Proc. Nati. Acad. Sci. USA Vol. 82, pp. 6114-6118, September 1985 Biochemistry Identification of intracellular degradation intermediates of by antiserum to the denatured (truncated peptides/immunoblot) ABRAHAM Z. REZNICK, LEAH ROSENFELDER, SHARONA SHPUND, AND DAVID GERSHON Department 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 us cally cross-reactive forms of enzyme molecules might be, at to identify intracellular degradation intermediates of fructose- least in part, intermediates of degradation. bisphosphate aldolase B (D-fructose-1,6-bisphosphate D- The present communication describes studies that led to -3-phosphate-, EC 4.1.2.13). This method the identification of seven peptides and three peptides in liver is based on the use of antibody against thoroughly denatured and kidney homogenates, respectively. These are antigeni- purified aldolase. This antibody has been shown to recognize cally cross-reacting, inactive forms of aldolase B (fructose- only denatured molecules, and it did not interact with "native" bisphosphate aldolase, D-fructose-1 ,6-bisphosphate D- enzyme. Supernatants (24,000 x g for 30 min) of liver and glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). It is sug- kidney homogenates were incubated with antiserum to dena- gested that they are normal degradation products of the tured enzyme. The antigen-antibody precipitates thus formed enzyme. were subjected to NaDodSO4/PAGE, followed by electrotrans- fer to nitrocellulose paper and immunodecoration with antise- MATERIALS AND METHODS rum to denatured enzyme and 1251-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of Animals. C57BL/6J female mice were maintained under the intact subunit) to 18,000, which cross-reacted antigenically specific pathogen-free conditions. Food was provided ad with denatured fructose-bisphosphate aldolase, could be iden- libitum. tified in liver. The longest three peptides were also present in Preparation of Native and Denatured Aldolase B. Enzyme kidney. The possibilit that these peptides were artifacts of was purified and assayed essentially as described (3). In brief, homogenization was ruled out as follows: 1251-labeled tagged livers were homogenized in 5 vol (wt/vol) of cold buffer A purified native aldolase was added to the buffer prior to liver (0.05 M Tris HCl/1 mM EDTA/1 mM 2-mercaptoetha- homogenization. The homogenates were then subjected to nol/0.25 M sucrose, pH 7.4), which contained 1 mM NaDodSO4/PAGE followed by autoradiography, and the la- phenylmethylsulfonyl fluoride (PhMeSO2F) and 15 ,ug of beled enzyme was shown to remain intact. This method is leupeptin per ml. The protease inhibitors were maintained in suggested for general use in the search for degradation prod- all subsequent stages of the purification procedure. ucts of other cellular proteins. Homogenates were centrifuged at 24,000 x g for 30 min and the supernatant was chromatographed on phosphocellulose The various stages that comprise the process of intracellular columns. More than 90% of the activity was retained on the protein degradation and the proteases involved have re- column and was eluted by 2.5 mM fructose bisphosphate. mained obscure despite extensive studies in many laborato- The void volume was retained for further analysis. A second ries (1, 2). So far no high molecular weight intermediates of passage through a phosphocellulose column usually sufficed intracellular degradation ofspecific native proteins have been to obtain pure preparations of the enzyme. The degree of identified in eukaryotes. The identification of such interme- purity was verified by NaDodSO4/PAGE. Denaturation of diates is obviously necessary in order to elucidate the the enzyme was achieved by suspending 1 mg of the purified mechanism of intracellular degradation of proteins. Several preparation in 1 ml of buffer containing 2% NaDodSO4 and previous experimental results from our laboratory have 2% 2-mercaptoethanol and boiling for 5 min. Complete indicated that a direct search for degradation intermediates of denaturation is an essential condition for the preparation of specific proteins might be feasible for the following reasons: antisera with absolute specificity for denatured molecules (i) because of a considerable slow down in degradation of that do not react with native molecules. "native" proteins and aberrant peptides in cells of aging Preparation of Antiserum to Denatured and Native Aldolase animals (3-5), it was possible to find an age-associated B. Antisera to native enzyme (ANE) were prepared as accumulation of catalytically altered enzyme molecules that described (3). Antisera to denatured enzyme (ADE) were retained antigenic cross-reactivity with native forms of the prepared as described elsewhere (8). In brief, immediately (e.g., see refs. 6 and 7); a small proportion of the after boiling in NaDodSO4 and 2-mercaptoethanol, the de- same forms was subsequently found in cells of young animals natured enzyme was emulsified in complete Freund's adju- (8); (ii) such altered enzyme molecules of liver cytosolic vant (1:1, vol/vol) and injected into rabbits in multiple superoxide dismutase, lens aldolase C and glyceraldehyde- intradermal sites. Animals were bled 10, 14, and 21 days later 3-phosphate dehydrogenase could be preferentially removed and antisera were tested for the presence of ADE by the from tissue homogenates by antibodies elicited against de- Ouchterlony double-diffusion test. Booster injections were natured forms of these enzymes (8-10); (iii) significantly, administered at 3-wk intervals until antibody titers were these antibodies did not interact with native enzyme mole- sufficiently high, at which point the animals were bled once cules (8); (iv) these findings suggested that altered antigeni- a week for as long as antibody titers remained high.

The publication costs of this article were defrayed in part by page charge Abbreviations: ADE, anti-denatured enzyme antibody; ANE, anti- payment. This article must therefore be hereby marked "advertisement" native enzyme antibody; PhMeSO2F, phenylmethylsulfonyl fluo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ride. 6114 Downloaded by guest on September 26, 2021 Biochemistry: Reznick et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6115 Immunotitration Studies With ANE With and Without overnight with ADE. The preparations were then centrifuged Previous Incubation in ADE. Livers of young and old mice and the supernatants, which still contained all the aldolase B were homogenized in 4 vol of buffer A containing 0.25 M enzyme activity, were subjected to immunotitration with sucrose (isotonic), PhMeSO2F, and leupeptin under condi- ANE. In parallel, aliquots of the same homogenates that had tions that facilitate the removal of intact lysosomes without not been incubated with ADE were also immunotitrated with disruption and prevent proteolysis (4). The homogenate was ANE. Comparison of the immunotitration curves of spun at 24,000 x g for 1 hr, and the resultant supernatant was homogenates with and without incubation with ADE taken used for further analysis. The enzyme activities of "young" from a representative experiment are depicted in Fig. 1. The and "old" preparations were adjusted to the same levels. results clearly show that incubation with ADE causes re- Supernatant (150-200 ,p1) was incubated with 600 ,u1 of ADE moval of cross-reacting material from "old" and to a small and 850 ,ul of buffer A. After overnight incubation at 40C and but easily discernible degree from "young" homogenates, centrifugation at 3000 x g for 30 min, the precipitates were which lacks catalytic activity and is apparently at least removed and aldolase activity was determined in the super- partially denatured. This is apparent from the fact that after natant. In all the experiments, it was repeatedly observed incubation with ADE less ANE is required to precipitate the that the entire activity remained in the supernatant after the same amount ofaldolase activity from the homogenates. The incubation with ADE. Immunotitration ofthis supernatant or removal of cross-reacting material by ADE from young ofhomogenates without incubation with ADE was performed homogenates has been consistently observed in several as described (6). preparations. The cross-reacting material precipitated with Identification of Antigenically Cross-Reactive Peptides in ADE from young homogenates in several preparations was Liver Homogenates. Homogenate supernatants (24,000 x g) used for all further analyses reported in this communication. were concentrated 4-fold over Amicon PM30 membranes. The immunotitrations with ANE depicted in Fig. 1 agree well The concentrates were incubated overnight with either ADE with previous experiments, which unequivocally demon- or ANE as is indicated below for the individual experiments. strated cross-reacting material in old aldolase preparations After centrifugation at 3000 x g, precipitates were washed (4, 6). once with buffer (10 mM Tris base, pH 8.0/10 mM Identification of Aldolase B Derived Altered Peptides in NaCl/1.5% Triton X-100) and twice with the same buffer Liver and Kidney Extracts. Liver and kidney homogenates devoid ofTriton X-100. The final precipitates were dissolved were spun at 24,000 X g for 60 min. The supernatant was in buffer with 2% NaDodSO4/2% 2-mercaptoethanol/5% bromophenol blue, and boiled for 5 min. Samples were then incubated with ADE and the resultant precipitate was pre- subjected to NaDodSO4/PAGE in 12.5% gels and protein pared for and subjected to NaDodSO4/PAGE. The peptide bands were stained with Coomassie brilliant blue. For de- patterns observed with Coomassie blue are depicted in Fig. tection of aldolase degradation products, gels were subjected 2A. Duplicates ofthe same preparation were transferred from to a procedure of electrotransfer to nitrocellulose paper and the NaDodSO4/polyacrylamide gel to nitrocellulose paper immunodecoration with ADE and 1251-labeled protein A (8, and immunodecorated with ADE and 125I-labeled protein A. 11). The autoradiographic patterns of this preparation are shown Labeling of Aldolase With "2SI. Todination of purified in Fig. 2B. In liver extracts, seven peptides ranging in aldolase with lactoperoxidase was carried out according to molecular weight from 38,000 to 18,000 (based on molecular the procedure of Morrison (12) with one modification. In- weight markers) are discerned (lane 2). One of these has an stead of adding all the H202 at the start of the reaction, five apparent size equivalent to the intact subunit (Mr, 38,000), aliquots of0.03% H202 solution were added to the incubation and six appear to be truncated forms that antigenically mixture at 2-min intervals with gentle shaking. The reaction was stopped by cooling to 4°C and by applying the reaction mixture to a phosphocellulose column equilibrated with 1000- buffer A. This procedure separated the "native" aldolase from the peroxidase, the free 1251, and any inactivated molecules of the enzyme. The iodinated aldolase was then eluted from the phosphocellulose column with 2.5 mM 90 fructose 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 with 10- ADE. The precipitate obtained after centrifugation was 830 washed and subjected to NaDodSO4/PAGE in parallel to purified aldolase B. The gels were stained with Coomassie 20 blue 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 placed 10 20 30 40 50 60 in the wells of a freshly prepared NaDodSO4/polyacrylamide Anti-native antiserum, Al gel. V8 protease (Sigma) at the concentrations indicated in the legend to Fig. 5, was then applied to the wells and FIG. 1. Immunotitration analysis of aldolase B in homogenates of incubated with the gel pieces containing the various peptides livers of young and old rats without ADE and after incubation with for 1 hr prior to electrophoresis. After electrophoresis, the ADE. Aliquots ofhomogenates adjusted to the same aldolase activity bands were stained with Coomassie blue for peptide identi- were incubated overnight with increasing amounts of ANE. After fication. centrifugation at 3000 x g for 30 min, activity was measured in the supernatant. Titration with ANE without incubation with ADE is depicted by solid lines. Aliquots of the same homogenates, adjusted RESULTS to the same aldolase activity, were first incubated overnight with ADE; after centrifugation, the supernatants, which still retained all Evidence for the Existence of Inactive Partially Denatured the aldolase activity, were subjected to immunotitration with ANE Molecules of Aldolase B in Liver Homogenates. Aliquots of (broken lines). A and *, aldolase B from young mice (5 months); E liver homogenates from young and old mice were incubated and *, aldolase B from old mice (26 months). Downloaded by guest on September 26, 2021 6116 Biochemistry: Reznick et al. Proc. Natl. Acad. Sci. USA 82 (1985) 1 23 1 23 pure, intact, and not denatured. The labeled enzyme was go w subjected to NaDodSO4/PAGE and autoradiography without any treatment (lanes 2 and 6), and after incubation and precipitation with ANE or ADE (lanes 3 and 4, respectively) or normal rabbit serum (lane 5). Unlabeled purified enzyme was run in lane 1. Fig. 3A shows the NaDodSO4/PAGE pattern obtained with Coomassie blue staining and Fig. 3B depicts its duplicate, which was subjected to autoradi- ography. It can be seen that the labeled enzyme is intact Aid-a.4A.~~~~~~3.Id-*9 38 (lanes 2 and 6), and fully recognized by ANE (lane 3), but it is neither precipitated with ADE nor with normal rabbit I'_ serum (lanes 4 and 5). The repurified iodinated enzyme, eluted from the phosphocellulose column, retained at least 85% of its catalytic activity. These results indicated that the labeled enzyme could serve as a good probe in the search for A B supposed homogenization-derived breakdown products. Liver was homogenized in buffer containing 1251I-labeled FIG. 2. Identification of aldolase (Ald) degradation intermediates aldolase B and was processed as described in Materials and in 24,000 x g supernatants ofliver and kidney homogenates. Proteins Methods. Aliquots of the resulting liver extract containing were precipitated directly from the supernatants by incubation with labeled aldolase B were incubated overnight with either ANE ADE. The precipitates were run in duplicate on NaDodSO4/POIY- acrylamide gels. (A) Coomassie blue staining. (B) Electrotransfer to or ADE and then spun at 5000 x g for 30 min. The precipitates nitrocellulose paper and immunodecoration with ADE and 12511 thus obtained were subjected to NaDodSO4/PAGE in par- labeled protein A of duplicates of the samples in A followed by allel to aliquots ofthe extract that were not incubated with the autoradiography. Lanes: 1, Mr 38,000 subunit of aldolase B; 2, antiserum. Duplicate preparations were then subjected to precipitates from liver homogenates; 3, precipitates from kidney either Coomassie blue staining (Fig. 4A) or autoradiography homogenates. Numbers on right represent M, x iO-1. (Fig. 4B). In these figures, lane 1 shows a mixture of pure labeled (8125 cpm per well) and unlabeled enzyme. Lane 2 cross-react with the full subunit when ADE is used. Such a shows labeled enzyme (8125 cpm per well) only, which could degree of cross-reactivity is not found when ANE is used not be discerned with Coomassie blue but is very obvious (unpublished observations). In kidney (lane 3), one observes after autoradiography. Lane 3 shows the labeled enzyme the three longest peptides that were found in liver extracts. (4000 cpm per well) in the x liver The is of an inactive 24,000 g supernatant of longest peptide part partially denatured homogenate. The labeled enzyme was the form of the enzyme because it is precipitated with ADE, introduced into which does not recognize the native form. This peptide is homogenization buffer, which contained 0.25 M sucrose and equal in size to the intact subunit and may be an early protease inhibitors, prior to tissue disruption and was sub- intermediate in degradation (see Discussion). jected to the homogenization procedure; 98% of the labeled The Fate of Exogenous 12,I-Labeled Aldolase B During Liver enzyme was recovered after this procedure. Lane 4 shows Homogenization. Cross-reacting peptides observed with another labeled enzyme aliquot (3800 cpm per well), which ADE were not produced as an artifact during homogenization was subjected to the same procedure as that in lane 3 except of the tissue. This was shown by adding '25I-labeled aldolase that the homogenization buffer did not contain PhMeSO2F to the homogenization buffer before liver disruption. It was and leupeptin. The recovery of labeled enzyme in this case necessary, however, to characterize the labeled enzyme prior to the Purified aldolase was homogenization experiments. 1 1 2 3 4 5 6 78 labeled with "25I and repurified by chromatography on a 2 3 4 5 6 7 8 phosphocellulose column. Fig. 3 depicts results of one of several control experiments that prove the labeled enzyme is

jser~ ~~~~~~~ 2 3 4 5 6 1 2 3 4 5 6 . -0 'P. I; On ,1 A

1 .

A.I i 4w MO * F4 40 t 4 A B

FIG. 4. NaDodSO4/PAGE of liver homogenates prepared in various buffers to which 'l25-labeled aldolase was added prior to A B homogenization. (A) Coomassie brilliant blue staining. (B) Autoradi- ography. Lanes: 1, mixture of '251-labeled and unlabeled purified aldolase; 2, '25I-labeled aldolase; 3, homogenate prepared with FIG. 3. NaDodSO4/PAGE of 125I-labeled aldolase after '25I-labeled aldolase in buffer A containing 0.25 M sucrose, leupeptin phosphocellulose repurification. (A) Coomassie brilliant blue stain- and PhMeSO2F; 4, homogenate prepared with 125I-labeled aldolase in ing. (B) 125I exposure on x-ray films. Lanes: 1, purified unlabeled buffer A with 0.25 M sucrose and without protease inhibitors. Lanes aldolase marker; 2, 125I-labeled purified aldolase; 3, 125I-labeled 5 and 6, homogenates prepared as in lanes 3 and 4, respectively, aldolase incubated and precipitated with ANE; 4, 125I-labeled en- incubated in the presence of ANE, and dissolved in NaDodSO4. zyme incubated and precipitated with ADE; 5, 125I-labeled aldolase Lanes 7 and 8, homogenates prepared as in lanes 3 and 4, respec- incubated and precipitated with normal rabbit serum; 6, duplicate of tively, incubated with ADE, and the precipitates dissolved in enzyme depicted in lane 2. NaDodSO4. Downloaded by guest on September 26, 2021 Biochemistry: Reznick et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6117 was 96%. The same supernatants were incubated with either homogenates (unpublished results). Our method became ANE or ADE centrifuged at 5000 x g for 30 min, and the possible after it was found that, if produced against totally precipitates were washed and run on NaDodSO4/polyacryl- denatured enzyme, ADE exclusively recognizes molecules amide gels. ANE precipitated the label (lanes 5 and 6) but no that are at least partially denatured, or peptides derived from label was precipitated with ADE (lanes 7 and 8). More than them that no longer possess native antigenic determinants. 90% of the label was precipitated by ANE and recovered in The partially denatured whole molecules and the truncated the bands depicted in lanes 5 (homogenization with inhibi- peptides share denatured domains, which are only recog- tors) and 6 (homogenization without inhibition). The results nized by ADE. The number of such epitopes in denatured of these studies clearly show that under the homogenization molecules is not known at present, but each of the truncated conditions routinely used in this laboratory and within the forms probably possesses at least one of them. ANE, on the limits of sensitivity of our detection method, the labeled other hand, recognizes undenatured epitopes common to the enzyme retains its full size and is recognized by ANE but not longer peptides, which may still maintain partial conforma- by ADE. The latter observation indicates that there is no tional integrity, and to the intact molecules. ANE, though, noticeable denaturation of the molecules during the prepa- does not recognize the shortest peptides (unpublished re- ration of liver extract. Yet, under the same conditions, one sults), because they appear to have lost all the native observes the altered and truncated forms ofthe enzyme in the epitopes. This has now been shown by us to be also the case homogenates (see below). for superoxide dismutase, glyceraldehyde-3-phosphate de- Characterization of the Mr 36,500 Peptide by V8 Proteolysis. hydrogenase, aldolase C, and glucose-6-phosphate dehy- The proteolytic pattern of the intact enzyme subunit and the drogenase, each with its own specific ADE (8). Mr 36,500 peptide is depicted in Fig. 5. The untreated M, ADE is a polyclonal antibody and thus consists of a 36,500 peptide and the intact aldolase subunit are shown in mixture of immunoglobulins, which recognize various amino lanes 1 and 4, respectively. One can see the peptide patterns acid sequences in the denatured molecules and not complex of the Mr 36,500 peptide (lanes 2 and 3) and the intact subunit conformational domains that comprise the antigenic deter- (lanes 5 and 6) after incubation with V8 protease at the minants of the native form. It might be argued that a concentrations indicated in the legend to Fig. 5. It can be seen possibility exists that the peptides recognized by ADE are that essentially nine peptides, which migrate in a very similar unrelated to aldolase B but share with it fortuitously identical fashion, could be obtained from both the intact subunit and short primary sequences (composed of a minimum of 5-6 from the Mr 36,500 peptide. There were minor differences in amino acids). Such a case was recently demonstrated for a pattern, which can probably be explained by the fact that the monoclonal antibody to p6Osrc, a Rous sarcoma-transforming Mr 36,500 peptide is a slightly truncated form of the aldolase protein (13). This monoclonal antibody, when used in "suf- subunit to start with. 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 very DISCUSSION low because, unlike monoclonal antibodies, it is heteroge- The experiments presented in this communication demon- neous and consists of a considerable number of antibody species with a very small chance, therefore, that one of them strate that intracellular degradation intermediates of a spe- is as specialized as a single monoclonal species (see ref. 14 for cific protein can be identified. This can be achieved by a discussion). In addition, the same pattern of peptide recog- method developed in our laboratory that combines incuba- nition has been obtained with five different ADE preparations tion of a tissue extract with ADE, electrotransfer to nitro- (unpublished results). Moreover, the peptide patterns ob- cellulose paper, and immunodecoration with ADE and 12511 tained by controlled proteolysis of the intact Mr 38,000 labeled protein A. A search in the void volume wash of liver subunit of the enzyme and the abundant Mr 36,500 peptide homogenate chromatographed on phosphocellulose columns identified by ADE indicates the existence of great similarity has yielded the same peptides as those found in the between the two proteins with slight variations (Fig. 5). The shorter peptides identified by ADE are currently being 1 2 3 4 5 6 studied in the same manner. It should also be noticed that one ADE-recognized peptide is equal in size to the intact subunit, while the rest of the peptides identified by ADE are shorter than the subunit of aldolase B. It seems reasonable to assume 38 L 36.5 A, that most if not all of these peptides are derived from aldolase B. The possibility that the seven peptides identified by ADE are artifacts produced during the homogenization of the tissue has been excluded. '25I-labeled native enzyme added to the buffer before liver homogenization remains intact throughout 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 these FIG. 5. Controlled proteolysis with S. aureus protease V8 of the preparations. On the other hand, 125I-labeled enzyme that Mr 36,500 peptide (of Fig. 2) and the intact aldolase subunit (Mr, was purposely denatured and then subjected to homogeni- 38,000). Bands of Mr 36,500 peptide and bands of aldolase subunit zation with the tissue was entirely degraded and could not be were cut out of stained gels and placed in wells of freshly prepared discerned by the means applied to the native form (8). NaDodSO4 gel. Lanes: 1 and 4, untreated samples of M, 36,500 Iodination did not render the enzyme more resistant to peptide and aldolase subunit, respectively; 2 and 3, the Mr 36,500 we have that native peptide was incubated with 0.25 gg and 0.5 ,ug of the protease, proteolysis because consistently found respectively; 5 and 6, the intact subunit (Mr, 38,000) was incubated enzyme retained full activity after its addition to the homog- "~tt enization buffer and subsequent exposure to tissue with 0.25 ,ug and 0.5 Ag of the protease, respectively. Samples were disrup- incubated with protease V8 for 30 min before the onset of electro- tion. phoresis. Gels were fixed and stained with Coomassie brilliant blue. A possibility exists that these peptides are nascent incom- Numbers on left represent Mr x lo-3. plete chains of aldolase B that were being synthesized at the Downloaded by guest on September 26, 2021 6118 Biochemistry: Reznick et al. Proc. Natl. Acad. Sci. USA 82 (1985) time of cell disruption by homogenization. We consider this Preliminary studies using the methods described in the possibility to be very remote for the following reasons: (i) present communication have recently yielded evidence of nascent peptides should not fall into distinct molecular sizes four glyceraldehyde-3-phosphate dehydrogenase-derived as appear on NaDodSO4/PAGE: they should rather show a peptides in liver homogenates. Several truncated peptides continuous spectrum of sizes; (ii) these peptides seem to be were also found for in muscle and more abundant in cells of old rather than young animals (8), homogenates. These preliminary results, taken together with despite the fact that the rate of protein synthesis is known to those presented here as "restriction maps" of enzyme be higher in the latter (15). molecules, suggest that the identification of the specific Several reports in the literature indicate that inactive proteases involved in intracellular protein degradation may aldolase B can be detected in livers of starved rabbits (16) and now be possible by further characterizing their specific in livers of leupeptin-treated mice (17-19). In the case of the cleavage sites in proteins. starved rabbit, it was suggested that the inactive enzyme is a slightly truncated form derived from cathepsin M activity (20, This work was supported by U. S. Public Health Service Grant 21). A similar truncated form was found in the rat irradiated AG-00459. with x-rays and was attributed to cathepsin B activity (22, 23). It is not known whether both ofthese truncated forms are 1. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem. actual early intermediates of the normal degradation path- 43, 835-869. 2. Hershko, A. & Ciechanover, A. (1982) Annu. Rev. Biochem. way. We suggest that they are probably produced under 51, 335-364. special stress conditions (starvation, destabilization of 3. Reznick, A. Z. & Gershon, D. (1979) Mech. Ageing Dev. 11, lysosomes by leupeptin and x-ray irradiation) that invoke the 403-415. activity of lysosomal enzymes. In the present work, the 4. Reznick, A. Z., Lavie, L. & Gershon, D. (1981) FEBS Lett. intermediates are most probably those produced normally 128, 221-224. during protein degradation in the cytosol, because they are 5. Lavie, L., Reznick, A. Z. & Gershon, D. (1982) Biochem. J. found in the post-lysosomal fraction (24,000 x g supernatant) 202, 47-51. of the liver and the kidney. 6. Gershon H. & Gershon, D. (1973) Proc. Natl. Acad. Sci. USA Isolation of this peptide, which has an apparent molecular 70, 909-913. 7. Gershon, D. (1979) Mech. Ageing Dev. 9, 189-196. weight of the intact subunit of aldolase B, should be of great 8. Reznick, A. Z., Dovrat, A., Rosenfelder, L., Shpund, S. & interest because it is probably an early form not yet cleaved Gershon, D. (1985) in Modification ofProteins During Ageing, that is earmarked for degradation. That this form is partially ed. Adelman, R. C. (Liss, New York), in press. denatured is obvious from the fact that it is recognized by 9. Dovrat, A. & Gershon, D. (1983) Biochim. Biophys. Acta 757, ADE. We suggest that it is perhaps a modified form of the 164-167. intact aldolase B molecule and that the modification leads to 10. Dovrat, A., Scharf, Y. & Gershon, D. (1984) Mech. Ageing denaturation of a domain or several domains in the enzyme Dev. 28, 187-191. 11. Nelson, N. (1983) Methods Enzymol. 97, 510-523. molecule. The formation of denatured domains is most 12. Morrison, M. (1974) Methods Enzymol. 32, 103-109. probably an essential step in making the protein available to 13. Nigg, E. A., Walter, G. & Singer, S. J. (1982) Proc. Nati. proteolytic attack, as the native form of enzymes is probably Acad. Sci. USA 79, 5939-5943. a poor substrate for proteases (unpublished observations). 14. Sperling, R., Francus, T. & Siskind, G. W. (1983) J. Immunol. An initial "nick" in the protein is followed by a further loss 131, 882-885. of conformational organization that renders the molecule 15. Rothstein, M. (1982) Biochemical Approaches to Aging (Aca- demic, New York), pp. 201-206. even more susceptible to proteolytic attack. This develop- 16. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., ment results in further cleavage of the molecule into smaller Melloni, E., Michetti, M. & Horecker, B. L. (1979) Proc. peptides, some of which are demonstrated for aldolase B in Natl. Acad. Sci. USA 76, 6323-6325. this paper, followed by progressive hydrolysis into amino 17. Kominami, E., Hashida, S. & Katunuma, N. (1981) Biochim. acids. Similar schemes on aldolase degradation and on Biophys. Acta 659, 378-389. 18. Kominami, E., Hashida, S. & Katunuma, N. (1981) Biochim. protein degradation in general were previously proposed by Biophys. Acta 659, 390-400. Bond and Offermann (24), Ballard (25), and Gershon et al. 19. Kominami, E., Hashida, S. & Katunuma, N. (1980) Biochem. (26). These proposals lacked basic information on the specific Biophys. Commun. 93, 713-719. intracellular proteases and their products that are involved in 20. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michette, M. & Horecker, B. L. (1982) Arch. Biochem. protein turnover in the cells. Aldolase-derived peptides ofMr Biophys. 214, 376-385. <18,000 were not detected in our studies. We can only 21. Pontremoli, S., Melloni, E., Michetti, M., Salamino, F., speculate that they are either degraded rapidly in the cytosol Sparatore, B. & Horecker, B. L. (1982) Proc. Natl. Acad. Sci. or they may enter the lysosomes for final hydrolysis. USA 79, 5194-51%. The pattern of aldolase B-derived peptides in kidney 22. Pote, M. S. & Altekar, W. (1980) Indian J. Biochem. Biophys. 17, 225-262. homogenates shows the presence of the three longest pep- 23. Pote, M. S. & Altekar, W. (1981) Biochim. Biophys. Acta 661, tides, which are identical to those observed in liver. This 390-400. finding, if further substantiated for other tissues, may indi- 24. Bond, J. S. & Offerman, M. K. (1981) Acta Biol. Germ. 40, cate a common basic proteolytic activity, at least for several 1365-1374. of the initial steps involved in 25. Ballard, F. J. (1977) Essays Biochem. 13, 1-37. normal protein degradation. 26. Gershon, D., Reznick, A. & Reiss, U. (1979) Physiology and Further studies are required to determine why the shorter Cell Biology ofAging, ed. Cherkin, A. (Raven, New York), pp. aldolase-derived peptides are not found in kidney. 21-26. Downloaded by guest on September 26, 2021