kinetic studies with transglutaminases · kinetic studies with transglutaminases the human blood...

Txa JOUILWI. OR DIOLOGICAL CHEMISTRY Vol. 247, No. 9,Issue of May 10,~~. 2798-2807. 1972

Printed in U.S.A.

Kinetic Studies with Transglutaminases

THE HUMAN BLOOD ENZYMES (ACTIVATED COAGULATION FACTOR XIII) AND THE GUINEA PIG HAIR FOLLICLE ENZYME

Soo IL CHUNG AND J. E. FOLK

(Received for publication, November 15, 1971)

From the Laboratory of Biochemistry, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014

SUMMARY

The transfer reaction of [W!]methylamine into the acetylated B chain of oxidized insulin as catalyzed by several transglutaminases has been studied at pH ‘7.5 in the presence of calcium ion. The only radioactive product of the transfer reaction with each transglutaminase was identified as peptide-bound y-glutamic acid methylamide. Initial velocity and product inhibition patterns for the human plasma and guinea pig hair follicle transglutaminases are consistent with a ping-pong mechanism, previously proposed for guinea pig liver transglutaminase (FOLK, J. E. (1969) J. BioZ. Chem.

244, 3707) in which acyl enzyme formed from peptide-bound glutamine may react with water (hydrolysis) or with a primary amine (transfer). Hydrolysis and isotope exchange in the presence of one substrate and one product add substan- tial support for this mechanism. The pattern of calcium activation of plasma transglutaminase in the transfer reaction is in accord with an equilibrium-ordered activation mechanism in which the first substrate, acetylated B chain of oxidized insulin, adds only to enzyme-metal complex. The similarity in the kinetic constants obtained for human plasma and platelet transglutaminases adds support to recent evidence that the catalytic subunits of the plasma and platelet zymogens and enzymes are closely related (SCHWARTZ, M. L., hzzo, S. V., HILL, R. L., AND MCKEE, P. A. (1971) J. Biol. Chem. 246, 5851).

Improved purification procedures for plasma and platelet protransglutaminases are presented.

Transglutaminase of guinea pig liver catalyzes hydrolysis and transfer at the carboxamide group of peptide-bound glutamine residues (1, 2). Kinetic findings are in accord with a ping-pong mechanism (Mechanism I)

~ZECHANISM I

for these reactions (3). In this modified ping-pong mechanism the spontaneous breakdown of F to E results in a pattern of intersecting lines in double reciprocal plots when either Q or R is the product measured and in a pattern of parallel lines in these plots when P is measured and plotted against A ; nonlinear plots are obtained when P formed is plotted against B (3, 4). This is in contrast to the simple ping-pong mechanism in which a pattern of parallel lines is obtained in double reciprocal plots when any product is measured. The kinetic findings define glutamine substrate as A, the first substrate to add to transglutaminase, and ammonia as P, the first product formed. The acyl enzyme intermediate, F, may react with water to form peptide-bound glutamic acid, R, (hydrolysis) or with a primary amine, B, to form the peptide-bound y-glutamic acid amide, Q (transfer). The order of substrate additions has been confirmed by a direct binding study (3). Hydrolysis and transfer by transglutaminase of several active esters was shown to proceed by the same mechanism (4). Isolation of a stable trimethylacetyl enzyme following incubation of transglutaminase with p-nitrophenyl trimethylacetate supplied evidence in support of the acyl enzyme theory (5).

The transglutaminases from a number of organs, e.g. adrenal, spleen, and kidney, and from muscle of the guinea pig have been found to be identical with that of liver as defined by immunoas- say, specific enzymatic assay, ion exchange chromatography, and gel filtration (6).

The term plasma transglutaminase has recently been assigned on the basis of enzymatic specificity to the blood enzyme that catalyzes the formation of e-(y-glutamyl)lysine cross-links between fibrin monomers to produce insoluble fibrin (7). This enzyme occurs in blood as an inactive zymogen, plasma protransglutaminase (blood coagulaticn Factor XIII), that is activated by thrombin (for review see Reference 7). This is in contrast to liver transglutaminase for which there is no indication of a zymogen form. Pronounced differences are evident in the physicochemical and immunological properties of the liver and blood enzymes (6).

As a portion of a continuing program to define and charac- terize transglutaminases of tissues and organs we have isolated a low molecular weight transglutaminase from guinea pig hair follicle (8). This enzyme displays distinctly different physicochemical and immunological properties from those of the blood and liver enzymes.

On the basis of the above findings it was deemed worthwhile to determine the kinetic mechanisms of the blood and hair follicle enzymes. The results presented here serve as evidence that the mechanisms for these transglutaminases are identical with that

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Issue of May 10, 1972 8. 1. Chung and J. l3. P’ollc 2799

of the liver enzyme, show differences in terms of kinetic parame- ters among these enzymes, and supply further evidence that the enzymatic action of each enzyme is directed toward glutamine residues only.

EXPERIME4TAL PROCEDURE

Materials

[‘*C]Methylamine-HCl (52 pCi per pmole) was obtained from Amersham-Searle. The radioactive amine was purified by diffusion into dilute HCI. The excess acid was removed under vacuum over PZOs and KOH. The amine was diluted to the desired specific radioactivity by addition of nonradioactive methylamine- HCl and adjusted to the desired concentration by addition of water.

The B chain from oxidized insulin (9) was isolated by an ion exchange procedure (10) and was acetylated with acetic anhydride in half-saturated sodium acetate (11). Acetylated B chain displayed less than 5% of the color with ninhydrin given by equivalent amounts of unacetylated B chain, Stock solutions of acetylated B chain were prepared by dissolving the material in water and carefully adjusting the pH of the solutions to 7.5. The concentrations of acetylated B chain in stock solutions were determined from amino acid analyses (Beckman model 117 ana- lyzer) of acid hydrolysates (6 N HCI at 106” for 24 hours in sealed tubes under Nz).

The sample of y-L-glutamic acid methylamide was kindly supplied by Dr. L. Hersh. P-n-Aspartic acid methylamide was prepared as follows. To the mixed anhydride prepared from benzyloxycarbonyl-L-aspartic acid-a-benzyl ester (Cycle Chemi- cal Corp.) and isobutyl chloroformate in dioxane was added an excess of 40% aqueous methylamine. Crystalline benzyloxycarbonyl-n-aspartic acid /3-methylamide-a-benzyl ester was obtained from ethyl acetate-pentane in 40% yield, m.p. 142”.

CdL~O~N~ (270.4)

Calculated: C 64.9, H 6.0, N 7.6 Found : C 64.9, H 6.0, N 7.6

The blocking groups were removed from the above derivative by hydrogenation with palladium black catalyst. Crystalline P-L-

aspartic acid methylamide was obtained from water-acetone in 80% yield, m.p. 250” with decomposition.

CJH~~NQO~ (146.2)

Calculated: C 41.1, H 6.9, N 19.2 Found : C 40.7, H 6.9, N 18.9

Dithiothreitol was obtained from Calbiochem; Bio-Gel A-0.5m (200 to 400 mesh) and A-5m (200 to 400 mesh) from Bio-Rad; leucine aminopeptidase from Worthington. Porcine chymotrypsin C was prepared by a published procedure (12). Purified human thrombin was a gift from Dr. D. L. Aronson. Millipore filters were 0.45 p pore size from Millipore Corp.

Guinea pig liver transglutaminase was prepared from fresh tissue by a published procedure (13). The purified enzyme showed 95 =t 5% of the reported specific activity when assayed by hydroxamate formation with the specific substrate benzyloxycarbonyl-L-glutaminylglycine (14). Enzyme concentration was determined by the use of the E,,, I% of 15.8 (14).

The transglutaminase of guinea pig hair follicle was prepared by a procedure described in detail elsewhere (8). In brief the

procedure involves gel filtration of hair follicle homogenate on 6% agarose, removal of contaminating proteins by passage through a column of DEAE-cellulose, chromatography on CM- cellulose,i and finally gel filtration on 10% agarose. The preparations of hair follicle transglutaminase, isolated and purified by this procedure, were judged 90 f 5% homogeneous by several criteria.

Plasma protransglutaminase (plasma coagulation Factor XIII) was isolated from 95% clottable human fibrinogen that was prepared from fresh acid citrate dextrose (USP Formula A) plasma by the glycine precipitation method (15). Fibrinogen prepared by this procedure contained 80 to 85% of the protransglutaminase initially present in the plasma from which it was prepared. This protransglutaminase-rich fibrinogen was subjected to the heat treatment of Loewy, Veneziale, and For- man (16) (exactly 3 min at 56”) and a partially purified protransglutaminase was obtained by the polyethylene glycol 6000 precipitation method of Kazama and Langdell (17). Further purification was obtained by gel filtration on 6% agarose (Bio- Gel A-5m) in 10 mM Tris-acetate, pH 7.5, containing 0.3 M

NaCl and 1 mM EDTA at 4”. Two peaks of protein with dis- tribution coefficients (Ko) of 0.27 and 0.35 were eluted from agarose. The first peak (K. of 0.27) to emerge from the column contained all of the potential enzymatic activity as measured by [14C]putrescine incorporation into casein after activation with thrombin (18, 19). Fractions of the highest and equal specific activities based on the absorbance at 280 nm were combined, dialyzed against 10 mM Tris-acetate, pH 7.5, containing 1 mM

EDTA, and lyophilized. The over-all recovery of potential enzymatic activity from fresh plasma ranged from 60 to 70% in several preparations. The dried zymogen preparations retained their full potential activity upon storage at -20” for as long as 6 months. Plasma protransglutaminase prepared by this procedure showed a single peak in the ultracentrifuge. A single band was observed on polyacrylamide gel electrophoresis. For the enzymatic experiments reported here an EiR of 15 was assumed. The zymogen (1 mg per ml) was fully activated by incubation with thrombin (2 U.S.P. units per ml) for 30 min at 37” in 0.1 M Tris-HCl, pH 7.5, containing 1 mM dithiothreitol. Solutions of the active enzyme were stored at 0” for periods up to 2 hours. No detectable loss in activity was observed during this time, the limit of duration of the kinetic experiments.

Platelet protransglutaminase (platelet coagulation Factor XIII) was isolated in the following manner. Platelet-concentrated plasma, obtained from the National Institutes of Health blood bank within 24 hours after preparation, was centrifuged for 1 min at 1,500 x g to remove residual erythrocytes. The platelets were washed three times with 10 mM Tris-acetate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA, by suspending them in the buffer mixture and centrifuging. The washed platelets (20 g, wet weight) were homogenized in 100 ml of 10 mM Tris-acetate, pH 7.5, containing 1 mM EDTA for 3 mm at 0” by the use of a Polytron PT 10 OD homogenizer (Brinkmann Instruments) at low speed. The homogenate was centrifuged for 1 hour at 105,000 x g. This and the following operations were carried out at O-4”. The clear supernatant fluid was applied to a column (2.5 x 25 cm) of DEAE-cellulose (DE-52, Whatman) that had beerrequilibrated with 5 mM Tris-acetate, pH 7.5, containing 1 mM EDTA. The chromatogram was developed by the use of a linear gradient of 0 to 0.5 M NaCl in the

1 The abbreviations used are: CM-cellulose, carboxymethyl- cellulose; PTH, phenylthiohydantoin.


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equilibration buffer. The zymogen was eluted between 0.12 and 0.2 M NaCl as measured by [i4C]putrescine incorporation into casein after activation by thrombin (18, 19). Precipitation of the protransglutaminase was affected by 0.4 saturation with (NH4&S04 (24.3 g/100 ml). The precipitate obtained by centrifugation for 30 min at 14,600 x g was dissolved in 2 to 4 ml of 10 mM Tris-acetate, pH 7.5, containing 0.3 M NaCl and 1 mM EDTA and subjected to gel filtration on a column (2.5 x 96 cm) of 6% agarose as outlined for the plasma zymogen. All of the potential enzymatic activity emerged from the column in the last protein peak (K,, 0.34) before the salt fraction. The fractions of highest and equal specific activities were combined and the zymogen was precipitated by 0.4 saturation with (NH&S04. After dialysis against 10 mM Tris-acetate, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA, the concentrated zymogen solution was stored frozen at -20”. Platelet protransglutaminase prepared by this method showed a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This zymogen was more labile than that from plasma. After frozen solutions were thawed the precipitate that usually appeared was removed by centrifugation before aliquots were taken for kinetic studies. The zymogen (1.5 mg per ml) was fully activated by incubation with thrombin (1 U.S.P. unit per ml) for 10 min at 0’ in 0.1 M

Tris-HCl, pH 7.5, containing 1 JIIM dithiothreitol. Solutions of active enzyme were used within 10 min after activation. No loss in activity was observed during this time. Attempts to activate the zymogen at higher temperatures and lower thrombin levels resulted in significant losses in enzymatic activity even during a lo-min period. For the present experiments an El,% of 15 was assumed.

Other materials and reagents have been described in previous publications (3, 4, 13).

Methods

Rate studies were carried out at 37” in 0.1 M Tris-chloride buffer, pH 7.5, containing 1 mM EDTA, 20 mM CaC12, and 1 mM dithiothreitol except where otherwise stated. For measurement of [‘4C]methylamine incorporation into the acetylated B chain of oxidized insulin reaction mixtures of 0.1 ml were incubated for periods of 1 to 20 min. Reactions were terminated by the addition of 5 ml of 5% trichloroacetic acid solution. The B chain and radioactive product were quantitatively collected on Milli- pore filters and freed of [14C]methylamine by washing four times with 5- to lo-ml portions of 5% trichloroacetic acid solution. The washed filters were dissolved in 10 ml of methanol-dioxane- counting fluid (20) and radioactivity was measured by the use of a Beckman LS-233 liquid scintillation spectrometer.

Release of ammonia was used as a measure of hydrolysis of the carboxamide group of the acetylated B chain of oxidized insulin. Reaction mixtures of 0.5 ml were incubated for periods of 10 to 30 min. Aliquots of 0.1 ml were removed into diffusion jars and reactions were terminated by addition of 0.5 ml of saturated Na2C03. Ammonia collected in dilute HzS04 in the usual manner was measured by Nesslerization.

Conditions were adjusted so that no more than 5% of the substrate of lowest concentration was converted to product within the reaction periods employed. Supplementary experiments showed that with each enzyme the amount of product formed was linear with time during the experimental period.

The nomenclature used is, in general, that of Cleland (21). For clarity, the definitions of certain kinetic constants for Mecha-

nism I, given elsewhere in terms of rate constants (3), are also listed in the footnote to Table II or in the text. The kinetic constant subscript i designates an inhibition constant, h the Michaelis constant for hydrolysis, and t the Michaelis constant for transfer.

Analysis of Results

Reciprocal velocities were first plotted graphically against the reciprocals of substrate concentration. These plots were linear in all cases. The data were fitted to Equation 1 assuming equal variance for the velocities.

VA V=K+A

All fits were performed by means of an interactive curve-fitting program, MLAB, developed at National Institutes of Health and running on the PDP-10 digital computer (22). Slopes (K/v) and intercepts (1/V) obtained from fits to Equation 1 were plotted graphically against the reciprocal of the nonvaried substrate concentration for initial velocity experiments, against the reciprocal of the activator concentration for activation experiments, or against the inhibitor concentration for inhibition experiments in order to determine the form of the over-all equation. Final estimates of kinetic constants were made by fitting the data points to the appropriate over-all equation. Data for initial velocity of [r4C]methylamine incorporation into acetylated B chain of oxidized insulin in each case were found to conform to Equation 2. Data conforming to linear competitive inhibition were fitted to Equation 3; those conforming to linear noncompetitive inhibition were fitted to Equation 4; those for activation were found to conform to equilibrium-ordered activation and were fitted to Equation 5.

(2)

VA ’ = K(1 + I/KiJ + A (3)

VA ’ = K(1 + I/.&J + (1 + I/KiJA

VAM ’ = K,K;, + K,M + AM

(5)

The points of the double reciprocal plots shown are the experimental values. The lines drawn through the points are those calculated from fits to Equation 1 except where stated otherwise.

RESULTS

Preliminary Substrate Specijicity Examinations-The glutamine peptide derivative, benzyloxycarbonyl-L-glutaminylglycine, and several active esters have proven to be valuable substrates for studies on the mechanism of guinea pig liver transglutaminase (3,4). It has been reported that the ethyl ester of benzyloxycarbonyl-n-glutaminylglycine does not act as a substrate for plasma transglutaminase (23). We have found that under conditions where benzyloxycarbonyl-n-glutaminylglycine serves as an efficient substrate for the liver enzyme (50 mM glutamine peptide and 1 InM [i4C]methylamine in 0.1 M Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 rnM dithiothreitol, and 20 mM CaClt at 37”) the plasma, platelet, and hair follicle transglutaminases show no catalytic activity toward this peptide derivative. No amine in-


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Issue of May 10, 1972 8. I. Chung and J, E. Polk 2801

corporation into the peptide was observed after 2 hours incubation at levels of enzymes as high as 0.2 mg per ml. Further, two active esters that function as efficient substrates for the liver enzyme were not hydrolyzed by, or observed to participate in transfer reactions with, the plasma, platelet, or hair follicle enzymes. These active esters, p-nitrophenyl acetate and benzyloxycarbonyl-oc-L-glutamyl(y-p-nitrophenyl ester)glycine, in which struc- tural features of both active ester and glutamine substrate are contained, were tested at high enzyme levels (0.02 to 0.2 mg per ml).

TABLE I Identijication of amino acid formed by [14C]methylamine incorpora-

tion into acetylated B chain of oxidized insulin by

transglutaminases Incorporation mixtures (0.5 ml) contained 1.8 mM acetylated

B chain of oxidized insulin, 3.6 mM [l%]methylamine (10 &i per pmole), 20 mM CaC&, 1 mM EDTA, and 1 mM dithiothreitol in 0.1 M Tris-HCl, pH 7.5. Thrombin-activated plasma and platelet protransglutaminases were used at levels of 0.2 and 0.08 mg per ml, respectively; hair follicle enzyme at 0.2 mg per ml. In- corporations were carried out for 3 hours at 37”. Following this incubation period the acetylated B chain and labeled product were separated from reagents by gel filtration on Sephadex G-25 (fine) in 0.1 M NHaHCOa. Estimates of percentage of product formed, made from ratios of radioactivity in the B chain region and in the salt region, were: 30’% with the plasma enzyme; 20% with the platelet enzyme; 20% with the hair follicle enzyme. The fractions containing the labeled product were combined, lyophilized, and taken up in 0.5 ml of 0.05 M Tris-HCl, pH 7.5. Chymotrypsin C (0.2 mg) was added. After 2 hours at 25” 0.1 ml of a solution of carboxypeptidase A (1 mg per ml in 10% LiCl) was added and digestion was allowed to proceed for 18 hours at 25”. Pronase (0.1 mg) was added. After 2 hours at 37” the digestion mixture was made 5 mM in MgCI, and 0.2 mg of leucine aminopeptidase was added. Digestion was continued for 5 hours at 37”. The digests were diluted to 5 to 6 ml, 1 g of dry Dowex 50 ion exchange resin (H+, X8, 20 to 50 mesh) was added and the mixtures were agitated gently until most of the radioactivity was removed from the supernatant fluids. The resin beads were washed well with water and the radioactive material was eluted with three 2-ml portions of 10% NHdOH. The combined eluates from each digest were taken to dryness in a stream of air and the residues dissolved in O.l-ml portions of HzO. Aliquots of 5 to 10 ~1 were applied to Whatman No. 1 filter paper for descending chromatography. Radioactivity was located on dried chromatograms by cutting 0.5-cm sections, covering the individual sections with counting fluid in counting vials, and subjecting them to scintillation counting. The recovery of radioactivity in the single area of the chromatograms after development approximated 100% of that in controls in which samples were applied to paper and counted without chromatographic development.

Amino acid

p-L-Aspartic acid methylamide. ...... r-L-Glutamic acid methylamide ...... In digests of B chain labeled by

Plasma enzyme. ................... Platelet enzyme .................... Hair follicle enzyme ................

Rp value

0.45 0.68 0.64 0.84

0.64 0.84 0.64 0.84 0.64 0.84

-

I C’

i

Phenol- IzO-CO& e&rated VHIOH 160:40: 1

-- 0.71 0.86

0.86 0.86 0.86

A survey of several higher molecular weight glutamine-containing peptides and derivatives showed that the B chain of oxidized insulin, which contains a single glutamine residue, serves as a substrate for all of the transglutaminases. That the B chain also contained a single asparagine adjacent to the glutamine offered a unique opportunity to determine with each of the enzymes whether their catalytic action is directed toward the carboxamide group of the glutamine residue only. The B chain was modified by acetylation in order to mask the a-amino group, as well as the single e-amino group of lysine, either or both of which might participate in the transfer reaction. An added ad- vantage in the use of the modified B chain was that it is quanti-

‘AC B CHAIN (mM!

FIG. 1. Initial velocity pattern for plasma transglutaminase with acetylated B chain of oxidized insulin (AC B chain) as varied substrate. The [14C]methylamine concentrations were: (1) 1, (.2), 0.5, (9) 0.33, (4) 0.25, (6) 0.2 mM. Initial velocities are given in millimicromoles of labeled acetylated B chain formed per min (per 0.1 mg of thrombin-activated proenzyme).

“AC B CHAIN (mh4)

FIG. 2. Initial velocity pattern for hair follicle transglutaminase with acetylated B chain of oxidized insulin as varied substrate. The [14C]methylamine concentrations were: (1) 1.16, (2) 0.58, (3) 0.387, (4) 0.29, (5) 0.23 mM. Initial velocities are given in millimicromoles of labeled acetylated B chain formed per min (per 0.1 mg of enzyme).


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2802 Kinetic Sludies with l’ransglutami?zases Vol. 247, No. 9

tatively precipitated by 5% trichloroacetic acid and, thus, is easily sepa,rated from soluble radioactive amine.

Identification of Product of Transfer Reaction-The site of [Wlethanolamine incorporation into the B chain of oxidized

0 75

r

- 1.0 -0.5 0 0.5 1.0 1.5 2.0 2.5

“AC 8 CHAIN (mM)

FIG. 3. Initial velocities of ammonia formation from acetylated B chain of oxidized insulin as catalyzed by plasma transglutaminase. Velocities are given in millimicromoles of ammonia liber- ated per min (per 66 pg of thrombin-activated proenzyme).

insulin by liver transglutaminase has been identified as the carboxamide group of the glutamine residue (2). In the present study (Table I) [14C]methylamine incorporation into the acetylated B chain by plasma, platelet, and hair follicle transglutaminases was examined. Digestion of the labeled B chain samples with chymotrypsin C and carboxypeptidase A, as outlined earlier (2), failed to release all of the amino acid that was formed by the transglutaminase-catalyzed [14C]methylamine incorporation. A portion of the radioactivity appeared in the area corresponding to y-glutamic acid methylamide in five thin layer chromatography systems in each of which y-glutamic acid methylamide and ,&aspartic acid methylamide moved to the identical position. However, in each case some radioactivity was observed below this area in the chromatograms. Therefore, digestion was continued with Pronase and leucine aminopeptidase until a single area of radioactivity corresponding to the methylamides was obtained in all of the thin layer systems. Chromatography of these digests on paper showed single radioactive areas of RF identical with y-glutamic acid methylamide (Table I).

Initial Velocity Patterns-When the acetylated B chain of oxidized insulin and methylamine were used as substrates and methylamine incorporation was measured, intersecting initial velocity patterns were obtained. Those for plasma transglutaminase and the hair follicle enzyme are shown in Figs. 1 and 2, respectively. The patterns for the liver and platelet enzymes were of identical form and also appeared to intersect on the horizontal

TABLE II Kinetic constants for transglutaminases with acetylated B chain of

oxidized insulin and methylamine at pH 7.6 and 87’

The constants are assigned on the basis of Mechanism I where A is acetylated B chain; B, methylamine; P, NHa; &, labeled acet- plated B chain. Values for V, were estimated from the equality, Kahvab = K,tVG; those for Kibb from the equality, K,lK:bb = KoiJ(bl. The two values recorded for Kdi, and V, for the plasma enzyme were obtained from the transfer and hydrolysis reactions, respectively. V, and V,b are expressed in millimicromoIes per min (per mg of enzyme or per mg of thrombin-activated zymogen in the case of the blood enzsmes).

Transglutaminase

Plasma (human)

Platelet (human) Hair follicle (guinea pig) Liver (guinea pig)

Constant”

&h &t Kibt Kbt V, vab

ma WtM eL?4 m‘H

2.28 f 0.26 1.85 f 0.21 0.74 f 0.09 0.62 5 0.08 175 f 25 142 f 11 1.70 f 0.35 139 f 17 1.90 ZlZ 0.45 2.15 f 0.37 0.58 rt 0.11 0.65 f 0.15 222 zk 50 240 f 22 0.49 f 0.04 0.47 * 0.03 1.04 f 0.1 1.07 f 0.06 40 f 3 38.3 f 1.2

0.075 zk 0.008 0.082 f 0.006 0.26 i 0.03 0.28 i 0.02 230 z!z 25 250 i 8

a The kinetic constants are defined as (3) K

a* = h(k2 + ka)

kdkl + ks) (2a)

K _ kdh + k3)

at - hoc3 + k7)

Kbt = &a + h)(ka +ks)

kdh + Jd

(2b)

(2c)

(2d)

(24


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Issue of M:ty 10, 1972 2803

4- 4

3

2

I

-2.0 -1.0 0 1.0 2.0 3.0 4.0 5.0

‘/CH3NH2 (mM 1

FIG. 4. Product inhibition pattern for plasma transglutaminase with [l%]methylamine as the varied substrate and ammonium chloride as the inhibitor. The acetylated B chain concentration was 0.5 mM. The ammonium chloride concentrations were: (1) 0, (2) 0.1, (8) 0.2, (4) 0.3 mM. Initial velocities are given in millimicromoles of labeled acetylated B chain formed per min (per 0.1 mg of thrombin-activated proenayme).

-1.0 0 IO 2.0 3.0

‘/AC B CHAIN (mM)

FIG. 5. Product inhibition pattern for plasma transglutaminase with acetylated B chain (AC B) as varied substrate and ammonium chloride as inhibitor. The [%]methylamine concentration was 0.33 mM. The ammonium chloride concentrations were: (1) 0, (2) 0.1, (3) 0.2, (4) 0.3 mM. Initial velocities are given in millimicromoles of labeled acetylated B chain formed per min (per 0.1 mg of thrombin-activated proenzyme).

axis. The kinetic constants obtained from fits to Equation 2 of the data obtained with each of these enzymes is given in Table II.

When the acetylated B chain of oxidized insulin was used as a substrate and ammonia liberation was measured, the substrate- velocity relationship of Fig. 3 was found with the plasma enzyme. The constants obtained by fits of the data of Fig. 3 to Equation 1 are given in Table II. The platelet, hair follicle, and liver enzymes were also observed to catalyze ammonia liberation from

TABLE III Transglutaminase-catalyzed 15N incorporation into glutamine carboxamide group of acetylated B chain of oxidized insulin

Incorporation mixtures (0.4 ml) contained 2.5 mM acetylated B chain and 50 mM 16NHdCl (Miles Laboratories, 98.1 atom %) in the buffer mixture given in Table I. The enzyme concentrations were: plasma and platelet transglutaminases, 0.1 and 0.05 mg of thrombin-activated proenzyme per ml, respectively; hair follicle enzyme, 0.1 mg per ml. Incubation was conducted at 37” for the time indicated and reactions were stopped by the addition of 0.1 ml of 1 M EDTA. The acetylated B chain and labeled product were separated from reagents by gel filtration on Sephadex G-25 (fine) in 0.05 M Tris-HCl, pH 7.5. The fractions containing the acetylated B chain were combined, lyophilized, and taken up in l-ml portions of water. Chymotrypsin C (0.25 mg) was added. After 2 hours at 25” a second and equal portion of chymotrypsin C and 0.1 ml of a solution of carboxypeptidase A (1 mg per ml in 10% LiCl) were added. Digestion was allowed to proceed for 3 additional hours at 25”. The digests were lyophilized and the dry residues were dissolved in 3-ml portions of a pyridine-water mixture (1:1 v/v) containing 2% triethylamine. Phenyl isothio- cyanate (0.3 ml) was added and the reaction mixtures were incubated under Nz for 2 hours at 37”. The solvents and reagents were removed under high vacuum at 60”. The residues were dissolved in 0.4 ml of trifluoroacetic acid and incubated under Nz at 40” for 15 min. Formed phenylthiocarbamylamino acids were extracted into ethylene chloride (12 ml) and the solvent was removed in a stream of Nz. Conversion to phenylthiohydantoins (PTH-amino acids) was carried out in 0.8 ml of 1 N HCl at 80” for 10 min under Nz. The PTH-amino acids were extracted with two 4-ml portions of ethyl acetate and the combined ethyl acetate extracts were taken to a small volume in a stream of NQ. The en- tire extract from each incubation was applied as a streak to a lo-cm thin layer chromatography plate (Brinkmann F-2, 0.25 mm) and each chromatogram was developed with chloroform-methanol (9O:lO). The area corresponding to PTH-glutamine (RF 0.3) was scraped from the dried plate, extracted with ethyl acetate, and the extract was taken to dryness in a stream of Ns. These samples were subjected to chemical ionization mass spectrometry in an MS-9 mass spectrometer by a published method (24). The percentage of isotope enrichment is defined as follows (25) where P refers to the height of the peak at m/e = P = 264 (M $ l)+

I rel = [(P -t 1)lPl100% y0 isotope enrichment = (labeled Irel) - (unlabeled Zrel)

Transglutaminase merit in PTH-

Plasma

Platelet

Hair follicle

Control (without enzymes)

min % 0 0 9 2.5

60 16.9 300 31.0

0 0 300 22.0

0 0 300 42.9

300 0.6

the acetylated B chain. The dependency of velocity on substrate concentration was not examined with these enzymes.

Product Inhibition Patterns-With each of the transglutaminases ammonium chloride showed linear competitive inhibit’ion when methylamine was the varied substrate. The pattern for this inhibition with the plasma enzyme is shown in Fig. 4. Ki,


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2804 Kit&c S’tudies wdh Transglu%aminasas Vol. 247, No, 9

values from fits to Equation 3 for the plasma and hair follicle enzymes were obtained at several concentrations of the acetylated B chain. These values were found in each case to be inde- pendent of the concentration of acetylated B chain. At 2,1, and

I

15 20 25

‘/Ac B CHAIN (mM)

FIG. 6. Inhibition pattern for plasma transglutaminase with acetvlated B chain (AC B) as varied substrate and benzvloxvcar- bonyl-n-glutaminyl&ycine as inhibitor. The [W]me<hyla”mine concentration was 1 mM. The benzyloxycarbonyl-n-glutaminylglycine concentrations were (1) 0, (2) 5, (3) 10, (4) 15 mikr. Initial velocities are given in millimicromoles of labeled acetylated B chain formed per min (per 0.1 mg of thrombin-activated proen- 5yme).

0.5 mM acetylated B chain values for Ki, of 0.115 5 0.004, 0.110 f 0.005, and 0.113 f 0.004 mM, respectively, were obtained with the plasma enzyme. Values for K;, for the hair follicle enzyme of 0.44 i 0.06 and 0.41 f 0.08 mM were obtained at 2 and 0.66 mM acetylated B chain, respectively.

With acetylated B chain as variable substrate, ammonium chloride gave simple linear noncompetitive inhibition with the plasma enzyme (Fig. 5). Values for Ki, and Kii from fits to Equation 4 appeared to vary with the concentration of methylamine. Ka and K;J values of 0.23 f 0.05 and 0.19 i 0.04 mM

were found at 0.33 mM methylamine whereas K,, and Kii values of 0.30 i 0.05 and 0.29 f 0.05 mM were obtained at 1 mM methylamine.

Isotope Exchange-The data of Table III clearly show an isotope exchange between 1SNH4Cl and the glutamine carboxamide- NH2 of the acetylated B chain as catalyzed by the blood and hair follicle enzymes.

In the procedure outlined in Table III for enzymatic release of free glutamine from acetylated B chain the use of leucine aminopeptidase was avoided because of possible contamination of this enzyme with the glutamine-degrading enzyme, glutaminase (26). Release of glutamine from the B chain of oxidized insulin was ac- complished previously with chymotrypsin C and carboxypeptidase A (27). The use of these enzymes proved satisfactory in the present experiment (Table III).

In chemical ionization mass spectrometry used for isotope identification (Table III), PTH-glutamine (glutamine phenylthiohydantoin) gives a clearly defined Q&I+ ion at m/e (M + l)+ 264 (24). Preliminary separation of PTH-glutamine from PTH- glutamic acid and the e-phenylthiocarbamyl derivative of PTH- lysine was important since the latter two compounds give ions at m/e 264 and m/e 265, respectively (24). This was readily ac-

3Or

I I t I t -0.5 0 0.5 1.0 1.5 2.0 2.5 -1.0 0 1.0 2.0 3.0

“AC EI CHAIN (mM) ‘/Ca++hM)

FIG. 7. Metal activation pattern for plasma transglutaminase thrombin and then freed of EDTA by passage through Sephadex (A) with acetylated B chain (AC B) as the varied substrate and G-25 in the assay buffer mixture. The enzyme was used at 0.1 Ca++ as the activator, and (B) with Ca* as the varied activator mg of thrombin-activated proenzyme per ml. Initial velocities and acetylated B chain as the substrate. The ]%Jmethylamine are given in millimicromoles of labeled acetylated B chain formed concentration was 1 mM. The CaClz concentrations in A were per min. This figure is a tracing from a Calcomp plotter repro- (f) 5, (2) 3.33, (a) 1, (4) 0.5, (b) 0.33 mM. The acetylated B chain duction of the graphic display showing the fit of the data points concentrations in B were (1) 2.5, (2) 1.25, (3) 0.835, (4) 0.625, (5) to the reciprocal forms of Equation 5. The lines were constructed 0.418 mM. The assays were conducted at 37” in 0.1 M Tris-HCl from values for the constants, K, = 1.73, V = 8.90, Kim = 1.66 containing 1 mM dithiothreitol. The enzyme was activated by obtained from the fit of the data points to Equation 5.


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Issue of May 10, 1972 8. I. Chung and J. E. Polk 2805

TABLE IV Inhibition of hair follicle transglutaminase by EDTA and activation

by Ca++ Assays were carried out at 37” in solutions of 0.66 mM acetylated

B chain and 0.33 rnM [l%]methylamine in 0.1 M Tris-HCI, pH 7.5, containing 10 PM dithiothreitol. The hair follicle enzyme was gel filtered on Sephadex G-25 in the assay buffer and was used at the level of 0.1 mg per ml.

Additions Initial velocity

m~mole/min

None ..................................... 2 1llM CaCL. ..............................

1mMEDTA.. ........................... 1 mM EDTA + 2 mu dithiothreitol.. ...... 1 mM EDTA + 2 mM CaCh. ..............

0.4 0.57 0 0 0.57

more reactants, whereas in sequential mechanisms exchange is observed only in the presence of all reactants (28). The hydrolysis of glutamine substrate, the acetylated B chain of oxidized insulin (e.g. Fig. 3), observed with each enzyme, defines this substrate as the first to add to enzyme in each case. Finally, inhibition by the product, ammonium chloride, is competitive with methylamine (e.g. Fig. 4) and noncompetitive with glutamine substrate (e.g. Fig. 5) as predicted by the reciprocal forms of the expressions for product, P, inhibition in Mechanism I (3).

(6)

complished by the single thin layer chromatographic system employed. Evidence for separation was the finding of the expected natural abundance of isotopes at m/e 265 (15.1%) in control and samples and the absence of ions at m/e 219 and at m/e 203 which are given by PTH-glutamic acid and the PTH-lysine derivative, 4 = I

K (‘+$$$)(l +&(I respectively (24). 2, Vaa \- Inhibition by Benzyloxycarbonyl-L-glutaminylglycine-Benzyl-

oxycarbonyl-n-glutaminylglycine apparently does not serve as a substrate for plasma transglutaminase (see “Preliminary Sub- strate Specificity Examinations”). This peptide derivative was observed to inhibit the plasma enzyme-catalyzed methylamine incorporation into acetylated B chain. Inhibition against acet- where ylated B chain was observed to be of the linear competitive type (Fig. 6). The value for Ki, obtained by a fit of the data of Fig. 6 to Equation 3 was 8.15 f 0.17 mM.

With the hair follicle enzyme, however, no inhibition by benzyloxycarbonyl-L-glutaminylglycine was observed at levels as high as 20 mM.

(7b)

Preliminary Calcium Activation Studies-The essential requirement for calcium ion in fibrin stabilization by plasma transglutaminase is well known (for review see Reference 7). The essential role of Ca++ for methylamine incorporation into the acetylated B chain was evident from early observations that omission of this metal from assay solutions resulted in total loss in enzyme activity. A kinetic experiment (Fig. 7) showed equilibrium-ordered activa,tion of plasma transglutaminase by Ca*. A value for K+,, for Ca++ of 1.66 =I= 0.09 mM was obtained by a fit of the data of Fig. 7 to Equation 5.

With hair follicle transglutaminase omission of Ca* from assay solutions in which EDTA was present resulted in a complete loss in enzyme activity. However, a significant restoration of activity occurred upon removal of EDTA as well as Ca+f (Table IV). Further investigation of the role of metal ion in hair follicle transglutaminase activation is underway.

DISCUSSION

The present findings are in accord with Mechanism I as that for each of the transglutaminases studies. The intersecting initial velocity patterns obtained with each enzyme, examples of which are given in Figs. 1 and 2, are as predicted by the steady state equation for this mechanism (Equation 2). Isotope exchange in the presence of one substrate and one product (Table III) defines the mechanism as ping-pong. In ping-pong mechanisms isotope exchange can take place in the absence of one or

and the other constants are defined in the legend to Table II. The correspondence of Kah and V, values for the plasma

enzyme (Table II) obtained from transfer and hydrolysis experiments (Fig. 1 and 3, respectively) is in agreement with Mech- nism I. The equation for initial rate of appearance of P, ammonia, from A, acetylated B chain, Equation 8, (3) reduces to Equation 1 (where K = Kah and V = V,) as B approaches 0.

VA v= / D\

K +AP+a ob / Q2\

(8)

(1+2-J In the initial velocity patterns for each enzyme the lines in-

tersect on the horizontal axis as a result of the equalities, Kah = Katr K;bb = Kbt, and V, = V,a. This may be a consequence of (a) equal rates of deacylation of acyl enzyme, F, to water and to added methylamine, B, i.e. ky = kg, when these steps are rate controlling, or (b) the rate-limiting nature of enzyme acylation for both hydrolysis and transfer, i.e. JGx < lo and kg. That the latter is the case for each enzyme is suggested by the product inhibition studies with plasma and hair follicle transglutaminases (Figs. 4, 5, and text). For example, Equation 6 predicts that the apparent Ki, values from fits to Equation 3 will vary with the concentration of the nonvaried substrate, except in the case where Ka = Kah. The apparent inhibitor constants in this


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2606 Kinetic Studies with Transglutanukases Vol. 247, No. 9

cnhe corres~~ond to

With each enzyme, the apparent I<;, values do not, vary with the nonvaried substrate, A. Thus, wit;h each enzyme, Rah = Ii;;,. It is apparent from the definitions of these constants, Equations 2n and icy, respectively, that this can be true only when lea << J67, kg, kl, at~l 1~2, i.e. where lea is rate-limiting.

If this is indeed the case, the recorded 1Iichnclis constants obtained with the acetylated 1% chain (Table 11) approximate true dissociation constants and maximum velocities are proportional to the rates of enzyme acylatiou. Indications of the rebtion- chips of sub&ate structure to binding and to csatalysis may be forthcoming from kinetic studies with other Aemicnlly or en- zymatically modified forms of the U chain of oxidized insulin. In this regard a single experiment with plaslna transglutaminase with unacetylated 13 chain and [1”C]methylamine gave the following constants: I‘&, 0.42 f 0.07 III&~; I<,,, 0.39 i 0.07 m&f; ICibb, 0.69 f 0.08 mM; Kbt, 0.68 f 0.09 mar; Vab, 21 f 2 mpmoles per min per mg of thrombin-activated zymogen. Comparison of this finding with the data of Table II indicates that one or both free amino groups in the 1: cshain may Ilave a lironouncetl influence on both binding and catalysis. ‘I’he competitive type inhibition of the plasma enzynle by benzyloxycarbo~lyl-L-glu- taminylglycinc (Fig. 6, Ki, = -8), which has no apparent substrate property, is consistent with a suggestion that ucplntion of this enzyme in a function of Ihe amino acids surrounding the glutamine residue. In the same line, the lack of both substrate aL1d inhibitor prollerties of the benz:ylosycarhotl~l tlilleptide for the hair follicle enzyme points up a llrobable requirement ol” certain amino aAds surrounding the glutamine residue for lii’oper binding.

The calcium ion activation study of plasm:~ t~niisglut:2liiiiiase defines an obligatory order of addition and release of metal ion alid first substrate. The data are consistent with Mechanism II, for ~vliich Equation 5 is

where

I( n

= ‘;,I + L-5 ii3

the steady state expression and ill which first substrate A, con- bines only with enzyme-met,al complex. Early data collected for calcium activation of guinea pig liver Imnsglutaminaw appeared to be in accord wit11 this mechanism (29). However, metal ion nctivat,ion of the liver enzyme has liroveii to he signifi- cantly more complicated than first estimated (30). Uecause of the prelinlinary nature of the l)resent, studies we prefer to offer t,his finding with the p&ma enzyme as a characteristic observed u&r the present esperimental condit’ion.

The term transgluta.rninase, first applied by Mycek ef nl. to the enzyme of liver origin, denotes a limited specificity (I). The present fincling that the glutamine residue, but not the adjacent asparagine residue, in the acetylated B chain of oxidized insulin is modified by each of the enzymes studied here (Table I), justi- fies at least the tentative classificat’ion of these enzymes as transglutaminases. The pronouncedly different kinetic constants for the blood, hair follicle, and liver enzymes (Table II) contrib- utes to a growing list of distinctions among these transglutaminases.

Schwartz et ccl. have recently esamined the subunit structures of human plasma and platelet protransglutalnirlases (blood coagulation Factor XIII) (31). The.y conclude that the plasma zymogen of approximately 310,000 molecular weight contains four subunits of two different types, designated a and b, each with a molecular weight of 81,000. Only one type subunit, Subunit n, decreases in molecular weight upon activation of the plasma zymogen with thrombin. The platelet proenzyme, on the other hand, contains only one type subunit that is indistinguishable from the plasma Subunit a 011 the basis of several physical and chemical crit,eria. A reported molecular weight of 150,000 to 200,000 for the platelet zymogen (32) has led Schwartz el nl. (31) to assume that this zymogen contains two CL subunits. The subunit structure described by these workers account for the immunological cross-reactivities of the plasma iJ,lltl platelet proenzymes described earlier (32). Schwartz cl al. (31) point out that uo function for the b subunits of plasma 1~rotrallsglntaIniiiase is evident from their studies. On the tacit assumption that the b subunits assume no catalytic function uporI thrombin activation, our present findings show close similarity in the enzymatic properties of the a subunits of plasma and platelet protransglutaminases. Indeed, we find identical Michaelis constants for the l&ma and platelet enzymes with the acetylatecl I3 chain of oxidized insulin and met~hylamine (Table II). Further, ac*cept- ing the extinction coefficients for the two zymogens as approximate, the maximum velocities per mg of thrombin-activated proenzymes fall into the expected range.

Acknowledgmenls-Our thanks are due to Dr. C. W. A. Xlne for carrying out the chemical ionization mass spectrometry em- llloyed for determination of ‘5N incorporation. The skillful technical assistance of Niss Norma K. Whetzel is acknowledged.

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Issue of May 10, 1972 8. I. Chung and J. E. Folk 2807

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Soo Il Chung and J. E. FolkFOLLICLE ENZYME

(ACTIVATED COAGULATION FACTOR XIII) AND THE GUINEA PIG HAIR Kinetic Studies with Transglutaminases: THE HUMAN BLOOD ENZYMES

1972, 247:2798-2807.J. Biol. Chem.

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