Proc. Nati. Acad. Sci. USAVol. 82, pp. 6114-6118, September 1985Biochemistry

Identification of intracellular degradation intermediates of aldolaseB by antiserum to the denatured enzyme

(truncated peptides/immunoblot)

ABRAHAM Z. REZNICK, LEAH ROSENFELDER, SHARONA SHPUND, AND DAVID GERSHONDepartment of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Communicated by Kenneth V. Thimann, May 28, 1985

ABSTRACT A method has been developed that enables usto identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This methodis based on the use of antibody against thoroughly denaturedpurified aldolase. This antibody has been shown to recognizeonly denatured molecules, and it did not interact with "native"enzyme. Supernatants (24,000 x g for 30 min) of liver andkidney homogenates were incubated with antiserum to dena-tured enzyme. The antigen-antibody precipitates thus formedwere subjected to NaDodSO4/PAGE, followed by electrotrans-fer to nitrocellulose paper and immunodecoration with antise-rum to denatured enzyme and 1251-labeled protein A. Sevenpeptides with molecular weights ranging from 38,000 (that ofthe intact subunit) to 18,000, which cross-reacted antigenicallywith denatured fructose-bisphosphate aldolase, could be iden-tified in liver. The longest three peptides were also present inkidney. The possibilit that these peptides were artifacts ofhomogenization was ruled out as follows: 1251-labeled taggedpurified native aldolase was added to the buffer prior to liverhomogenization. The homogenates were then subjected toNaDodSO4/PAGE followed by autoradiography, and the la-beled enzyme was shown to remain intact. This method issuggested for general use in the search for degradation prod-ucts of other cellular proteins.

The various stages that comprise the process of intracellularprotein degradation and the proteases involved have re-mained obscure despite extensive studies in many laborato-ries (1, 2). So far no high molecular weight intermediates ofintracellular degradation of specific native proteins have beenidentified in eukaryotes. The identification of such interme-diates is obviously necessary in order to elucidate themechanism of intracellular degradation of proteins. Severalprevious experimental results from our laboratory haveindicated that a direct search for degradation intermediates ofspecific proteins might be feasible for the following reasons:(i) because of a considerable slow down in degradation of"native" proteins and aberrant peptides in cells of aginganimals (3-5), it was possible to find an age-associatedaccumulation of catalytically altered enzyme molecules thatretained antigenic cross-reactivity with native forms of theenzymes (e.g., see refs. 6 and 7); a small proportion of thesame forms was subsequently found in cells of young animals(8); (ii) such altered enzyme molecules of liver cytosolicsuperoxide dismutase, lens aldolase C and glyceraldehyde-3-phosphate dehydrogenase could be preferentially removedfrom tissue homogenates by antibodies elicited against de-natured forms of these enzymes (8-10); (iii) significantly,these antibodies did not interact with native enzyme mole-cules (8); (iv) these findings suggested that altered antigeni-

cally cross-reactive forms of enzyme molecules might be, atleast in part, intermediates of degradation.The present communication describes studies that led to

the identification of seven peptides and three peptides in liverand kidney homogenates, respectively. These are antigeni-cally cross-reacting, inactive forms of aldolase B (fructose-bisphosphate aldolase, D-fructose-1 ,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). It is sug-gested that they are normal degradation products of theenzyme.

MATERIALS AND METHODSAnimals. C57BL/6J female mice were maintained under

specific pathogen-free conditions. Food was provided adlibitum.

Preparation of Native and Denatured Aldolase B. Enzymewas purified and assayed essentially as described (3). In brief,livers were homogenized in 5 vol (wt/vol) of cold buffer A(0.05 M Tris HCl/1 mM EDTA/1 mM 2-mercaptoetha-nol/0.25 M sucrose, pH 7.4), which contained 1 mMphenylmethylsulfonyl fluoride (PhMeSO2F) and 15 ,ug ofleupeptin per ml. The protease inhibitors were maintained inall subsequent stages of the purification procedure.Homogenates were centrifuged at 24,000 x g for 30 min andthe supernatant was chromatographed on phosphocellulosecolumns. More than 90% of the activity was retained on thecolumn and was eluted by 2.5 mM fructose bisphosphate.The void volume was retained for further analysis. A secondpassage through a phosphocellulose column usually sufficedto obtain pure preparations of the enzyme. The degree ofpurity was verified by NaDodSO4/PAGE. Denaturation ofthe enzyme was achieved by suspending 1 mg of the purifiedpreparation in 1 ml of buffer containing 2% NaDodSO4 and2% 2-mercaptoethanol and boiling for 5 min. Completedenaturation is an essential condition for the preparation ofantisera with absolute specificity for denatured moleculesthat do not react with native molecules.

Preparation of Antiserum to Denatured and Native AldolaseB. Antisera to native enzyme (ANE) were prepared asdescribed (3). Antisera to denatured enzyme (ADE) wereprepared as described elsewhere (8). In brief, immediatelyafter boiling in NaDodSO4 and 2-mercaptoethanol, the de-natured enzyme was emulsified in complete Freund's adju-vant (1:1, vol/vol) and injected into rabbits in multipleintradermal sites. Animals were bled 10, 14, and 21 days laterand antisera were tested for the presence of ADE by theOuchterlony double-diffusion test. Booster injections wereadministered at 3-wk intervals until antibody titers weresufficiently high, at which point the animals were bled oncea week for as long as antibody titers remained high.

Abbreviations: ADE, anti-denatured enzyme antibody; ANE, anti-native enzyme antibody; PhMeSO2F, phenylmethylsulfonyl fluo-ride.

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Immunotitration Studies With ANE With and WithoutPrevious Incubation in ADE. Livers of young and old micewere homogenized in 4 vol of buffer A containing 0.25 Msucrose (isotonic), PhMeSO2F, and leupeptin under condi-tions that facilitate the removal of intact lysosomes withoutdisruption and prevent proteolysis (4). The homogenate wasspun at 24,000 x g for 1 hr, and the resultant supernatant wasused for further analysis. The enzyme activities of "young"and "old" preparations were adjusted to the same levels.Supernatant (150-200 ,p1) was incubated with 600 ,u1 of ADEand 850 ,ul of buffer A. After overnight incubation at 40C andcentrifugation at 3000 x g for 30 min, the precipitates wereremoved and aldolase activity was determined in the super-natant. In all the experiments, it was repeatedly observedthat the entire activity remained in the supernatant after theincubation with ADE. Immunotitration of this supernatant orofhomogenates without incubation with ADE was performedas described (6).

Identification of Antigenically Cross-Reactive Peptides inLiver Homogenates. Homogenate supernatants (24,000 x g)were concentrated 4-fold over Amicon PM30 membranes.The concentrates were incubated overnight with either ADEor ANE as is indicated below for the individual experiments.After centrifugation at 3000 x g, precipitates were washedonce with buffer (10 mM Tris base, pH 8.0/10 mMNaCl/1.5% Triton X-100) and twice with the same bufferdevoid of Triton X-100. The final precipitates were dissolvedin buffer with 2% NaDodSO4/2% 2-mercaptoethanol/5%bromophenol blue, and boiled for 5 min. Samples were thensubjected to NaDodSO4/PAGE in 12.5% gels and proteinbands were stained with Coomassie brilliant blue. For de-tection of aldolase degradation products, gels were subjectedto a procedure of electrotransfer to nitrocellulose paper andimmunodecoration with ADE and 1251-labeled protein A (8,11).Labeling of Aldolase With "2SI. Todination of purified

aldolase with lactoperoxidase was carried out according tothe procedure of Morrison (12) with one modification. In-stead of adding all the H202 at the start of the reaction, fivealiquots of0.03% H202 solution were added to the incubationmixture at 2-min intervals with gentle shaking. The reactionwas stopped by cooling to 4°C and by applying the reactionmixture to a phosphocellulose column equilibrated withbuffer A. This procedure separated the "native" aldolasefrom the peroxidase, the free 1251, and any inactivatedmolecules of the enzyme. The iodinated aldolase was theneluted from the phosphocellulose column with 2.5 mMfructose bisphosphate. It was tested for purity by NaDod-S04/PAGE and autoradiography.Limited Proteolysis of Aldolase B With Staphylococcus

aureus V8 Protease. Liver homogenate was incubated withADE. The precipitate obtained after centrifugation waswashed and subjected to NaDodSO4/PAGE in parallel topurified aldolase B. The gels were stained with Coomassieblue and the bands were identified as aldolase B subunit (Mr,38,000) and the largest truncated peptide (Mr, -36,500)derived from the enzyme were cut out of the gels and placedin the wells of a freshly prepared NaDodSO4/polyacrylamidegel. V8 protease (Sigma) at the concentrations indicated inthe legend to Fig. 5, was then applied to the wells andincubated with the gel pieces containing the various peptidesfor 1 hr prior to electrophoresis. After electrophoresis, thebands were stained with Coomassie blue for peptide identi-fication.

RESULTSEvidence for the Existence of Inactive Partially Denatured

Molecules of Aldolase B in Liver Homogenates. Aliquots ofliver homogenates from young and old mice were incubated

overnight with ADE. The preparations were then centrifugedand the supernatants, which still contained all the aldolase Benzyme activity, were subjected to immunotitration withANE. In parallel, aliquots of the same homogenates that hadnot been incubated with ADE were also immunotitrated withANE. Comparison of the immunotitration curves ofhomogenates with and without incubation with ADE takenfrom a representative experiment are depicted in Fig. 1. Theresults clearly show that incubation with ADE causes re-moval of cross-reacting material from "old" and to a smallbut easily discernible degree from "young" homogenates,which lacks catalytic activity and is apparently at leastpartially denatured. This is apparent from the fact that afterincubation with ADE less ANE is required to precipitate thesame amount of aldolase activity from the homogenates. Theremoval of cross-reacting material by ADE from younghomogenates has been consistently observed in severalpreparations. The cross-reacting material precipitated withADE from young homogenates in several preparations wasused for all further analyses reported in this communication.The immunotitrations with ANE depicted in Fig. 1 agree wellwith previous experiments, which unequivocally demon-strated cross-reacting material in old aldolase preparations(4, 6).

Identification of Aldolase B Derived Altered Peptides inLiver and Kidney Extracts. Liver and kidney homogenateswere spun at 24,000 X g for 60 min. The supernatant wasincubated with ADE and the resultant precipitate was pre-pared for and subjected to NaDodSO4/PAGE. The peptidepatterns observed with Coomassie blue are depicted in Fig.2A. Duplicates ofthe same preparation were transferred fromthe NaDodSO4/polyacrylamide gel to nitrocellulose paperand immunodecorated with ADE and 125I-labeled protein A.The autoradiographic patterns of this preparation are shownin Fig. 2B. In liver extracts, seven peptides ranging inmolecular weight from 38,000 to 18,000 (based on molecularweight markers) are discerned (lane 2). One of these has anapparent size equivalent to the intact subunit (Mr, 38,000),and six appear to be truncated forms that antigenically

1000-

90

830

20

10-

10 20 30 40 50 60Anti-native antiserum, Al

FIG. 1. Immunotitration analysis of aldolase B in homogenates oflivers of young and old rats without ADE and after incubation withADE. Aliquots ofhomogenates adjusted to the same aldolase activitywere incubated overnight with increasing amounts of ANE. Aftercentrifugation at 3000 x g for 30 min, activity was measured in thesupernatant. Titration with ANE without incubation with ADE isdepicted by solid lines. Aliquots of the same homogenates, adjustedto the same aldolase activity, were first incubated overnight withADE; after centrifugation, the supernatants, which still retained allthe aldolase activity, were subjected to immunotitration with ANE(broken lines). A and *, aldolase B from young mice (5 months); Eand *, aldolase B from old mice (26 months).

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1 23go w

1 23

Aid-a.4AId-*9 38.~~~~~~3.I'_

A B

FIG. 2. Identification of aldolase (Ald) degradation intermediatesin 24,000 x g supernatants of liver and kidney homogenates. Proteinswere precipitated directly from the supernatants by incubation withADE. The precipitates were run in duplicate on NaDodSO4/POIY-acrylamide gels. (A) Coomassie blue staining. (B) Electrotransfer tonitrocellulose paper and immunodecoration with ADE and 12511labeled protein A of duplicates of the samples in A followed byautoradiography. Lanes: 1, Mr 38,000 subunit of aldolase B; 2,precipitates from liver homogenates; 3, precipitates from kidneyhomogenates. Numbers on right represent M, x iO-1.

cross-react with the full subunit when ADE is used. Such adegree of cross-reactivity is not found when ANE is used(unpublished observations). In kidney (lane 3), one observesthe three longest peptides that were found in liver extracts.The longest peptide is part of an inactive partially denaturedform of the enzyme because it is precipitated with ADE,which does not recognize the native form. This peptide isequal in size to the intact subunit and may be an earlyintermediate in degradation (see Discussion).The Fate of Exogenous 12,I-Labeled Aldolase B During Liver

Homogenization. Cross-reacting peptides observed withADE were not produced as an artifact during homogenizationof the tissue. This was shown by adding '25I-labeled aldolaseto the homogenization buffer before liver disruption. It wasnecessary, however, to characterize the labeled enzyme priorto the homogenization experiments. Purified aldolase waslabeled with "25I and repurified by chromatography on aphosphocellulose column. Fig. 3 depicts results of one ofseveral control experiments that prove the labeled enzyme is

2 3 4 5 6 1

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pure, intact, and not denatured. The labeled enzyme wassubjected to NaDodSO4/PAGE and autoradiography withoutany treatment (lanes 2 and 6), and after incubation andprecipitation with ANE or ADE (lanes 3 and 4, respectively)or normal rabbit serum (lane 5). Unlabeled purified enzymewas run in lane 1. Fig. 3A shows the NaDodSO4/PAGEpattern obtained with Coomassie blue staining and Fig. 3Bdepicts its duplicate, which was subjected to autoradi-ography. It can be seen that the labeled enzyme is intact(lanes 2 and 6), and fully recognized by ANE (lane 3), but itis neither precipitated with ADE nor with normal rabbitserum (lanes 4 and 5). The repurified iodinated enzyme,eluted from the phosphocellulose column, retained at least85% of its catalytic activity. These results indicated that thelabeled enzyme could serve as a good probe in the search forsupposed homogenization-derived breakdown products.

Liver was homogenized in buffer containing 1251I-labeledaldolase B and was processed as described in Materials andMethods. Aliquots of the resulting liver extract containinglabeled aldolase B were incubated overnight with eitherANEorADE and then spun at 5000 x g for 30 min. The precipitatesthus obtained were subjected to NaDodSO4/PAGE in par-allel to aliquots ofthe extract that were not incubated with theantiserum. Duplicate preparations were then subjected toeither Coomassie blue staining (Fig. 4A) or autoradiography(Fig. 4B). In these figures, lane 1 shows a mixture of purelabeled (8125 cpm per well) and unlabeled enzyme. Lane 2shows labeled enzyme (8125 cpm per well) only, which couldnot be discerned with Coomassie blue but is very obviousafter autoradiography. Lane 3 shows the labeled enzyme(4000 cpm per well) in the 24,000 x g supernatant of liverhomogenate. The labeled enzyme was introduced into thehomogenization buffer, which contained 0.25 M sucrose andprotease inhibitors, prior to tissue disruption and was sub-jected to the homogenization procedure; 98% of the labeledenzyme was recovered after this procedure. Lane 4 showsanother labeled enzyme aliquot (3800 cpm per well), whichwas subjected to the same procedure as that in lane 3 exceptthat the homogenization buffer did not contain PhMeSO2Fand leupeptin. The recovery of labeled enzyme in this case

1 2 3 4 5 6 7 8

jser~ ~~~~~~~ -0.,1 A

2 3 4 5 6

MO *

A

IA.

1 2 3 4 5 6 78

'P.

B

A B

FIG. 3. NaDodSO4/PAGE of 125I-labeled aldolase afterphosphocellulose repurification. (A) Coomassie brilliant blue stain-ing. (B) 125I exposure on x-ray films. Lanes: 1, purified unlabeledaldolase marker; 2, 125I-labeled purified aldolase; 3, 125I-labeledaldolase incubated and precipitated with ANE; 4, 125I-labeled en-zyme incubated and precipitated with ADE; 5, 125I-labeled aldolaseincubated and precipitated with normal rabbit serum; 6, duplicate ofenzyme depicted in lane 2.

FIG. 4. NaDodSO4/PAGE of liver homogenates prepared invarious buffers to which 'l25-labeled aldolase was added prior tohomogenization. (A) Coomassie brilliant blue staining. (B) Autoradi-ography. Lanes: 1, mixture of '251-labeled and unlabeled purifiedaldolase; 2, '25I-labeled aldolase; 3, homogenate prepared with'25I-labeled aldolase in bufferA containing 0.25 M sucrose, leupeptinand PhMeSO2F; 4, homogenate prepared with 125I-labeled aldolase inbuffer A with 0.25 M sucrose and without protease inhibitors. Lanes5 and 6, homogenates prepared as in lanes 3 and 4, respectively,incubated in the presence of ANE, and dissolved in NaDodSO4.Lanes 7 and 8, homogenates prepared as in lanes 3 and 4, respec-tively, incubated with ADE, and the precipitates dissolved inNaDodSO4.

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was 96%. The same supernatants were incubated with eitherANE or ADE centrifuged at 5000 x g for 30 min, and theprecipitates were washed and run on NaDodSO4/polyacryl-amide gels. ANE precipitated the label (lanes 5 and 6) but nolabel was precipitated with ADE (lanes 7 and 8). More than90% of the label was precipitated by ANE and recovered inthe bands depicted in lanes 5 (homogenization with inhibi-tors) and 6 (homogenization without inhibition). The resultsof these studies clearly show that under the homogenizationconditions routinely used in this laboratory and within thelimits of sensitivity of our detection method, the labeledenzyme retains its full size and is recognized by ANE but notby ADE. The latter observation indicates that there is nonoticeable denaturation of the molecules during the prepa-ration of liver extract. Yet, under the same conditions, oneobserves the altered and truncated forms ofthe enzyme in thehomogenates (see below).

Characterization of the Mr 36,500 Peptide by V8 Proteolysis.The proteolytic pattern of the intact enzyme subunit and theMr 36,500 peptide is depicted in Fig. 5. The untreated M,36,500 peptide and the intact aldolase subunit are shown inlanes 1 and 4, respectively. One can see the peptide patternsof the Mr 36,500 peptide (lanes 2 and 3) and the intact subunit(lanes 5 and 6) after incubation with V8 protease at theconcentrations indicated in the legend to Fig. 5. It can be seenthat essentially nine peptides, which migrate in a very similarfashion, could be obtained from both the intact subunit andfrom the Mr 36,500 peptide. There were minor differences inpattern, which can probably be explained by the fact that theMr 36,500 peptide is a slightly truncated form of the aldolasesubunit to start with.

DISCUSSION

The experiments presented in this communication demon-strate that intracellular degradation intermediates of a spe-cific protein can be identified. This can be achieved by amethod developed in our laboratory that combines incuba-tion of a tissue extract with ADE, electrotransfer to nitro-cellulose paper, and immunodecoration with ADE and 12511labeled protein A. A search in the void volume wash of liverhomogenate chromatographed on phosphocellulose columnshas yielded the same peptides as those found in the

1 2 3 4 5 6

38 L

36.5 A,"~ttFIG. 5. Controlled proteolysis with S. aureus protease V8 of the

Mr 36,500 peptide (of Fig. 2) and the intact aldolase subunit (Mr,38,000). Bands of Mr 36,500 peptide and bands of aldolase subunitwere cut out of stained gels and placed in wells of freshly preparedNaDodSO4 gel. Lanes: 1 and 4, untreated samples of M, 36,500peptide and aldolase subunit, respectively; 2 and 3, the Mr 36,500peptide was incubated with 0.25 gg and 0.5 ,ug of the protease,respectively; 5 and 6, the intact subunit (Mr, 38,000) was incubatedwith 0.25 ,ug and 0.5 Ag of the protease, respectively. Samples wereincubated with protease V8 for 30 min before the onset of electro-phoresis. Gels were fixed and stained with Coomassie brilliant blue.Numbers on left represent Mr x lo-3.

homogenates (unpublished results). Our method becamepossible after it was found that, if produced against totallydenatured enzyme, ADE exclusively recognizes moleculesthat are at least partially denatured, or peptides derived fromthem that no longer possess native antigenic determinants.The partially denatured whole molecules and the truncatedpeptides share denatured domains, which are only recog-nized by ADE. The number of such epitopes in denaturedmolecules is not known at present, but each of the truncatedforms probably possesses at least one of them. ANE, on theother hand, recognizes undenatured epitopes common to thelonger peptides, which may still maintain partial conforma-tional integrity, and to the intact molecules. ANE, though,does not recognize the shortest peptides (unpublished re-sults), because they appear to have lost all the nativeepitopes. This has now been shown by us to be also the casefor superoxide dismutase, glyceraldehyde-3-phosphate de-hydrogenase, aldolase C, and glucose-6-phosphate dehy-drogenase, each with its own specific ADE (8).ADE is a polyclonal antibody and thus consists of a

mixture of immunoglobulins, which recognize various aminoacid sequences in the denatured molecules and not complexconformational domains that comprise the antigenic deter-minants of the native form. It might be argued that apossibility exists that the peptides recognized by ADE areunrelated to aldolase B but share with it fortuitously identicalshort primary sequences (composed of a minimum of 5-6amino acids). Such a case was recently demonstrated for amonoclonal antibody to p6Osrc, a Rous sarcoma-transformingprotein (13). This monoclonal antibody, when used in "suf-ficiently high concentrations" cross-reacted with three cyto-skeletal proteins and another unidentified intracellular pro-tein. The probability that such is the case with ADE is verylow because, unlike monoclonal antibodies, it is heteroge-neous and consists of a considerable number of antibodyspecies with a very small chance, therefore, that one of themis as specialized as a single monoclonal species (see ref. 14 fordiscussion). In addition, the same pattern of peptide recog-nition has been obtained with five different ADE preparations(unpublished results). Moreover, the peptide patterns ob-tained by controlled proteolysis of the intact Mr 38,000subunit of the enzyme and the abundant Mr 36,500 peptideidentified by ADE indicates the existence of great similaritybetween the two proteins with slight variations (Fig. 5). Theshorter peptides identified by ADE are currently beingstudied in the same manner. It should also be noticed that oneADE-recognized peptide is equal in size to the intact subunit,while the rest of the peptides identified by ADE are shorterthan the subunit of aldolase B. It seems reasonable to assumethat most if not all of these peptides are derived from aldolaseB.The possibility that the seven peptides identified by ADE

are artifacts produced during the homogenization of thetissue has been excluded. '25I-labeled native enzyme added tothe buffer before liver homogenization remains intactthroughout the procedure and is recovered with 96-98%efficiency (Fig. 4). It is recognized by ANE but not by ADE.Even after long autoradiographic exposure times, no trun-cated forms of the native molecules were observed in thesepreparations. On the other hand, 125I-labeled enzyme thatwas purposely denatured and then subjected to homogeni-zation with the tissue was entirely degraded and could not bediscerned by the means applied to the native form (8).Iodination did not render the enzyme more resistant toproteolysis because we have consistently found that nativeenzyme retained full activity after its addition to the homog-enization buffer and subsequent exposure to tissue disrup-tion.A possibility exists that these peptides are nascent incom-

plete chains of aldolase B that were being synthesized at the

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time of cell disruption by homogenization. We consider thispossibility to be very remote for the following reasons: (i)nascent peptides should not fall into distinct molecular sizesas appear on NaDodSO4/PAGE: they should rather show acontinuous spectrum of sizes; (ii) these peptides seem to bemore abundant in cells of old rather than young animals (8),despite the fact that the rate of protein synthesis is known tobe higher in the latter (15).

Several reports in the literature indicate that inactivealdolase B can be detected in livers of starved rabbits (16) andin livers of leupeptin-treated mice (17-19). In the case of thestarved rabbit, it was suggested that the inactive enzyme is aslightly truncated form derived from cathepsinM activity (20,21). A similar truncated form was found in the rat irradiatedwith x-rays and was attributed to cathepsin B activity (22,23). It is not known whether both ofthese truncated forms areactual early intermediates of the normal degradation path-way. We suggest that they are probably produced underspecial stress conditions (starvation, destabilization oflysosomes by leupeptin and x-ray irradiation) that invoke theactivity of lysosomal enzymes. In the present work, theintermediates are most probably those produced normallyduring protein degradation in the cytosol, because they arefound in the post-lysosomal fraction (24,000 x g supernatant)of the liver and the kidney.

Isolation of this peptide, which has an apparent molecularweight of the intact subunit of aldolase B, should be of greatinterest because it is probably an early form not yet cleavedthat is earmarked for degradation. That this form is partiallydenatured is obvious from the fact that it is recognized byADE. We suggest that it is perhaps a modified form of theintact aldolase B molecule and that the modification leads todenaturation of a domain or several domains in the enzymemolecule. The formation of denatured domains is mostprobably an essential step in making the protein available toproteolytic attack, as the native form of enzymes is probablya poor substrate for proteases (unpublished observations).An initial "nick" in the protein is followed by a further lossof conformational organization that renders the moleculeeven more susceptible to proteolytic attack. This develop-ment results in further cleavage of the molecule into smallerpeptides, some of which are demonstrated for aldolase B inthis paper, followed by progressive hydrolysis into aminoacids. Similar schemes on aldolase degradation and onprotein degradation in general were previously proposed byBond and Offermann (24), Ballard (25), and Gershon et al.(26). These proposals lacked basic information on the specificintracellular proteases and their products that are involved inprotein turnover in the cells. Aldolase-derived peptides ofMr<18,000 were not detected in our studies. We can onlyspeculate that they are either degraded rapidly in the cytosolor they may enter the lysosomes for final hydrolysis.The pattern of aldolase B-derived peptides in kidney

homogenates shows the presence of the three longest pep-tides, which are identical to those observed in liver. Thisfinding, if further substantiated for other tissues, may indi-cate a common basic proteolytic activity, at least for severalof the initial steps involved in normal protein degradation.Further studies are required to determine why the shorteraldolase-derived peptides are not found in kidney.

Preliminary studies using the methods described in thepresent communication have recently yielded evidence offour glyceraldehyde-3-phosphate dehydrogenase-derivedpeptides in liver homogenates. Several truncated peptideswere also found for aldolase A in muscle and brainhomogenates. These preliminary results, taken together withthose presented here as "restriction maps" of enzymemolecules, suggest that the identification of the specificproteases involved in intracellular protein degradation maynow be possible by further characterizing their specificcleavage sites in proteins.

This work was supported by U. S. Public Health Service GrantAG-00459.

1. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem.43, 835-869.

2. Hershko, A. & Ciechanover, A. (1982) Annu. Rev. Biochem.51, 335-364.

3. Reznick, A. Z. & Gershon, D. (1979) Mech. Ageing Dev. 11,403-415.

4. Reznick, A. Z., Lavie, L. & Gershon, D. (1981) FEBS Lett.128, 221-224.

5. Lavie, L., Reznick, A. Z. & Gershon, D. (1982) Biochem. J.202, 47-51.

6. Gershon H. & Gershon, D. (1973) Proc. Natl. Acad. Sci. USA70, 909-913.

7. Gershon, D. (1979) Mech. Ageing Dev. 9, 189-196.8. Reznick, A. Z., Dovrat, A., Rosenfelder, L., Shpund, S. &

Gershon, D. (1985) in Modification ofProteins During Ageing,ed. Adelman, R. C. (Liss, New York), in press.

9. Dovrat, A. & Gershon, D. (1983) Biochim. Biophys. Acta 757,164-167.

10. Dovrat, A., Scharf, Y. & Gershon, D. (1984) Mech. AgeingDev. 28, 187-191.

11. Nelson, N. (1983) Methods Enzymol. 97, 510-523.12. Morrison, M. (1974) Methods Enzymol. 32, 103-109.13. Nigg, E. A., Walter, G. & Singer, S. J. (1982) Proc. Nati.

Acad. Sci. USA 79, 5939-5943.14. Sperling, R., Francus, T. & Siskind, G. W. (1983) J. Immunol.

131, 882-885.15. Rothstein, M. (1982) Biochemical Approaches to Aging (Aca-

demic, New York), pp. 201-206.16. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B.,

Melloni, E., Michetti, M. & Horecker, B. L. (1979) Proc.Natl. Acad. Sci. USA 76, 6323-6325.

17. Kominami, E., Hashida, S. & Katunuma, N. (1981) Biochim.Biophys. Acta 659, 378-389.

18. Kominami, E., Hashida, S. & Katunuma, N. (1981) Biochim.Biophys. Acta 659, 390-400.

19. Kominami, E., Hashida, S. & Katunuma, N. (1980) Biochem.Biophys. Commun. 93, 713-719.

20. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B.,Michette, M. & Horecker, B. L. (1982) Arch. Biochem.Biophys. 214, 376-385.

21. Pontremoli, S., Melloni, E., Michetti, M., Salamino, F.,Sparatore, B. & Horecker, B. L. (1982) Proc. Natl. Acad. Sci.USA 79, 5194-51%.

22. Pote, M. S. & Altekar, W. (1980) Indian J. Biochem. Biophys.17, 225-262.

23. Pote, M. S. & Altekar, W. (1981) Biochim. Biophys. Acta 661,390-400.

24. Bond, J. S. & Offerman, M. K. (1981) Acta Biol. Germ. 40,1365-1374.

25. Ballard, F. J. (1977) Essays Biochem. 13, 1-37.26. Gershon, D., Reznick, A. & Reiss, U. (1979) Physiology and

Cell Biology ofAging, ed. Cherkin, A. (Raven, New York), pp.21-26.

Proc. Natl. Acad. Sci. USA 82 (1985)

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