Biochemical and Molecular Roles of Nutrients

Protective Effect of Ascorbic Acid on the Breakdown of Proteins Exposed to Hydrogen Peroxide in Chicken Skeletal Muscle1 Department of Animal Sciences, Rutgers University, Hew Brunswick,

NJ 08903

trophils stimulated to undergo a respiratory burst (Chance et al. 1979, Halliwell and Gutteridge 1985). Living organisms are also continuously exposed to UV radiation and to other external sources of oxidants present in dietary constituents, in naturally occurring radioactive gases such as radon, and in such environ mental pollutants as car exhaust and cigarette smoke (Ames 1983, Cross et al. 1987). When antioxidants are unable to counteract the oxidants and their products, damage to amino acids, proteins, nucleic acids and lipids can occur. Oxidants have been linked to the development or propagation of cancer, cataracts, strokes, heart attacks and the aging process (Ames 1983, Cross et al. 1987, Halliwell and Gutteridge 1985). Increasing the intake of such dietary antiox idants as a-tocopherol, ß-carotene and ascorbic acid may provide protection against cellular degeneration by oxidants and free radicals (Pauling 1970). Ascorbic acid functions as both a reducing and a chelating agent (Kroneck et al. 1982, Marteli 1982). It has been shown to scavenge free radicals and is an important component of the antioxidative defense mechanism in cells and tissue (for a review, see Bendich et al. 1986). Ascorbic acid can prevent the initiation of lipid peroxidation by aqueous peroxyl radicals (Frei et al. 1989) and is thought to regenerate vitamin E by reacting with the vitamin E radical (Packer et al. 1979). Levels of ascorbate (140 umol/L)

ABSTRACT Ascorbic acid is believed to protect cells from oxidative damage by reacting with oxygen-derived free radicals. We investigated whether ascorbic acid would affect the rate of breakdown of skeletal muscle proteins in extracts exposed to hydrogen peroxide. As corbic acid (20 mmol/L) alone had little or no effect on the rate of ATP-independent or ATP-dependent breakdown of proteins in chicken skeletal muscle. Pretreatment of chicken skeletal muscle extracts with 10 mmol/L H2Ü2resulted in a complete loss of ATPdependent proteolysis and a significant increase (14- to 15-fold) in the rate of ATP-independent protein breakdown. Ascorbic acid (20 mmol/L) did not prevent H2O2 (10 mmol/L) from inactivating the ATP-de pendent proteolytic pathway in skeletal muscle. However, ascorbic acid (20 mmol/L) prevented the H2O2-induced increase in the ATP-independent pro teolysis of endogenous muscle proteins. Ascorbic acid also slowed the rate of hydrolysis of exogenously added [3H]superoxide dismutase exposed to \\2®2 and in hibited the enhanced degradation of [3H]lysozyme and H2C>2-treated [3H]superoxide dismutase by the proteo lytic systems exposed to H2Û2. Thus ascorbic acid seems to inhibit the H2U2-induced increase in ATPindependent proteolysis 1) by preventing damage to pro teins by H-¿O-¿ resulting in a decreased supply of sub strates for the ATP-independent degradative system and 2) by preventing activation of the proteolytic enzymes that participate in the energy-independent degradation of H2O2-treated proteins. J. Nutr. 122: 2087-2093, 1992. INDEXING KEY WORDS:

•ascorbic acid •hydrogen peroxide •skeletal muscle protein breakdown •chickens ^his work was conducted during the tenure of an established investigatorship of the American Heart Association awarded to J.M.F and was supported by research grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, USDA, American Heart Association, Rutgers University and the New Jersey Agricultural Experiment Station, which is supported by State funds. To whom correspondence should be addressed. Abbreviations used: DTPA, diethylene triamine pentaacetic acid; E-64, £funs-expoxysuccinyl-L-leucylamido-(4-guanidino) butane; SOD, Superoxide dismutase.

Active oxygen species produced in the body are usually rendered harmless by endogenous enzymatic and nonenzymatic antioxidative defenses. Such antioxidative enzymes as Superoxide dismutase (SOD),3 glutathione peroxidase and catalase help maintain low levels of oxidants that are normally produced by, for example, respiring mitochondria and by neu0022-3166/92

$3.00 ©1992 American

Institute

of Nutrition.

Received 9 March 1992. Accepted 25 June 1992. 2087

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OKSANA M. GECHA ANDJULIE M. FAGAN2

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MATERIALS AND METHODS Animals and reagents. Day-old male White Leghorn chicks (Avian Services, Frenchtown, NJ) were housed in wire-floored 32-35°C brooder chambers with a 12-h light:dark photoperiod in an environmentally controlled room. Chicks were al lowed ad libitum access to food (Country Broiler Maker Agway, Flemington, NJ) and water. A protocol describing the care and use of the birds was reviewed and approved by the Rutgers University Institutional Review Board for the Use and Care of Animals. Dithiothreitol and Tris base were obtained from Research Organics (Cleveland, OH); frans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) was purchased from Cambridge Research Biochemicals (Wilmington, DE). Diethylene triamine pentaacetic acid (DTPA), EGTA, EDTA, ATP, 2-mercaptoethanol, ubiquitin and ascorbic acid were purchased from Sigma Chemical (St. Louis, MO). Ascorbic acid was prepared daily as a 1 mol/L stock solution in 50 mmol/L sodium phosphate buffer (pH 7.3) containing 0.1 mmol/L DTPA and was neutralized to pH 7.3. Bovine erythrocyte cupro-zinc SOD (EC 1.15.1.1) was obtained from Calbiochem (La Jolla, CA), and egg white lysozyme (EC 3.2.1.17) was purchased from

Miles Laboratories (West Haven, CT). Lysozyme and SOD were tritiated by reductive methylation with [3H]formaldehyde (NEN Products, Du Pont, Wil mington, DE) and sodium cyanoborohydride (Jenthoft and Dearborn 1979). [3H]Lysozyme was denatured by treatment overnight at 37°Cwith 6 mol/L guanidine, 0.1 mmol/L EDTA, 1 mmol/L EGTA, and the sulfhydryl groups were then alkylated at room temper ature for 30 min with 0.2 mol/L iodoacetamide. Treated samples were then dialyzed against several changes of H2O (pH 10) over 2 d. Preparation of skeletal muscle extracts and fraction II. Pectoralis muscle (400 g) from 11-mo-old White Leghorn male chickens was diced, ground in a pre-chilled meat grinder and homogenized for 1 min in a Waring blender in three volumes (wt/v) of 20 mmol/L Tris-HCl buffer (pH 8) containing 1 mmol/L 2-mercaptoethanol, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 0.05 mmol/L E-64 and 12 g/L glycerol. The homogenate was adjusted to pH 7 with 1 mol/L Tris base and then centrifuged at 15,000 x g for 30 min. The pellet was discarded and the supernatant (crude extract) was either dialyzed over 2 d against several changes of buffer containing 10 mmol/L Tris-HCl (pH 7.6), 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1 mmol/ L EGTA, 10 mmol/L KC1 and 240 g/L glycerol, or applied to a 70-mL diethylaminoethyl-cellulose (Whatman, DE-52) column (3 cm x 24 cm) equilibrated with 20 mmol/L Tris-HCl (pH 7.05), 0.5 mmol/L dithiothreitol, 0.1 mmol/L EDTA, 0.1 mmol/ L EGTA, and 240 g/L glycerol. Proteins bound to the resin and eluted with 0.5 mol/L NaCl (fraction II) were dialyzed over 2 d against several changes of buffer containing 10 mmol/L Tris-HCl (pH 7.6), 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 10 mmol/L KC1 and 120 g/L glycerol. Treatment of proteins with H¿O2and ascorbic acid. Skeletal muscle crude extracts and fraction-II were pretreated with 0.25 mmol/L sodium azide for 10 min at 4°C to inactivate endogenous catalase. Crude extract (18.6 mg/mL) and fraction-II (1.3 mg/ mL) were then incubated for l h at 37°C in the presence of 5 mmol/L ATP with 10 mmol/L H2O2, 20 mmol/L ascorbic acid or both. Treated extracts were then dialyzed against three changes of 10 mmol/L Tris-HCl (pH 7.6) buffer containing 1 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA and 240 g/L glycerol in order to remove the ATP, H2Û2 and as corbic acid before assaying for protein breakdown. pHjSuperoxide dismutase (2 mg) was incubated for 1 h at 37°C with H2Û2 (10 mmol/L) and/or ascorbic acid (20 mmol/L) and was then dialyzed against H2Û (pH 7.6) containing 0.25 mmol/L sodium azide over 2 d to remove the H2Û2 and ascorbic acid. When treating proteins with both H2Û2 and ascorbic acid, the protein was preincubated with ascorbic acid on ice 5 min before the addition of H2Û2-

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in human plasma were reported to quench the oxidants (e.g., Superoxide radicals, H2Û2, hypochlorous acid) released from activated polymorphonuclear leu kocytes (Frei et al. 1989). High concentrations of as corbic acid in the ocular lens may also help prevent the formation of cataracts induced by photo-oxidative damage (Varma et al. 1982). Recently it has been shown that the total ascorbic acid content in lung alveolar macrophages was twofold higher in human smokers and in hamsters exposed to cigarette smoke than in nonsmoking controls (McGowan et al. 1984). These increased levels of ascorbate may be an adaptive response to protect against the oxidizing agents present in cigarette smoke. In the present investigation we examined the effect of ascorbic acid on the degradation of skeletal muscle proteins exposed to H2Û2- Proteins damaged by the oxidant phenylhydrazine have been shown to be rapidly removed from red blood cells by a soluble ATP-independent proteolytic system (Fagan et al. 1986). Alternatively, a soluble ATP-requiring proteo lytic pathway in reticulocytes was found to hydrolyze other types of abnormal proteins, such as those formed as a result of mutation, nonenzymatic glycosylation, spontaneous deamidation or errors in biosynthesis (reviewed in Goldberg and St. John 1976). We therefore examined the effect of H2Û2 and ascorbic acid on the ATP-dependent and ATP-in dependent proteolytic systems in chicken skeletal muscle.

ASCORBIC ACID AND H2O2-INDUCED

Determination of protein breakdown. Proteolysis of endogenous proteins in dialyzed extracts was deter mined by measuring the release of tyrosine from skeletal muscle proteins. The protein concentration of the crude extracts and fraction-II was determined by the method of Bradford (1976) with bovine serum albumin as a standard. Crude extracts (3 mg) or fraction-II (0.6 mg) were incubated for l h at 37°Cin

RESULTS Previous results have shown that hemoglobin damaged by oxidants was rapidly degraded by an energy-independent proteolytic system in erythroid cells (Fagan et al. 1986). Similarly, skeletal muscle proteins treated with H2Ü2 were degraded more rapidly than untreated proteins in the absence of ATP in soluble extracts of skeletal muscle. Exposure of chicken skeletal muscle lysate to 10 mmol/L H2C>2 resulted in a 14- to 15-fold increase in the ATPindependent degradation of skeletal muscle proteins (Table 1). The concentration of H2Û2 used in these studies is far in excess of what is considered to be physiological (10-9-lQ-7 mol/L, Oshino et al. 1973), although these calculations are based on a number of simplifying assumptions that may or may not be valid. Lower concentrations (0.01-5 mmol/L) of H2Û2 were also found to increase proteolysis. However, we chose to expose muscle extracts to the higher concen trations of H2Û2 because the effect of H2Û2 on pro teolysis was more variable (>5% variation within

TABLE

2089

1

Effect of IÌ22 '""' ascorbic acid on protein breakdown in chicken skeletal muscle lysate1 Protein degradation Treatment

of lysate

ATP-independent

ATP-dependent

h894400±±± NoneAscorbicH2O2

Ascorbicacidacid+ H2O2921041442178±±± ±pmol101654* 3Tyr/1.5 'Proteolysis

was measured

following

* ±1700 0**

an incubation

of the

treated lysates (0.3 mg) in the presence or absence of ATP and ubiquitin as described in Materials and Methods. ATP-dependent proteolytic activity was calculated by subtracting the rate of protein breakdown observed in the absence of ATP from the rate of proteolysis observed in the presence of ATP. Values are means ± SEM.Effects of H2O2 and ascorbic acid on protein degradation were analyzed by one-way ANOVA and tested by Fisher's protected least significant difference test. 'P < 0.05 compared with no treatment.

some treatment groups) and the stimulation of degra dation was not as great (two- to fivefold) at the lower concentrations (data not shown). When skeletal muscle lysates were pre-treated with 20 mmol/L as corbic acid and 10 mmol/L H2Û2, ATP-independent protein breakdown increased by only one- to twofold (Table 1). Concentrations (0.02-10 mmol/L) of as corbic acid in the physiologic range [1-5 mg/100 g skeletal muscle wet wt (0.05-0.3 mmol/L, Hornig, 1975), values that are influenced by the age, and physiological and nutritional status of the animal] were also found to inhibit the ATP-independent degradation of skeletal muscle proteins exposed to lower concentrations of H2Û2 (0.01-5 mmol/L) (data not shown). The maximal protective effect of ascorbic acid was observed when a molar excess of ascorbic acid (to H2Û2) was present. This indicated that as corbic acid, when present in molar excess to H2Û2, can markedly suppress the H2C>2-induced increase in ATP-independent protein breakdown. In contrast, pretreatment of skeletal muscle lysate with H2Û2 seemed to inactivate the ATP-dependent proteolytic system, and the inclusion of ascorbic acid did not restore the H2C>2-induced loss of ATP-dependent pro teolytic activity (Table 1). Pretreatment of skeletal muscle lysate with ascorbic acid alone also seemed to inhibit (but not as completely as with H2Û2)the ATPdependent degradation of endogenous muscle proteins (Table 1). Although we do not fully understand the various mechanisms by which ascorbic acid can act, it is possible that ascorbate, which can undergo an oxidation-reduction reaction with formation of a free radical intermediate, may have become oxidized in the extract, thereby damaging the very labile ATPdependent proteolytic system. Alternatively, the as corbate, which can also act as a chelating agent, may

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0.5 mL of buffer containing 50 mmol/L Tris-HCl (pH 8), 10 mmol/L MgCl2, 1 mmol/L dithiothreitol, in the presence or absence of 5 mmol/L ATP and 7.5 ug of ubiquitin. Reactions were terminated by the addition of trichloroacetic acid (72 g/L final concentration). After centrifugation to precipitate the protein, tyrosine release from muscle protein was measured in the supernatant by a sensitive fluorometric assay (Waalkes and Udenfriend 1957). Protein breakdown of exogenous substrates was determined by incubating skeletal muscle extracts (0.3 mg) or fraction-II (0.1-0.24 mg) with radiolabeled substrate [3H]lysozyme, 5 ug, 2511 Bq/ug; [3H]SOD, 5 ug, 776 Bq/ug) in 0.2 mL of the above buffer. After a 1-h incubation, samples were placed on ice and reac tions stopped by the addition of 575 uL of 100 g/L trichloroacetic acid and 25 uL of 100 g/L bovine serum albumin. Samples were then centrifuged at 3000 x g for 15 min and the acid-soluble peptides were determined by liquid scintillation counting. All experiments were repeated at least three times to ensure reproducibility, and the assays were conducted in duplicate or triplicate. Rates of proteolysis under the various conditions were analyzed by one-way ANOVA and Fisher's protected least significant difference (P < 0.05) test.

MUSCLE PROTEOLYSIS

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TABLE 2 Effect of //_>0j and ascorbic acid on proteolysis of endogenous and exogenous proteins by skeletal muscle fractioa-II proteinsTreatment

Endogenous

fraction-nNoneAscorbicH2O2 of

Ascorbicacidacid+ H2Oj-ATP4494302145

14± 31± 65± 10± 0* 0*+ QO"*" ± 0*[3H]Lysozyme-ATP4004411021 409±±±±pmol131012*67+ATP+Tyr/1.5 0ubiquitin± 365±±± 11+ATP+ubiquitinng/h10749800 432± ±6085*

'The degradation of endogenous proteins by treated fraction-n (0.24 mg) and of (^H)lysozyme by treated fraction-n (0.1 mg) was determined following a 1-h incubation at 37'C as described in Materials and Methods. Data represent means ±SEMand the effects of hfyOj and ascorbic acid were analyzed by one-way ANOVA and tested by Fisher's least significant different test. *P < 0.05 compared with no treatment and *P < 0.05 compared with ascorbic acid treatment.

have bound a metal essential for the ATP-, ubiquitindependent proteolytic pathway. Thus, H2Ü2and as corbic acid affect both the ATP-dependent and the ATP-independent proteolytic systems in skeletal muscle but they seem to affect each system in a different manner. To examine further the effect of ascorbic acid on protein breakdown and the proteolytic systems in chicken skeletal muscle exposed to H2Û2, fraction-II was preincubated with H2U2 (10 mmol/L) with or without ascorbic acid (20 mmol/L). Chicken skeletal muscle fraction-II contains -95% of the proteolytic activity but only 7% of the soluble proteins in chicken skeletal muscle lysate (Fagan and Waxman 1989). Native endogenous skeletal muscle proteins and exogenous proteins ((3H]lysozyme) were degraded by untreated fraction-II in the absence and presence of ATP and ubiquitin (Table 2). Ubiquitin was added because this 8500-Da heat-stable polypeptide has been shown to covalently conjugate to e amino groups of lysine residues on proteins, which marks them for degradation by an ATP-dependent proteolytic pathway (reviewed by Hershko and Ciechanover 1982). Pretreatment of fraction-II with 20 mmol/L ascorbic acid did not alter the rate of ATP-in dependent or ATP-dependent proteolysis (Table 2). As was shown for the breakdown of endogenous proteins in the lysate, H2Û2 (10 mmol/L) completely inhibited the ATP-dependent system but significantly increased by one- to fourfold the ATP-independent protein breakdown of endogenous muscle proteins and of [3H]lysozyme by chicken skeletal muscle fraction-II (Table 2). These findings suggest that H2Û2 may Õ) damage skeletal muscle proteins and make them sub strates for the ATP-independent proteolytic system; 2) activate proteases that participate in the energyindependent pathway (as evidenced by the increased rate of degradation of [3H]lysozyme that had not been exposed to ^02); and 3) inactivate the ATP-de pendent proteolytic pathway. Addition of ascorbic

acid blocked the r^Oj-induced increase in ATP-in dependent proteolysis of endogenous skeletal muscle proteins and of exogenous proteins, but it did not overcome the H2U2-induced inactivation of the ATPdependent proteolytic pathway. To investigate further the effect of ascorbic acid on preventing H2U2-induced damage to proteins and their susceptibility to hydrolysis by the ATP-in dependent proteolytic system in skeletal muscle, we pretreated [3H]SOD with 10 mmol/L H2Û2 in the presence and absence of 20 mmol/L ascorbic acid, removed the H2Û2 and ascorbic acid by dialysis, and then incubated the treated [3H]SOD with skeletal muscle lysate or fraction-II. The H2O2-treated [3H]SOD was previously shown to be rapidly degraded in rabbit, bovine and human red blood cell extracts (Salo et al. 1990). Likewise, we found that H2Ü2treated [3H]SOD is more rapidly degraded than un treated [3H]SOD by an ATP-independent proteolytic process in skeletal muscle lysate and fraction-II (Table 3). The addition of 5 mmol/L ATP and 7.5 ug of ubiquitin did not further increase the rate of degra dation of H2O2-treated [3H]SOD to acid-soluble peptides (data not shown). [3H]Superoxide dismutase treated with both ascorbic acid and H2Û2 was hydrolyzed at a much slower rate than the [3H]SOD treated with H2Û2 alone (Table 3). Therefore, ascorbic acid seems to partially prevent H2Ü2from modifying pro teins that are substrates for the ATP-independent pro teolytic system in skeletal muscle. In addition to blocking the H2U2-induced posttranslational modification of proteins, we also present evidence that suggests that ascorbic acid prevents activation by H2Û2 of the ATP-independent proteo lytic system in skeletal muscle. [3H]Lysozyme, which is a good substrate for the ATP-dependent system (Fagan et al. 1987, Fagan and Waxman 1989), was degraded in the absence of ATP more rapidly by skeletal muscle fraction-II that had been treated with H2Û2 than both control fraction-II or fraction-II

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h5425250

ASCORBIC ACID AND H2O2-INDUCED

TABLE 3 ATP-independent hydrolysis of [3H]superoxide dismutase (SOD) treated with HjOi and ascorbic acid by chicken skeletal muscle ¡ysate and fraction-II1 Treatment

of [3HJSOD

Fraction-II

Lysate ng/2.5

acidH202Ascorbic acid + H2O21024

46861± 252090± 80'1460 ± ±240+700

9558 ± 321725± 315*1081 ± ± 89

'Chicken skeletal muscle lysate |0.3 mg| or fraction-II (.24 mg) were incubated for l h at 37°Cwith native [3H]SOD, H2O2-treated [3H]SOD, ascorbic acid-treated [3H]SOD or ascorbic acid and H2O2treated [3H]SOD, and the hydrolysis of [3H]SOD to acid-soluble peptides was determined as described in Materials and Methods. Values represent means ±SEM. Data were analyzed by one-way ANOVA and tested by Fisher's protected least significant difference test. 'P < 0.05 compared with no treatment and +P < 0.05 compared with ascorbic acid treatment.

treated with H2Û2 and ascorbic acid (Table 2). We also tested whether Ü2U2can activate the ATP-in dependent degradative pathway and the role of as corbic acid in this process by using H2C>2-treated [3H]SOD, a good substrate for the energy-independent system. In preliminary experiments, we found that exposure of skeletal muscle fraction-II to low concen trations of H2Ü2(0.01 mmol/L) maximally activated the ATP-independent degradation of H2U2-treated [3H]SOD and that high concentrations of H2U2 (1-10 mmol/L) decreased the stimulation of proteolysis by 5-50%. The hydrolysis of H2O2-treated [3H]SOD by fraction-II was significantly (P < 0.05) stimulated fol lowing treatment of fraction-II with 0.01 mmol/L H2O2 (596 ng H2O2-[3H]SOD/h) compared with native fraction-II (476 ng H2O2-[3H]SOD/h) or fraction-II ex posed to both 0.02 mmol/L ascorbic acid and 0.01 mmol/L H2O2 (468 ng H2O2-[3H]SOD/h). Therefore, using H2C>2-[3H]SOD as substrate, we found that as corbic acid prevented the H2O2-induced activation of the ATP-independent degradative system in skeletal muscle. These data suggest that ascorbic acid can inhibit much of the H2U2-induced increase in protein breakdown by the ATP-independent system by pro tecting proteins from damage by H2Û2 and by preventing activation of the energy-independent degradative system.

DISCUSSION In bacterial and mammalian cells the removal of short-lived proteins and of some abnormal proteins containing amino acid analogues is carried out by a nonlysosomal energy-requiring proteolytic pathway

2091

(Hershko and Ciechanover 1982). In reticulocyte ex tracts (Waxman et al. 1987) and in skeletal muscle and liver (Fagan et al. 1987) the ATP-dependent pro teolytic system has been found to be extremely labile. When extracts containing the proteolytic enzymes are diluted or incubated at 37 or 42°Cin the absence of ATP, the rate of ATP-dependent proteolysis is decreased while the ATP-independent degradative pathway seems to be unaffected (Fagan et al. 1986, Hershko et al. 1979). Our results indicate that the ATP-dependent proteolytic system in skeletal muscle may also be irreversibly inactivated by H2O2 and that ascorbic acid is unable to prevent the loss of ATPdependent activity by H2Ü2.Therefore, it seems that the ATP-dependent proteolytic system is more labile to conditions such as dilution, heat and Hf)^ than the ATP-independent degradative pathway. Both the ATP-dependent and the ATP-independent proteolytic pathways are thought to be responsible for degrading certain types of abnormal proteins that may form within the cell as a result of mutation, denaturation, biosynthetic error or postsynthetic modifi cation (e.g., oxidative damage). In erythroid cells, pro teins damaged by the oxidant phenylhydrazine were rapidly hydrolyzed in the absence of ATP (Fagan et al. 1986). Exposure of erythroid cell lysates to H2Û2 also resulted in an increased breakdown of proteins by the ATP-independent proteolytic pathway (Fagan et al. 1985). Similarly, we found that skeletal muscle pro teins treated with H2Û2 were degraded up to 15-fold more rapidly than untreated proteins by an ATP-independent proteolytic process. Free radicals and oxidants can damage proteins, peptides and amino acids (Davies et al. 1987, Gross and Sizer 1959, Levine 1983). Oxidants and active oxygen species may therefore affect the rate of protein degradation in tissue and cells by altering cell pro teins and rendering them substrates for intracellular proteolysis (Davies et al. 1987, Stadtman 1986, Wolff et al. 1986). Similarly, we found that [3H]SOD treated with H2Û2 was degraded by an ATP-independent pro teolytic process more rapidly than native [3H]SOD and [3H]SOD that had been first exposed to ascorbic acid then to H2O2. Thus ascorbic acid may act to prevent H2Û2 from damaging proteins and thereby limit the availability of substrates for the ATP-in dependent degradative system. In addition to preventing post-synthetic modifi cation of proteins by H2Û2, ascorbic acid also seems to block the activation by H2Û2 of specific proteases involved in hydrolyzing these substrates. The pro teases in skeletal muscle that can be activated by H2Û2 have not yet been identified, but these could include lysosomal hydrolases, membrane-bound neutral proteases and cytosolic enzymes. In erythroid cells, two cytoplasmic proteases were purified which hydrolyzed oxidatively damaged proteins, the multicatalytic proteinase (EC 3.4.24.5) and the metallo

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hNoneAscorbic

MUSCLE PROTEOLYSIS

GECHA AND FAGAN

2092

ACKNOWLEDGMENT We are grateful to Marilyn Schwartz sistance in preparing the manuscript.

for her as

LITERATURE CITED Ames, B. N. (1983) Dietary carcinogens and anticarcinogens. Science (Washington, DC) 221: 1256-1264. Bendich, A., Machlin, L. ]., Scandurra, O., Burton, G. W. & Wayner, D.D.M. (1986) The antioxidant role of vitamin C. Adv. Free Radicals Biol. Med. 2: 419^44. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248-254.

Chance, B., Sies, H. & Boveris, A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59: 527-605. Cross, C. E., Halliwell, B., Borish, E. T., Pryor, W. A., Ames, B. N., Saul, R. L., McCord, J. M. & Karman, D. (1987) Oxygen radicals and human disease. Ann. Intern. Med. 107: 526-545. Dahlmann, B., Rutschmann, M., Kuehn, L. & Reinauer, H. (1985) Activation of the multicatalytic proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate. Biochem. f. 228: 171-177. Davies, K.J.A., Lin, S. W. & Pacifici, R. E. (1987) Protein damage and degradation by oxygen radicals. J. Biol. Chem. 262: 9914-9920. Fagan, J. M. & Waxman, L. (1989) A novel ATP-requiring protease from skeletal muscle that hydrolyzes non-ubiquitinated pro teins. J. Biol. Chem. 264: 17868-17872. Fagan, J. M. & Waxman, L. (1991) Purification of a protease in red blood cells that degrades oxidatively damaged haemoglobin. Bi ochem J. 277: 779-786. Fagan, J. M., Waxman, L. & Goldberg, A. L. (1985) Proteins damaged by hydrogen peroxide are rapidly degraded by a process which is independent of ATP and ubiquitin. 13th Int. Congr. Biochem. abs. no. M0278, p. 76, Amsterdam, The Netherlands. Fagan, J. M., Waxman, L. & Goldberg, A. L. (1986) Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin. J. Biol. Chem. 261: 5705-5713. Fagan, J. M., Waxman, L. & Goldberg, A. L. (1987) Skeletal muscle and liver contain a soluble ATP+ubiquitin-dependent proteo lytic system. Biochem. J. 243: 335-343. Frei, B., England, L. & Ames, B. N. (1989) Ascorbate is an out standing antioxidant in human blood plasma. Proc. Nati. Acad. Sci. U.S.A. 86: 6377-6381. Goldberg, A. L. & St. lohn, A. C. (1976| Intracellular protein degra dation in mammalian and bacterial cells: part 2. Annu. Rev. Biochem. 45: 747-803. Gross, A. J. & Sizer, I. W. (1959) The oxidation of tyramine, tyrosine, and related compounds by peroxidase. J. Biol. Chem. 234: 1611-1614. Halliwell, B. & Gutteridge, I.M.C. (1985) The importance of free radicals and catalytic metal ions in human diseases. Mol. Aspects Med. 8: 89-193. Hershko, A. & Ciechanover, A. (1982) Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem. 51: 335-364. Hershko, A., Ciechanover, A. & Rose, I. A. (1979) Resolution of the ATP-dependent proteolytic system from reticulocytes: a com ponent that interacts with ATP. Proc. Nati. Acad. Sci. U.S.A. 76: 3107-3110. Hornig, D. (1975) Distribution of ascorbic acid, metabolites and analogues in man and animals. Ann. N.Y. Acad. Sci. 258: 103-118. Jenthoft, N. & Dearborn, D. G. (1979) Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 254: 4359^365. Kroneck, P.M.H., Armstrong, F. A., Merkle, H. & Marchesini, A. (1982) Ascorbate oxidase: molecular properties and catalytic activity. In: Ascorbic Acid: Chemistry, Metabolism, and Uses (Seib, P. A. & Tolbert, B. M., eds.), pp. 223-248. Advances in Chemistry Series, 200, ACS, Washington, DC. Levine, R. L. (1983) Oxidative modification of glutamine synthetase. I. Biol. Chem. 258: 11823-11827. Marteli, A. E. (1982) Chelates of ascorbic acid. In: Ascorbic Acid: Chemistry, Metabolism, and Uses (Seib, P. A. & Tolbert, B. M., eds.), pp. 153-178. Advances in Chemistry Series, 200, ACS, Washington, DC. McGowan, S. E., Parenti, C. M., Hoidal, J. R. &. Niewoehner, D. E. (1984) Ascorbic acid content and accumulation by alveolar mac rophages from cigarette smokers and nonsmokers. f. Lab. Clin. Med. 104: 127-134. Oshino, N., Chance, B., Sies, H. & Bucher, T. (1973) The role of

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insulinase (EC 3.4.22.11) (Fagan and Waxman 1991). The metallo insulinase seemed to play a key role in removing oxidatively damaged proteins from erythroid cells. The multicatalytic proteinase also degraded oxidatively damaged proteins following acti vation by ammonium sulfate (Fagan and Waxman 1991). There is evidence to suggest that this enzyme can exist in extracts in a latent (or inactivated state) and an activated state. Several compounds and condi tions have been reported to activate the multicata lytic proteinase (e.g., freezing in the absence of glycerol, heat, ATP, urea, sodium dodecyl sulfate, fatty acids, polylysine, JV-ethylmaleimide) (Dahlmann et al. 1985, Tanaka et al. 1986). Thus, it is possible that H2Û2 can activate this enzyme and result in a more rapid clearance of proteins that have been postsynthetically modified and that would otherwise ac cumulate in the cell. Our observations indicate that ascorbic acid may play a vital role as an antioxidant in skeletal muscle. It seems that ascorbic acid has the ability to quench H2Û2 and thereby prevent both the H2U2-induced modifications of intracellular proteins and the acti vation of the ATP-independent proteolytic system. Although our data indicate that ascorbic acid can overcome the effects of H2Û2 on ATP-independent proteolysis in chicken skeletal muscle, ascorbic acid does not protect the ATP-dependent proteolytic system from being inhibited or inactivated by H2Û2. The mechanism by which ascorbic acid prevents the H2U2-induced damage to proteins and prevents the activation of the ATP-independent proteolytic system but does not block the deleterious effects of F^Oi on the ATP-dependent proteolytic pathway in skeletal muscle is unclear. An understanding of this mechanism may stimulate the development of food processing practices that help to maintain freshness of poultry and other foods and the development of com pounds that may be used to treat tissue and organs to help protect them from damage due to active oxygen species.

ASCORBIC ACID AND H2O2-INDUCED

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generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors. Arch. Biochem. Biophys. 154: 117-131. Packer, J. E., Slater, T. F. & Willson, R. L. (1979) Direct observation of a free radical interaction between vitamin E and vitamin C. Nature (Lond.) 278: 737-738. Pauling, L. (1970) Ascorbic acid and other diseases. In: Vitamin C, the Common Cold and the Flu, pp. 187-196. W. H. Freeman and Company, New York, NY. Salo, D. C., Pacifici, R. E., Lin, S. W., Giulivi, C. & Davies, K.J.A. (1990) Superoxide dismutase undergoes proteolysis and fragmen tation following oxidative modification and inactivation. J. Biol. Chem. 265: 11919-11927. Stadtman, E. R. (1986) Oxidation of proteins by mixed-function oxidation systems: implication in protein turnover, ageing and neutrophil function. Trends Biochem. Sci. 11: 11-12.

MUSCLE PROTEOLYSIS

Protective effect of ascorbic acid on the breakdown of proteins exposed to hydrogen peroxide in chicken skeletal muscle.

Ascorbic acid is believed to protect cells from oxidative damage by reacting with oxygen-derived free radicals. We investigated whether ascorbic acid ...
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