-Pro-Leu-Val-Thr Arg-Leu-Leu-Gln-Gly-Leu-Val-NH, His-Ser-Gln-Gly-Thr-Phe Z-Gln-Gly [Glne]-LH-RH: p-Glu-His-TrpSer-Tyr-Gly-Leu-Gln-ProGly-NH, [Gln4]-Neurotensin: p-Glu-Leu-Tyr-Gln-Asn-Lys-Pro-ArgArg-Pro-Tyr-Ile-Leu
KERSH
1 Substrates
Mobile
Transglutaminase” Retention time (min)’
phaseb
40-100% B, (15 min) 35% B 50% 35% 50%
B B c
lo-loo% B (20 min) lo-100% B (20 min)
’ Reaction conditions described under Materials and Methods. b The mobile phase consists of a pair of solvents: A/B or C/D. A, water containing methanol; D, unbuffered water. ’ Retention time given for product formed from peptide and monodansylcadaverine. d No product detected with these assay conditions. Peptide concentration was 0.66
available as substrates (19-21). The assay described in this report is applicable to the evaluation of other transglutaminase enzymes through the use of suitable synthetic substrates. By changing the elution solvent from methanol/water to acetonitrile containing 0.1% TFA/water with 0.1% TFA, the assay described with Z-Gln-Gly as substrate can be applied to evaluate various peptides as substrates for transglutaminase. Substance P, reported as a transglutaminase substrate (22,23), was determined to have an apparent K,,, of 0.17 f 0.02 mM (Table 1). Gradient elution is used for the separation of the product with substance P and monodansylcadaverine as substrates. With these conditions, monodansylcadaverine is not retained on the column, but elutes at 2.7 min. Didansylcadaverine, the external standard, has a retention time of 8.7 min. The amount of product fluorescence measured was linear with respect to time until the concentration of substrate, substance P, decreased significantly. An apparent K,,, of 0.48 f 0.02 mM was determined for cY-endorphin and separation of the product from the transglutaminasecatalyzed incorporation of monodansylcadaverine into cw-endorphin is carried out with isocratic conditions as shown in Fig. 4. In these reactions, no correction was made for intramolecular or intermolecular crosslinking reactions, and in fact, with longer reaction times, insoluble, fluorescent material formed. At a concentration of substrate (0.62 InM) and enzyme (50 pg/ml) that results in the formation of large amounts of product as determined by relative fluorescence with substance P and a-endorphin as substrates, no product could be determined with either [Gin*]-neurotensin or [Gin’]-luteinizing hormone-releasing hormone as substrate. With conditions in which all of the substrate, substance P, was
for
0.1%
8.7
0.17 f 0.02
5.7
0.48 + 0.02
4.5 4.5 3.8
6.7
9.1 34.7
No product
detectedd
No product
detectedd
TFA,
B, acetonitrile
containing
0.1%
f 0.6 k 2.2 + 2.4
TFA,
C,
mM.
used (0.1 mg enzyme, 12 h reaction time), no product could be detected with [Gln8]-luteinizing hormone-releasing hormone as substrate. Under these conditions, with [Gln4]-neurotensin as substrate, insoluble fluorescent material formed, but no soluble fluorescent product was detected. Two synthetic peptides, ArgLeu-Leu-Gln-Gly-Leu-Val-NH, and His-Ser-GlnGly-Thr-Phe, were found to be substrates (Table 1). We have developed a model system for the evaluation of potential inhibitors of transglutaminase based on the assay with Z-Gln-Gly and monodansylcadaverine as
a3 .? z 5 K
25
5
I
I
I
1
I
I
0
2
4
6
8
1012
Time
I
(min)
FIG. 4. HPLC elution profile of reaction product with a-endorphin and MDC as substrates and DDC as external standard. Mobile phase consists of 35% acetonitrile (0.1% TFA) in water (0.1% TFA).
FLUOROMETRIC
TABLE
CHROMATOGRAPHIC
2
Aldehydes as Inhibitors of the Transglutaminase Reaction” % Inhibition Compound
10
Propanal Butanal Pentanal Hexanal 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal 2-Ethylbutanal 2-Methylpentanal 2,2-Dimethylpropanal 3,3-Dimethylbutanal a Conditions
as described
mM
5mM
100 100 100 100 100 100 100 100 100 100 80 under
Materials
50 19 24 24 40 42 90 95 95 65
16 and Methods.
ASSAY
FOR
275
TRANSGLUTAMINASE
sylcadaverine as substrates (Table 2). At 5 InM aldehyde concentrations, 2-ethylbutanal and 2-methylpentanal were most effective. While the aldehydes tested clearly inhibited transglutaminase, the results do not parallel those of the specificity of alkyl amide substrates or the inactivation pattern observed with alkyl isocyanates (3,27). Branched-chain amides in which branching occurs at the (Y or fl position to the carboxamide do not function as substrates, and structurally related, branched, alkyl isocyanates are not effective active sitedirected inhibitors while the straight-chain compounds are. Further studies are required to determine the mechanism of enzyme inhibition by aldehydes. ACKNOWLEDGMENTS The authors thank Dr. J. E. Folk for his helpful discussions. preliminary report of this work has been presented (2829).
A
REFERENCES
substrates. Serine in the presence of borate is an effective inhibitor of y-glutamyltranspeptidase (not a transglutaminase), an enzyme that acts on y-glutamyl compounds as substrates and catalyzes the utilization of glutathione (24). This inhibition is thought to act through the formation of a borate bridge complex between the serine hydroxyl and the enzyme active site nucleophile. In an effort to form an analogous complex with transglutaminase, serine was used to replace glutamine in the dipeptide substrate Z-Gln-Gly (Z-Ser-Gly). However, no inhibition of guinea pig liver transglutaminase activity was observed with a substrate concentration of Z-Gln-Gly of 5 mM and 10 mM Z-L-Ser-Gly in the presence of 10 mM sodium borate. In other, preliminary studies using this assay, we have demonstrated that alkyl aldehydes can inhibit the guinea pig liver enzyme. A variety of aldehyde compounds, structurally related to the acyl portion of substrates, are extremely effective inhibitors of serine and thiol proteases. Such inhibitors often result in the formation of hemiacetal or thiohemiacetal species with the enzyme (25,26). Although not a protease, transglutaminase has an active site thiol that participates in the formation of an acyl-enzyme thioester intermediate during the course of the reaction in a manner analogous to that of a thiol protease such as papain. Therefore, a series of aldehydes was tested for inhibition of transglutaminase. Multiple sets of reactions were carried out and the results from a typical set of data are shown in Table 2. Control reactions include those with no inhibitor, those with no enzyme, and controls with no substrate. From the controls, it was apparent that no significant amount of Schiff base formed from aldehyde and monodansylcadaverine under the conditions used. At concentrations of 10 InM aldehyde, both straight-chain aldehydes, such as butanal, pentanal, and hexanal, and compounds branched at the a-carbon inhibited the action of transglutaminase with Z-Gln-Gly and monodan-
1. Clarke, D. D., Mycek, M. J., Neidle, Arch. Biochem. Biophys. 79, 338-354.
A., and Waelsch,
H. (1959)
2. Folk,
J. E., and Finlayson, J. S. (1977) in Advances in Protein Chemistry (Anfinsen, C. B., Edsall, J. T., and Richards, F. M., Eds.), Vol. 31, pp. 1-133, Academic Press, New York.
3. Folk, J. E. (1983) Adu. Enzymol. Relat. Areas Mol. Biol. 54, l-56. 4. Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9-35.
5. Folk,
J. E., and Chung, S. I. (1985) in Methods (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. Academic Press, Orlando, FL.
6. Matacic, mun.
S., and Loewy, 30,356-362.
A. G. (1968)
Biochem.
in Enzymology 113, pp. 358-375, Biophys.
Res. Com-
7. Lorand,
L., Rule, N. G., Ong, H. H., Furlanetto, R., Jacobsen, A.. Downey, J., Oner, N., and Bnmer-Lorand, J. (1968) Biochemistry 7, 1214-1223.
8. Pisano, J. J., Finlayson, 160,892-893. 9. Rice, R. H., and Green,
J. S., and Peyton, H. (1977)
Cell
M. P. (1968)
Science
11, 417-422.
10. Williams-Ashman, H. G. (1984) Mol. Cell. Biochem. 58, 51-61. 11. Mosher, D. F., and Schad, P. E. (1979) J. Clin. Znuest. 64, 781-
787. 12. Siefring, G. E., Jr., Apostol, A. B., Velasco, (1978) Biochemistry 17, 2598-2604. 13. Birckbichler, and Carter,
P. T., and Lorand,
L.
P. J., Orr, G. R., Patterson, M. K., Jr., Conway, E., H. A. (1981) Proc. Natl. Acad. Sci. USA 78, 5005-
5008. 14. Lorand,
L., and Campbell,
L. K. (1971)
Anal.
Biochem.
44,207-
220. 15. Ando, Y., Imamura, S., Yamagata, Y., Kikuchi, T., Murachi, and Kannagi, R. (1987) J. Biochem. 101, 1331-1337. 16. Takagi, J., Saito, Y., Kikuchi, T., and Inada, Y. (1986) Anal. them. 153,295-298.
T., Bio-
17. Jeon,
W. M., Lee, K. N., Birckbichler, P. J., Conway, E., and Patterson, M. K., Jr. (1989) Anal. Biochem. 182,170-175.
18. Cleland, W. W. (1979) in Methods in Enzymology (Purich, D. L., Ed.), Vol. 63, pp. 103-138, Academic Press, New York. 19. Gorman, J. J., and Folk, J. E. (1980) J. Biol. Chem. 255,419-427. 20. Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-
2715. 21. Ichinose, 369-371.
A., Tamaki,
T., and Aoki,
N. (1983)
FEBS
Lett.
153,
276
FINK,
SHAO, AND KERSH
22. Gorman, J. J., and Folk, J. E. (1980) J. Biol. Chem. 255, 11751180. 23. Porta, R., Esposito, C., Metafora, S., Pucci, P., Malorni, A., and Marino, G. (1988) Anal. Biochem. 172,499-503. 24. Tate, S. S., and Meister, A. (1978) Proc. Natl. Acad. Sci. USA 75, 4806-4809.
25. Westerik, J. OX., and Wolfenden, 8195-8197.
R. (1972) J. Biol. Chem.
247,
26. Lewis, C. A., Jr., and Wolfenden, R. (19’77) Biochemistry 16, 4890-4895. 27. Gross, M., Whetzel, N. K., and Folk, J. E. (1975) J. Bid. Chem. 250, 7693-7699. 28. Fink, M. L., and Kersh, G. J. (1988) J. Cell Biol. 107,839a. [Abstract] 29. Fink, M. L., Shao, Y. Y., and Shey, J. (1990) J. Cell Biol. 111, 320a. [Abstract]
