Age-dependent effects of treadmill exercise during a period of inactivity.

The objective of this study was to investigate the effect of a treadmill exercise protocol to prevent muscle weakness, atrophy and alterations in calcium regulation in adult, old and very old rats. Adult (7-12 months), old (29-30 months) and very old (34-36 months) F344BNF(1) rats were randomly assigned to weight bearing (WB), weight bearing exercise (WBX), non-weight bearing (NWB) and non-weight bearing exercise (NWBX) groups. The WB group was considered the sedentary-control animals. NWB rats were hindlimb unweighted for 14 days. WBX and NWBX groups were exercised on a treadmill for approximately 15 min four times daily. The contractile properties [diameter, peak active force (P(0)), specific tension (P(0)/CSA)] of single myosin heavy chain type II fibers and Ca regulation [Ca(2+) dependent ATPase activity] were determined. Fiber diameter reduced by 24% in the very old rats with NWB. P(0) and P(0)/CSA declined in the young adult and very old rats with NWB. NWBX attenuated these changes in the young and very old rats. Ca(2+) dependent ATPase activity increased with treadmill exercise during non-weight bearing in the young animals. In conclusion, the treadmill exercise is beneficial in attenuating the non-weight bearing-induced changes in the individual MHC type II muscle fibers of the gastrocnemius muscle.

Activation of the sarcoplasmic reticulum Ca2+-ATPase induced by exercise.

Prolonged exercise has been shown to cause disruption of intracellular calcium homeostasis in skeletal muscle, which is normally maintained by the sarcoplasmic reticulum (SR) Ca2+-ATPase. We have investigated the response of this enzyme to increased intracellular calcium levels by investigating the functional and physical characteristics of the SR Ca2+-ATPase and membrane lipids following 2 h of treadmill running and throughout a period of post-exercise recovery. The Ca2+-ATPase of SR membranes purified from exercised rats shows increases in enzymatic activity correlating with post-exercise recovery time. Corresponding increases in active Ca2+-ATPase pump units are observed, as measured by the concentration of phosphorylated enzyme intermediate formed from ATP. However, catalytic turnover rates of the Ca2+-ATPase are unchanged. Using spin-label electron paramagnetic resonance to assess both membrane fluidity and associations between individual Ca2+-ATPase polypeptide chains, we find no exercise-induced alterations in membrane dynamics which could explain the observed increases in Ca2+-ATPase activity. Nor do we find evidence for altered membrane purification as a result of exercise. We suggest that the cell responds to the challenge of increased cytosolic calcium levels by increasing the proportion of functional SR Ca2+-ATPase proteins in the membrane for the rapid restoration of calcium homeostasis.

Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging.

We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0-20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca(2+)-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca(2+)-ATPase and to its association with proteins.

In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals.

Sarcoplasmic reticulum (SR) Ca-ATPase of young adult (5 months) and aged (28 months) Fischer 344 male rat skeletal muscle was analyzed for posttranslational modifications as a result of biological aging and their potential functional consequences. The significant differences in the amino acid composition were a 6.8% lower content of sulfhydryl groups and a ca. 4% lower content of Arg residues of the Ca-ATPase from old as compared to young rats. Based on a total of 24 Cys residues the difference in protein thiols corresponds to a loss of 1.5 mol Cys/mol Ca-ATPase as a result of in vivo aging. The loss of Cys residues was not accompanied by a loss of enzyme activity though the 'aged' Ca-ATPase was more sensitive to heat inactivation, aggregation, and tryptic digestion. A comparison of the total sulfhydryl content of all SR proteins present revealed a 13% lower amount for SR vesicles isolated from aged rats. Compared to the alterations of Cys and Arg, there was only a slight and probably physiologically insignificant increase of protein carbonyls with aging, i.e. from 0.32 to 0.46 mol carbonyl groups per mol of Ca-ATPase. When SR vesicles from young rats were exposed to AAPH-derived peroxyl radicals, there was a loss of ca. 1.38 x 10(-4) M total SR sulfhydryl groups per 4 mg SR protein/ml (corresponding to ca. 25%) and a loss of 9.6 x 10(-5) M Ca-ATPase sulfhydryl groups (corresponding to ca. 31%) per 1.6 x 10(-5) M initiating peroxyl radicals, indicating that the stoichiometry of sulfhydryl oxidation was > or = 6 oxidized thiols per initiating AAPH-derived peroxyl radical. Besides Cys, the exposure to AAPH-derived radicals caused a slight loss of Ca-ATPase Arg, Met, and Ser residues. Most importantly, the SR Ca-ATPase exposed to this low concentration of peroxyl radicals displayed physical and functional properties quantitatively comparable to those of SR Ca-ATPase isolated from aged rats, i.e. no immediate loss of activity, increased susceptibility to heat inactivation, aggregation, and tryptic digestion. Moreover, a comparison of kinetically early tryptic fragments by HPLC-electrospray MS and N-terminal sequencing revealed that similar peptide fragments were produced from 'aged' and AAPH-oxidized Ca-ATPase which were not (or kinetically significantly later) generated from the 'young' Ca-ATPase, suggesting some conformational changes of the Ca-ATPase as a result of aging and AAPH-exposure. All except one of these peptides originated from locations remote from the nucleotide-binding and calcium-binding sites. The latter results suggest that aging and AAPH-exposure may target similar Cys residues, mainly at locations remote from the nucleotide-binding and calcium-binding sites, rationalizing the fact that Cys oxidation did not immediately cause inactivation of the Ca-ATPase. Our results provide a quantitative estimate of a net concentration of reactive oxygen species, here peroxyl radicals, which induces physical and chemical alterations of the SR Ca-ATPase quantitatively comparable to those induced by in vivo aging.

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle.

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase.

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca-ATPase of rat skeletal muscle during aging: a peroxynitrite-mediated process?

The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCA1a. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of O2. These data suggest the vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue implies an age-associated increase in susceptibility to oxidation by this species.

Age-related chemical modification of the skeletal muscle sarcoplasmic reticulum Ca-ATPase of the rat.

Much emphasis has been placed on the description of age-related changes in skeletal muscle physiology. The present paper summarizes the chemical characterization of age-related post-translational modifications of the rat skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase isoforms SERCA1 and SERCA2a obtained from 5- and 28-month-old male Fischer 344 rats. Whereas the SERCA1 isoform shows an age-dependent loss of Cys and Arg, the SERCA2a isoform displays a loss of Cys but also a significant accumulation of 3-nitrotyrosine. The in vitro exposure of SR vesicles particularly rich in SERCA1 (>90%) from 5-month-old rats to low levels of peroxyl radicals yielded SR vesicles with physical properties of the SR Ca-ATPase identical to those observed for the SR Ca-ATPase obtained from 28-month-old rats. The peroxyl radical-modified SR Ca-ATPase showed a loss of Cys and Arg but also of Ser and Met, indicating that peroxyl radicals, though a good model oxidant to generate 'aged' SR vesicles, may not be the only oxidant responsible for the chemical modification of the SR Ca-ATPase in vivo. In fact, efficient thiol modification of the SERCA1 was also observed after the exposure to peroxynitrite. Peroxynitrite selectively nitrated the tyrosine residues of the SERCA2a isoform even in the presence of an excess of SERCA1. Thus, peroxynitrite may be responsible for the age-dependent modification of the SR Ca-ATPase in vivo.

Effects of dietary protein on enzyme activity following exercise-induced muscle injury.

PURPOSE: The objective of this investigation was to determine the effects of varying levels of dietary protein on the postexercise increase in serum and muscle enzyme activity normally observed following exercise-induced muscle injury. METHODS: Serum creatine kinase (CK), serum aspartate aminotransferase (AST), and muscle glucose-6-phosphate dehydrogenase (G-6-PD) activities were measured in rats fed for 10 d on high (50%), normal (12%), or low (4%) protein diets following a single bout of eccentric exercise (treadmill running at 16 m.min(-1), -16 degrees incline, 90 min). RESULTS: The exercise intervention resulted in significant increases in serum CK and AST activities in all diet groups. Serum CK demonstrated peak activity immediately postexercise with increases reaching 910+/-94, 594+/-53, and 283+/-52 IU.L(-1) for animals on high, normal, and low protein diets, respectively. Similarly, peak postexercise AST activity for high, normal, and low protein diets reached 193+/-10, 147+/-3, and 162+/-9 IU.L(-1), respectively. The exercise intervention resulted in increases in muscle G-6-PD activity for all diet groups; however, LP rats demonstrated significantly lower values than NP or HP rats. CONCLUSIONS: These data show that dietary protein intake can significantly effect both serum and muscle enzyme activity following acute exercise-induced muscle injury.

Human retinal pigment epithelium proteome changes in early diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE. METHODS: The RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n=6) and from eyes of age-matched control donors (n=17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry. RESULTS: Analysis of 325 spots on 2-D gels identified 31 spots that were either up- or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism. CONCLUSIONS/INTERPRETATION: Changes identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.

Altered turnover of calcium regulatory proteins of the sarcoplasmic reticulum in aged skeletal muscle.

We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 +/- 0.4, 6.3 +/- 1.3, and 8.3 +/- 1.3 days, respectively. A half-life of 14.5 +/- 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 +/- 5% and 25 +/- 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 +/- 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.

Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)-Lys(148).

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.

Electron paramagnetic resonance: a high-resolution tool for muscle physiology.

Electron paramagnetic resonance: a high-resolution tool for muscle physiology. Exerc. Sport Sci. Rev., Vol. 29, No. 1, pp 3-6, 2001. Skeletal muscle function can be altered by changes in protein structure and motion. Electron paramagnetic resonance (EPR) paired with site-directed spin labeling has been used to study the relationships between (a) muscle force and myosin structure and (b) muscle relaxation and Ca-ATPase motion and structure.

Protein half-lives of calmodulin and the plasma membrane Ca-ATPase in rat brain.

We report the half-lives for two proteins involved in the regulation of intracellular calcium in the brain: the plasma membrane Ca-ATPase and its regulatory protein, calmodulin. [14C]-labeled leucine was injected into seven month old adult Fischer 344 rats and the time-dependent appearance and loss of radioactivity was monitored in both the serum and proteins from the brains of rats sacrificed from 4 hours to 13 days after injection. Experimental data obtained for calmodulin and the plasma membrane Ca-ATPase are best described by theoretical curves accounting for leucine reutilization that assume apparent half-lives of 18 (+/-2) hours and 12 (+/-1) days, respectively.

Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle.

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.

Protein half-lives of two subunits of an NMDA receptor-like complex, the 71-kDa glutamate-binding and the 80-kDa CPP-binding protein.

We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997, Biochem. Biophys. Res. Commun. 237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (+/- 2.4) and 18 (+/- 1.2) hours for CBP and GBP, respectively.

Repair of oxidized calmodulin by methionine sulfoxide reductase restores ability to activate the plasma membrane Ca-ATPase.

We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.

Selective degradation of oxidized calmodulin by the 20 S proteasome.

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.