Proc. Natl. Acad. Sci. USAVol. 77, No. 8, pp. 4564-4568, August 1980Biochemistry

oy-Glutamylamine cyclotransferase: Specificity towardE-(L-y-glutamyl)-L-lysine and related compounds*

(y-glutamylamines and polyamines/5-oxo-L-proline/kidney/transglutaminase product metabolism)

MARY LYNN FINK, SOO IL CHUNG, AND J. E. FOLK

Laboratory of Biochemistry, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205

Communicated by C. B. Anfinsen, May 5,1980

ABSTRACT 'y-Glutamylamine cyclotransferase, an enzymethat catalyzes the conversion of L-y-glutamylamines to freeamines and 5-oxo-L-proline, was found in rab it kidney and invarious other tissues. The specificity of this enzyme indicatesthat it functions in the metabolism of products of transglutam-inase action; among its substrates are F4L-'y-yglutamyl}L-lysinea derivative of this peptide in which the a-amino and car xygroups of the lysine moiety are blocked, and L-'y-glutamyl-polyamine derivatives.The E-(y-glutamyl)lysine crosslink, formed in proteins by thecatalytic action of transglutaminase (reaction 1),

-Gin- + -Lys-- -- + NWL [1]

occurs in various tissues and organisms (for review, see ref. 1).Despite an accumulation of information on the production anddistribution of this crosslink and of some evidence as to its bi-ological functions, virtually nothing is known of its metabolicfate. The resistance of the crosslink to hydrolysis by proteolyticenzymes is the basis for its isolation (2, 3). The end product ofexhaustive in vitro proteolysis is the dipeptide E-(y-glutamyl)-lysine. There are reports that f-(L-'y-glutamyl)-L-lysine canreplace lysine in supporting the growth of rats (4, 5) and chicks(5). The observations that this peptide passes unchangedthrough the intestinal wall of everted sacs of rat small intestinein vitro (6) and that, of the homogenates of rat organs tested,those of kidney cause, by far, the most rapid release of lysinefrom the peptide (6, 7) form a basis for the conclusion that hy-drolysis of e-(Qy-glutamyl)lysine occurs in vivo, largely in thekidney (6, 7).We report here the partial purification and characterization

of an enzyme from rabbit kidney for which E-(L-'y-glutam-yl)-L-lysine and other L-y-glutamylamines are substrates.Evidence is presented-that, although lysine and free amines areproducts of catalysis by this enzyme, the reaction proceeds notthrough hydrolysis but rather by way of cyclic transfer withproduction of 5-oxo-L-proline (synonyms: pyroglutamic acid,5-pyrrolidone-2-carboxylic acid) (reaction 2).

O H11 IC N RICH2

C'H.,

H-- NH2('O()H

(I,)

H.2C CH2-~

0,C<CHH COOH + R-NH2

H(L

On this basis, we propose the name y-glutamylamine cyclo-transferase for this enzyme. Its reaction is analogous to that(reaction 3) catalyzed by 'y-glutamylamino acid cyclotrans-feraset (for review, see ref. 8).

O H R

l lI X>C--C0CH2 H

CH2

H-C-NH2

COGH

(L,L)

H2C -CH2 RI IC;CH-COOH + H2N -COOH

O HH

(L)

[3]

(L)Here we report the initial findings of a study designed to

elucidate the catabolism of e-(y-glutamyl)lysine crosslinks andother biological products of transglutaminase action.

EXPERIMENTALMaterials. The following compounds were obtained from

commercial sources as follows: 5-oxo-L-proline, e-acetyl-L-lysine, and L-glutamic acid, Aldrich; L-glutamine, L-'y-glu-tamyl-L-glutamine, f.-alanine, E-formyl-L-lysine, and L-'y-glutamyl-p-nitroanilide, Sigma; and L-,y-glutamyl-L-methionine and Nl-(L-'y-glutamyl)putrescine-HCI, Vega(Tucson, AZ). E-(L-y-Glutamyl)-L-lysine (9) and N',N4-bis(L--y-glutamyl)putrescine (10) were synthesized by the citedmethods. The mixture of the N' and N8 forms of (L-'y-glu-tamyl)spermidine, N',N8-bis(L--y-glutamyl)spermidine,N1-(L-'y-glutamyl)spermine, and N'1,N 12-bis(L-y-gluta-myl)spermine were prepared via the transglutaminase-cata-lyzed transfer reaction between the appropriate polyamine andbenzyloxycarbonyl-L-glutaminyl-L-leucine, followed by re-moval of the leucine residue and the benzyloxycarbonyl groupas described (10). A similar approach was used for preparation

* Presented in part at the 178th National Meeting of the American|I Chemical Society, Washington, D.C., Sept. 9-14, 1979, Biol. 061

abstr.t For clarity, the enzyme designated y-glutamyl cyclotransferase[(5-L-glutamyl)-L-amino acid 5-glutamyltransferase (cyclizing), EC2.3.2.41 will be referred to herein as y-glutamylamino acid cyclo-transferase.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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of the methyl, ethyl, n-butyl, isobutyl, and 2-(4-imidazolyl)ethyl'y-amides of L-glutamic acid.For each preparation, intermediates were purified by

high-performance liquid chromatography using the systemsdescribed (10, 11). a-(L-y-Glutamyl)-L-lysine, E-(L-'y-gluta-myl)-D-lysine, E-(D- 7-glutamyl)-L-lysine, L- y-glutamyl-B-alanine, a-N-acetyl-E-(L-'y-glutamyl)-L-lysine methyl ester,a-N-acetyl-E-['y-(L-a-glutamylglycine)]-L-lysine methyl ester,a-N-acetyl-E-[y-(L-a-glutamyl-L-valine ethyl ester)]-L-lysinemethyl ester, N-['y-(L-a-glutamylglycylglycylglycine)]iso-butylamine, E-(L-f3-aspartyl)-L-lysine, E-(D,L-3-aminoglutar-yl)-L-lysine, D,L-f3-aminoglutaryl-L-a-aminobutyric acid,L-'y-glutamylisopropylamine, and N-[5-(L--y-glutamyl)ami-nopentyl]-5-dimethylaminonaphthalene-1-sulfonamide (L-'y-glutamyldansylcadaverine) were synthesized by conventionalmethods. The procedures for synthesis, together with pertinentanalytical data, will be published elsewhere.Methods. Enzyme activity was measured in reaction mix-

tures (final volume 0.06 ml), containing 6.66 mM E-(L-'y-glu-tamyl)-L-lysine and enzyme in 66.6 mM sodium phosphatebuffer at pH 7.5 and 370C. Reactions were terminated after15-60 min by addition of 0.06 ml of 10% trichloroacetic acid.Precipitated protein was removed by centrifugation, and lysinewas separated in an ion exchange chromatographic system (10)using a single buffer (sodium citrate buffer, 0.26 M in Na+, pH4.8, containing 0.55% ethanol) at 660C for elution and o-phthalaldehyde for detection. Quantitation was with an in-linecomputing integrator (System I, Spectra-Physics, Santa Clara,CA). Retention times for substrate and lysine were 145 and 555sec, respectively. Appropriate controls were run in all cases.Protein was determined by the method of Lowry et al. (12) withbovine serum albumin as standard. One unit of enzyme activityis defined as the amount required to catalyze the formation of1 Mmol of lysine from E-(L-'y-glutamyl)-L-lysine per hr underthe above conditions.The properties of other compounds as substrates for the en-

zyme were examined under the above conditions, except thatthese compounds were tested at 3.33 mM unless indicatedotherwise. Amine, amino acid, or polyamine product wasmeasured by using the ion exchange chromatographic systemwith a buffer or combination of buffers (10) found to give anadequate separation of substrate and product. ,B-Alanine andglutamine were separated from the respective 'y-glutamylpeptides with a conventional amino acid analyzer. N-(5-Ami-nopentyl)-5-dimethylamino-1-naphthalenesulfonamide (mo-nodansylcadaverine), the fluorescent product of enzyme actionon L-'y-glutamyldansylcadaverine, was separated from thissubstrate by passage of portions of reaction mixtures (withoutaddition of trichloroacetic acid) through 0.45 X 7cm columnsof MN-polyamid CC6 (Mackerey Nagel, Duiren, West Ger-many). Elution was with 1% aqueous pyridine that had beenadjusted to pH 5.6 with acetic acid. Monodansylcadaverine waseluted first, well ahead of the substrate, and was quantitatedby measurement of its relative fluorescence in a spectrofluo-rometer (excitation and emission at 360 and 460 nm, respec-tively). L-y-Glutamyldansylcadaverine was found to be a usefulsubstrate for testing for, and locating fractions of, enzymicactivity during enzyme purification. For this purpose, sepa-ration of fluorescent substrate and product was accomplishedby thin-layer chromatography on polyamide sheets using theabove solvent and other conditions as described (13).

Purification of the Enzyme. Step 1. Freshly excised orfrozen kidneys from New Zealand White rabbits were ho-mogenized for 3-5 min in 2 vol of 0.25 M sucrose with a Poly-tron homogenizer (Brinkmann). The supernatant obtained uponcentrifugation at 100,000 X g for 1 hr was filtered through

cheesecloth. This step and subsequent (NH4)2SO4 precipitationswere carried out at 4VC. Chromatography steps were conductedat room temperature.

Step 2. The supernatant obtained from 20 g of kidney tissuewas applied to a column (3 X 11 cm) of DEAE-cellulose. Thecolumn was washed with 200 ml of 5 mM sodium phosphatebuffer at pH 7.5 (buffer A) and the enzyme was eluted at a flowrate of 5 ml/min with a 300-ml linear gradient establishedbetween buffer A and 100mM sodium phosphate buffer at pH7.5. The fractions containing activity (eluted at about 50 mMbuffer) were combined.

Step 3. Solid (NH4)2SO4 (31 g/100 ml) was added and theprecipitate obtained upon centrifugation for 25 min at 20,000X g was discarded. Solid (NH4)2SO4 (22 g/100 ml) was addedto the supernatant and the precipitate collected by centrifu-gation was dissolved in a minimal volume of buffer A. Thissolution was chromatographed on a column (1.5 X 45 cm) ofSephadex G-100 in buffer A at a flow rate of 12 ml/hr. Thepeak of activity emerged at 52 ml, near the end of the de-scending shoulder of a large protein peak. Only those fractionsof specific activity >9 units/mg of protein were combined andused in the next step.

Step 4. Solid (NH4)2SO4 (66 g/100 ml) was added and theprecipitate obtained was dissolved in a minimal volume ofbuffer A. This solution was chromatographed on a column (1.25X 40 cm) of Sephadex G-50 in buffer A at a flow rate of 12ml/hr. The peak of activity emerged at 23 ml. Fractions con-taining activity were combined and used for most of the studiesreported here.

Specific activities of the initial homogenate and of the frac-tions obtained in each of the above steps were 0.18, 0.24, 0.36,10.2, and 32 units/mg of protein, respectively. An approxi-mately 180-fold purification was achieved. The overall yieldof activity, however, was only 5-10%, primarily as a result ofretaining only those fractions of highest specific activity in step3. Enzyme prepared in this manner showed no noticeable lossin activity over a period of several weeks when stored atOOC.

RESULTSProducts of the Action of y-Glutamylamine Cyclotrans-

ferase on e(L-'y-Glutamyl)L-Lysine. When c-(L-'y-gluta-myl)-L-lysine was incubated with the enzyme for variouslengths of time and portions of the reaction mixtures were ex-amined by ion exchange chromatography using either o-phthalaldehyde (10) or ninhydrin (conventional amino acidanalyzer) for detection, the only product observed was lysine.However, when samples of deproteinized reaction mixtureswere hydrolyzed with 6 M HCl (1050C for 18 hr) prior toanalysis, stoichiometric amounts of lysine and glutamic acidwere found. The data given in Table 1 establish that 5-oxo-L-proline is a product of the reaction. No 5-oxoproline was formedeither from L-glutamic acid or from L-glutamine upon incu-bation with enzyme; both amino acids remained unchanged.These observations are strong evidence that the enzyme cata-lyzes the cyclic transfer depicted in reaction 2.

'y-Glutamylamine Cyclotransferase and y-GlutamylaminoAcid Cyclotransferase Are Separate Enzymes. Although thesetwo enzymes catalyze similar reactions (reactions 2 and 3, re-spectively), several facts serve to define them as separate en-zymes with distinctly different specificities. (i) a1-(L-'y-Glu-tamyl)-L-lysine is a substrate for -y-glutamylamino acid cy-clotransferase (16); E-(L--y-glutamyl)-L-lysine is not [E-(L-y-glutamyl)-L-lysine was unchanged and no lysine was pro-duced during its incubation with a highly purified preparationof Py-glutamylamino acid cyclotransferase from rat kidney (0.

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Table 1. 5-Oxo-L-proline produced by -y-glutamylaminecyclotransferase reaction

Product ofStandard 5-oxo- enzymic

Method (values recorded) L-proline reaction

Thin-layer chromatography* onsilica gel in n-propanol/conc.NH40H, 7:3 (RF) 0.31 0.31

Chemical ionization massspectrometryt [protonatedmolecular ion (MH+), NH4+addition ion (M + 18)] 130,147 130,147

Optical rotations ([a]25 c = 0.01in water) +775 +787

Incubation of E-(L-'y-glutamyl)-L-lysine with enzyme was allowedto proceed until analysis by ion exchange chromatography showedthat substrate had been completely consumed. The reaction mixturewas deproteinized with trichloroacetic acid. The supernatant wasextracted with ether and was freed of lysine by passage through asmall column of Dowex 50-x8(H+) in water.* Detection was with a chlorine-tolidine reagent (14).t Mass spectrometry was carried out on a Finnigan 1015 D instrumentequipped with a chemical ionization source. Ammonia was the re-agent gas. Another sample was analyzed after conversion to themethyl ester by the use ofHCl in methanol; in this case, analysis wasby gas chromatography/mass spectrometry in an LKB 2091 gaschromatograph/mass spectrometer with an LKB SE-30 column.Retention times, parent ions, and major ions were found to beidentical for the methyl esters ofstandard 5-oxo-L-proline and theproduct of the enzymic reaction. The pattern of degradation of themethyl ester of 5-oxo-proline was the same as that reported (15).Rotations were measured with a Cary 60 recording spectropolari-meter and a 0.05-dm light path cell. The specific rotations ([alval-ues) were calculated on the basis of concentrations determined fromglutamic acid formed upon acid hydrolysis and measured by aminoacid analysis. The values at 260 nm were selected as representativevalues. The optical rotatory dispersion ofauthentic 5-oxo-L-prolineand that of the product of the enzymic reaction were found to beidentical within experimental error.

W. Griffith and A. Meister, personal communication)]. This isin agreement with the well-established specificity of the aminoacid cyclotransferase toward only L-,y-glutamyl a-L-aminoacids (including L-y-Glu-L-,y-Glu-NH-R in which the natureof the R moiety has relatively little effect on activity) (8, 17).(fi) The lysine-producing activity toward E-(L-'y-glutamyl)-L-lysine and that toward a-(L-'y-glutamyl)L-lysine were foundin different salt fractions of crude rabbit kidney homogenate.Whereas 70% of the total activity toward the c-lysine peptideprecipitated between 0% and 45% (NH4)2SO4, only 8% of thetotal activity toward the a-lysine peptide was found in thisfraction; >90% of the activity for the latter peptide was pre-cipitated above 60% saturation. (iii) The activities toward the'y-glutamyl lysine peptides were partially separated byDEAE-cellulose chromatography of the supernatant fromrabbit kidney homogenate. Upon stepwise elution with sodiumphosphate buffer at pH 7.5, activities toward theE- and a-lysinepeptides emerged, respectively, between 30 and 50 mMphosphate. (iv) L-13-Aminoglutaryl-L-a-aminobutyric acid isan effective inhibitor of y-glutamylamino acid cyclotransferase(17,18). DL-(3-Aminoglutaryl-L-a-aminobutyric acid at 20mMprovided almost complete inhibition of lysine release froma-(L-'y-glutamyl)-L-lysine by crude rabbit kidney homogenatebut caused no reduction in the rate of production of lysine fromE-(L-,y-glutamyl)L-lysine by the homogenate. In addition, theactivity of purified y-glutamylamine cyclotransferase towardE-(L-Y-glutamyl)-L-lysine was unaffected by addition of 20mMD,L-f3-aminoglutaryl-L-a-aminobutyric acid to the assaymixture. (v) The purified y-glutamylamine cyclotransferase

showed no release of lysine from a-(L-y-glutamyl)-L-lysineafter a 60-min incubation at 1 unit/ml.

Substrate Specificity of y-Glutamylamine Cyclotrans-ferase. The activity of the purified enzyme preparation wastested toward various -y-glutamylamines and related com-pounds. The rate of product formation given for each substratein Table 2 is that obtained at a single initial substrate concen-tration. An apparent Km value (mean ± SEM) for E-(L-'y-glu-tamyl)-L-lysine of 2.3 + 0.1 X 10-4 M was estimated from avelocity-versus-concentration experiment. At saturating levelsof this substrate the enzyme displayed a broad pH optimumbetween about 6.8 and 8.5 (phosphate buffer below pH 7.5;borate buffer above pH 7.5); activity decreased gradually abovepH 8.5. The following compounds were not acted upon by theenzyme under the conditions used to obtain the values in Table2: e-(D-y-glutamyl)-L-lysine, e-(L-f3-aspartyl)-L-lysine,E-(D,L-13-aminoglutaryl)-L-lysine, L-glutamine, a-N-acetyl-E-[L-a-glutamylglycine)]-L-lysine methyl ester, a-N-ac-etyl-E-|y-L-a-glutamyl-L-valine ethyl ester)]-L-lysine methylester, N-[-'y-(L-a-glutamylglycylglycylglycine)]isobutylamine,E-acetyl-L-lysine, e-formyl-L-lysine, and L-'y-glutamyl-p-nitroanilide.

As pointed out above, lysine is not formed froma-(L-y-glutamyl)-L-lysine by the purified enzyme preparation.However, because this peptide is a relatively poor substrate for,y-glutamylamino acid cyclotransferase (16), we chose to testfor the presence of this enzyme in the y-glutamylamine cy-clotransferase preparation by the use of more sensitive specificsubstrates. L--y-Glutamyl-L-methionine and L-'y-glu-

Table 2. Substrate specificity of y-glutamylaminecyclotransferase

Product,Substrate ,umol/hr/mg

E-(L-'y-Glutamyl)-L-lysine 32E-(L-y-Glutamyl)-D-lysine 54.4a-N-Acetyl-E-(L-'y-glutamyl)-L-lysine methyl

ester 36.8L-y-Glutamyldansylcadaverine 12.8L-y-Glutamylmethylamine 4.8L-,y-Glutamylethylamine 9.6L-y-Glutamylisopropylamine 11.2L-y-Glutamyl-n-butylamine 30.4L-'y-Glutamylisobutylamine 27.2L-y-Glutamyl-fl-alanine 20.8L-y-Glutamyl-2-(4-imidazolyl)ethylamine

('y-glutamylhistamine) 4.8L-'y-Glutamylputrescine 30.4N',N4-bis(L-'y-Glutamyl)putrescine *N1- and N84(L-'y-Glutamyl)spermidinett 11N',NS- bis(L-'y-Glutamyl)spermidinet §N1-(L-y-Glutamyl)sperminet 26N',Nl2-bis(L-y-Glutamyl)sperminet *

The reactions were conducted with 1.0 unit of enzyme/ml. Incu-bations were for 10-60 min. The amine or amino acid product wasmeasured in each case by the appropriate method.* Product formation was rapid. Rates were not measured because twoproducts, N-(y-glutamyl)amine and amine, were formed. One ofthese, the N-(y-glutamyl)amine, is also a substrate.

t A mixture of approximately equimolar amounts of the N' and N5forms was tested. Examination of the chromatographic pattern ofsubstrate remaining after the incubation period showed that aboutequal amounts of each form had been consumed.The initial substrate concentration was -1 mM.

§ Product formation was rapid. Rates were not measured becausethree products, the N' and N8 forms of N-(-y-glutamyl)spermidineand spermidine, were formed. Two of these, the N-('y-glutamyl)-spermidines, are also substrates.

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tamyl-L-glutamine are reported to be excellent substrates forthe amino acid cyclotransferase (16, 17). Using these substratesand assuming a specific activity for rabbit enzyme in the rangeof that reported for highly purified rat kidney enzyme (17), weestimate <0.4% impurity of y-glutamylamino acid cyclo-transferase in the purified preparation of 'y-glutamylaminecyclotransferase. Recently, we have obtained enzyme in whichno activity toward either y-glutamylmethionine or y-glu-tamylglutamine can be detected. The final step in this purifi-cation (chromatography on Sephacryl S-200, superfine) yieldsthe y-glutamylamino acid cyclotransferase-free material.However, only minimal separation of the two enzymic activitiesis achieved and only a minute fraction of the total 'y-gluta-mylamine cyclotransferase is obtained free of amino acid cy-clotransferase activity.

Distribution of y-Glutamylamine Cyclotransferase. Table3 summarizes the results of a preliminary examination of thedistribution of the enzyme in rabbit tissues. For comparativepurposes the relative levels of y-glutamylamino acid cyclo-transferase activity in the various tissues, as estimated fromactivity toward a-(L-'y-glutamyl)L-lysine, are also given in thetable. Activity of both enzymes was highest in kidney. Somedifferences in the distributions of the two activities are apparent.Notable is the relatively low 'y-glutamylamine cyclotransferaseactivity in skin. In those mammals examined to date, high levelsof y-glutamylamino acid cyclotransferase activity were foundin skin (8).

DISCUSSIONThe data presented here are evidence for the presence of anenzyme in kidney and other mammalian tissues that catalyzesthe release of free amines from L--y-glutamylamines with theconcomitant cyclization of the glutamyl moiety to 5-oxo-L-

Table 3. '-Glutamylamine cyclotransferase andY-glutamylamino acid cyclotransferase activities of

several rabbit tissuesActivity, % of kidney value

Amine Amino acidTissue cyclotransferase* cyclotransferaset

Kidney 100 100Liver 11 35Intestine 13 10Heart 3 4Lung 6 16Brain 12 19Testis 19 21Skin 3 21Spleen 3 11Pancreas 24 33Adrenal 11 14Thyroid 15 10

The fresh tissues, with the exception of skin, were homogenized at00C with 2 vol of 0.25 M sucrose. The homogenates were centrifugedfor 30 min at 30,000 X g, and the supernatants thus obtained wereused for determination of activity. The skin was frozen, lyophilized,pulverized, and then homogenized in 2 ml of sucrose solution per gof freshly excised skin used. Assays were conducted with 6.66 mMsubstrate. The activity values are expressed as those relative to thatof kidney and are based on wet weight of tissue.* Substrate was E-(L--y-glutamyl)-L-lysine; rate with kidney homog-enate, 27 ,mol/hr per g of wet tissue.

t Substrate was a-(L-y-glutamyl)-L-lysine; rate with kidney ho-mogenate, 42 ,mol/hr per g of wet tissue. With rat kidney homog-enate the rate of reaction varied greatly depending on the substrate.Rates for -glutamyl derivatives of L-glutamine and L-methioninewere found to be on the order of 10-fold higher than the rate ob-served for a-(L-'y-glutamyl)-L-lysine (16).

proline (reaction 2). The resemblance of this y-glutamylaminecyclotransferase reaction to that catalyzed by y-glutamylaminoacid cyclotransferase (reaction 3) is evident. There are distinctdifferences in the substrate specificities of the two enzymes,however, Whereas the amino acid cyclotransferase acts onlyon L-y-glutamyl-L-a-amino acids (8, 19), the amine cyclo-transferase exhibits less stringent requirements but apparentlydoes not act on y-glutamyl a-amino acids.

Difficulties were encountered in obtaining y-glutamylaminecyclotransferase free of amino acid cyclotransferase activity.Some of these difficulties may be related to the fact that'y-glutamylamino acid cyclotransferase exists in isozymic forms(16) and that changes in the substrate specificity and in theprotein structure of this enzyme apparently occur during pu-rification and storage (16).The distribution of y-glutamylamine cyclotransferase ac-

tivity in various animal tissues suggests a specific metabolicfunction for this enzyme. Our interest in the catabolism ofe-(y-glutamyl)lysine crosslinks and other products of the cat-alytic action of transglutaminases led us to the present findings.There is no indication that E-(y-glutamyl)lysine crosslinks arecleaved without prior degradation of the crosslinked proteins,and it seems unlikely that cleavage of crosslinks could occurthrough the reverse action of transglutaminases because thisreaction is slow and inefficient (20, 21). The in vitro proteolyticdegradation of transglutaminase-crosslinked proteins yieldse-(,y-glutamyl)lysine (2, 3), and polyamine-protein conjugatesformed in svo by transglutaminase action yield y-glutamyl-polyamine derivatives-upon in vitro proteolytic digestion (10).We have considered the idea that transglutaminase-modifiedproteins may be degraded in mammalian tissues in such amanner that substrates for y-glutamylamine cyclotransferaseare formed rapidly and preferentially. y-Substituted glutamylamino acids and peptides-e.g., a-N-acetyl-E-[y-(L-a-glu-tamylglycine)]-L-lysine methyl ester and N-ky-(L-a-gluta-mylglycylglycylglycine)]isobutylamine-are not substratesfor the enzyme. Furthermore, because it may be concluded thatthe a-amino group in yv-glutamyl substrates is required in un-bound form for cyclization, conversion of protein-bound'v-substituted glutamic acid residues to substrates for the aminecyclotransferase would require hydrolysis of the peptide bondsat both the a-carboxyl and a-amino groups of these residues.Such a preferential hydrolytic reaction by the concerted actionof pancreatic chymotrypsin C and carboxypeptidase A has beendescribed (22). a-N-Acetyl-e-(L-'y-glutamyl)-L-lysine methylester is a derivative of E-(y-glutamyl)lysine in which both thea-amino and carboxyl groups of lysine are blocked. The factthat this compound functions as a substrate for y-glutamyl-amine cyclotransferase (Table 2) suggests that conversion ofprotein e-(Qy-glutamyl)lysine crosslinks to substrates for thisenzyme requires hydrolytic cleavage of only those peptidebonds connected to the glutamic acid portion of the cross-link.

We thank Dr. Alton Meister for advice and encouragement and Mr.William E. Comstock, Dr. Henry M. Fales, and Mr. Noel F. Whittakerfor mass spectral analyses.

1. Folk, J. E. & Finlayson, J. S. (1977) Adv. Prot. Chem. 31, 1-144.

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4. Mauron, J. (1970) J. Int. Vitaminol. 40,209-227.5. Waibel, P. E. & Carpenter, K. J. (1972) Br. J. Nutr. 27, 509-

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15. EPA/NIH Mass Spectral Data Base (1978) Chem. Abstr. No.4931-66-2. NSRDS-NBS63 (GPO, Washington, DC), Vol. 1,p. 384.

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6424-6435.22. Folk, J. E. & Cole, P. W. (1965) J. Biol. Chem. 240, 2951-

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