Abstract
Creatine kinase catalyzes the reversible transfer of the gamma phosphate from ATP to creatine forming the high energy compound creatine phosphate. Muscle creatine kinase (CKm) activity maintains energetic homeostasis as variations in energy requirements dictate that ATP be readily available. Recent studies suggest that CKm activity is altered during aging. Proteomic analyses have shown that CKm is 3-nitrotyrosine (3-NT) modified and carbonylated in aged rodent skeletal muscle. However, it remains unknown if these modifications affect its structure and activity. To address this we characterized oxidatively modified CKm from the quadriceps of young, middle-aged, and aged mice. Our data indicate that 3-NT modified and carbonylated CKm are found predominantly in aged muscle and that it exists in high molecular weight oligomers and insoluble protein aggregates. CKm from middle-aged and aged mouse quadriceps also exhibits structural instability that may account for its reduction in function. These structural and functional changes correlate with the differential protein modifications. Interestingly, the majority of the age-related changes in enzyme activity and protein stability occurred by middle age. Our studies indicate that the age-associated oxidative and nitrative modification of CKm results in a decrease in its activity and may cause structural changes that promote oligomerization and aggregation.
Highlights
Creatine kinase (CK) is an essential enzyme found in tissues with periodic fluctuations in energetic requirements, such as skeletal muscle, cardiac muscle and the brain [1]
CKm protein that was greater than 95% pure (Figure 1A, lanes 4-6) was obtained from all three age groups using an additional hydroxyapatite chromatography step; these samples were used in circular dichroism (CD) and limited proteolytic digestion studies
Our structural studies using CD spectrometry and limited chymotrypsin digestion are consistent with our hypothesis that the structural alteration due to oxidative modification may be a factor that affects CKm enzyme function
Summary
Creatine kinase (CK) is an essential enzyme found in tissues with periodic fluctuations in energetic requirements, such as skeletal muscle, cardiac muscle and the brain [1]. CK catalyzes the reversible transfer of the gamma phosphate from ATP to creatine forming creatine phosphate (CrP) and ADP. The cycling of creatine and CrP play an important homeostatic role as CK catalyzes the synthesis of ATP from CrP and ADP when energy requirements are high, such as during exercise. Creatine phosphate pools are replenished as CK catalyzes the reverse reaction [1]. In its activated form acidification of the microenvironment stimulates its binding with M-line proteins [4] where it supplies ATP coupled with myofibrillar actin-activated Mg2+-ATPase [5, 6]. Recent studies have shown that a negative regulation of CKm occurs through its oxidation O-CKm www.impactaging.com
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