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Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: The ‘phosphocreatine circuit’ for cellular energy homeostasis

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Biochemical Journal 281 (Pt1) :21.40 (1992) Impact Factor 4.40
... In addition, levels of creatine kinase in both gastrocnemius homogenate and serum were assessed. Creatine kinase is a key enzyme in maintaining energy reserves in skeletal muscle (Wallimann et al., 1992;Watchko et al., 1996). It does so by transferring the gamma phosphate from ATP to creatine to form creatine phosphate in a reversible reaction. ...
... There is a clear need for a study investigating the short-and longterm health effects of embedded metal fragments in females especially with respect to metal distribution patterns and potential fetal effects. One of the functions of creatine kinase is to maintain intracellular energy levels in skeletal muscle (Wallimann et al., 1992;Watchko et al., 1996). Although there are no reports in the literature on the effect of embedded metals on creatine kinase activity in skeletal muscle, there are indications that environmental metal contamination can affect activity. ...
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Injuries suffered in armed conflicts often result in wounds with embedded metal fragments. Standard surgical guidance has been to leave fragments in place except under certain circumstances; meaning that individuals may carry these retained fragments for their lifetime. Because of advancements in weapon design and the use of improvised explosive devices, the list of metals that could be found in a wound is extensive. In most cases the toxicological properties of these metals when embedded in the body are not known. To assess the potential damage embedded metals may cause to surrounding tissue, we utilized a rodent model to investigate the effect of a variety of military-relevant metals on markers of oxidative damage. The metals tested included tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium. Herein we report our findings on creatine kinase activity, lipid and protein oxidation, total antioxidant capacity, and glutathione levels in gastrocnemius homogenates from Sprague-Dawley rats surgically implanted with metal pellets for periods up to 12 months. Not all embedded metals affected the measured markers equally. However, metal-associated effects were seen at various times for muscle and serum creatinine levels, protein oxidation, total antioxidant capacity, and glutathione levels. No metal-induced effects on lipid peroxidation were observed. Taken together, these data suggest that subtle oxidative damage may be occurring in the muscle surrounding an embedded metal and indicates the need for medical surveillance of those individuals wounded by metal shrapnel.
... Upregulated activity of TORC1 has been observed in lymphoblasts from CFS cells, associated with insufficient ATP synthesis and irregularly high expression of mitochondrial proteins (Missailidis et al. 2020). Moreover, reduced levels of creatinine kinase (CK) in the serum of patients with CFS may suggest lower cellular CK presence (responsible for cellular regulation of ATP levels) (Nacul et al. 2019), that may also contribute towards insufficient ATP synthesis (Wallimann et al. 1992). ...
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Chronic fatigue syndrome (CFS) is a pathological state of extreme tiredness that lasts more than six months and may possess an impact on the social, emotional, or occupational functioning of an individual. CFS is characterized by profound disabling fatigue associated with infectious, rheumatological, and neurological symptoms. The current pharmacological treatment for CFS does not offer a complete cure for the disease, and none of the available treatments show promising results. The exact mechanism of the pathogenesis of the disease is still unknown, with current suggestions indicating the overlapping roles of the immune system, central nervous system, and neuroendocrine system. However, the pathological mechanism revolves around inflammatory and oxidative stress markers. Polyphenols are the most abundant secondary metabolites of plant origin, with potent antioxidant and anti-inflammatory effects, and can exert protective activity against a whole range of disorders. The current review is aimed at highlighting the emerging role of polyphenols in CFS from both preclinical and clinical studies. Numerous agents of this class have shown promising results in different in vitro and in vivo models of chronic fatigue/CFS, predominantly by counteracting oxidative stress and the inflammatory cascade. The clinical data in this regard is still very limited and needs expanding through randomized, placebo-controlled studies to draw final conclusions on whether polyphenols may be a class of clinically effective nutraceuticals in patients with CFS. Graphical abstract
... Remarkably, the ATP-consuming CK/PCr cycle are promoting energy expenditure in skeletal muscle, while in other tissues with high energy and/or fluctuating requirements, such as oxidative striated muscles, brain, and neuronal cells, the mitochondrial mtCK is located in the intermembrane space and is tightly coupled to ATP synthesis and respiratory chain activity via the ANT [154,155], consuming ATP and releasing PCr and ADP. Thus, mtCK maintains a high local ADP/ATP ratio and high phosphorylating rates in mitochondria (For review, see [156]). ...
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Understanding temperature production and regulation in endotherm organisms becomes a crucial challenge facing the increased frequency and intensity of heat strokes related to global warming. Mitochondria, located at the crossroad of metabolism, respiration, Ca2+ homeostasis, and apoptosis, were recently proposed to further act as cellular radiators, with an estimated inner temperature reaching 50°C in common cell lines. This inner thermogenesis might be further exacerbated in organs devoted to produce consistent efforts as muscles, or heat as brown adipose tissue, in response to acute solicitations. Consequently, pathways promoting respiratory chain uncoupling and mitochondrial activity, such as Ca2+ fluxes, uncoupling proteins, futile cycling, and substrate supplies, provide the main processes controlling heat production and cell temperature. The mitochondrial thermogenesis might be further amplified by cytoplasmic mechanisms promoting the over-consumption of ATP pools. Considering these new thermic paradigms, we discuss here all conventional wisdoms linking mitochondrial functions to cellular thermogenesis in different physiological conditions.
... The existence of a link between aerobic glycolysis and tumorigenesis has been known for several decades ever since the "Warburg effect" [14]. Aerobic glycolysis is universally involved in rapid ATP synthesis [14], meanwhile numerous previous studies have reported that uMtCK together with cytosolic CK is essential for maintaining the normal function of cells, possibly through a continuous delivery of high-energy phosphates to the site of ATP utilization [15,16]. Although the essential role of aerobic glycolysis in cancers has been widely investigated, the involvement of uMtCK in glycolysis in GC remains to be elucidated. ...
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Background Ubiquitous mitochondrial creatine kinase (uMtCK) transfers high-energy phosphates from mitochondrially generated ATP to creatine to generate phosphocreatine. uMtCK overexpression has been reported in several malignant tumors, however, the clinical significance and impact of uMtCK in gastric cancer (GC) has not been comprehensively studied. Methods We first examined uMtCK expression in GC by quantitative real-time PCR and western blot assays. Then the clinicopathological significance of aberrant uMtCK expression was determined by immunohistochemical staining in a GC tissue microarray. Kaplan–Meier analysis was used for survival analysis. The biological functions of uMtCK in GC cells were explored by wound-healing, transwell assays and glucose metabolism assays in vitro as well as a liver metastasis model by spleen injection in nude mice in vivo. Results We verified that the expression of uMtCK was substantially elevated in GC tissues, significantly associating with a poorer prognosis in GC patients, especially for those with advanced stage. In univariate and multivariate analyses, uMtCK expression emerged as an independent prognostic factor for both disease-free survival and overall survival. Functionally, we demonstrated that uMtCK promoted glycolysis in GC cells and facilitated their migration, invasion and liver metastasis in vitro and in vivo. Mechanistically, uMtCK enhanced GC progression in a HK2-dependent glycolysis via acting the JNK-MAPK/JUN signaling pathway. Conclusions uMtCK could serve as a novel independent prognostic biomarker as well as potential therapeutic target for GC patients, particularly for GC patients with an advanced UICC stage and tumor recurrence.
... 2 This conversion and production of ATP occurs faster than oxidative phosphorylation and glycolytic processes. 3 Creatine supplementation increases brain creatine content and the ratio of phosphocreatine to ATP. 4,5 Further, creatine attenuates reactive oxygen species via facilitation of mitochondrial ATP coupling or by scavenging radical species in an acellular setting. 6 There is a growing body of literature on the neurobehavioral and physiological effects of creatine supplementation. ...
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Context From an energy perspective, the brain is very metabolically demanding. It is well documented that creatine plays a key role in brain bioenergetics. There is some evidence that creatine supplementation can augment brain creatine stores, which could increase memory. Objective A systematic review and meta-analysis of randomized controlled trials (RCTs) was conducted to determine the effects of creatine supplementation on memory performance in healthy humans. Data Sources The literature was searched through the PubMed, Web of Science, Cochrane Library, and Scopus databases from inception until September 2021. Data Extraction Twenty-three eligible RCTs were initially identified. Ten RCTs examining the effect of creatine supplementation compared with placebo on measures of memory in healthy individuals met the inclusion criteria for systematic review, 8 of which were included in the meta-analysis. Data Analysis Overall, creatine supplementation improved measures of memory compared with placebo (standard mean difference [SMD] = 0.29, 95%CI, 0.04–0.53; I2 = 66%; P = 0.02). Subgroup analyses revealed a significant improvement in memory in older adults (66–76 years) (SMD = 0.88; 95%CI, 0.22–1.55; I2 = 83%; P = 0.009) compared with their younger counterparts (11–31 years) (SMD = 0.03; 95%CI, −0.14 to 0.20; I2 = 0%; P = 0.72). Creatine dose (≈ 2.2–20 g/d), duration of intervention (5 days to 24 weeks), sex, or geographical origin did not influence the findings. Conclusion Creatine supplementation enhanced measures of memory performance in healthy individuals, especially in older adults (66–76 years). Systematic Review Registration PROSPERO registration no. 42021281027.
... It is striking that so much recent work on the physiological role of creatine and hArg has been performed in the mouse, while most of the data on creatine biosynthesis via AGAT and GAMT expression are derived from rat (22)(23)(24) and humans. There is therefore a need to determine whether the biosynthetic expression profiles and creatine accumulation patterns as previously described are also applicable to mice. ...
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Organisms obtain creatine from their diet or by de novo synthesis via AGAT (L-arginine:glycine amidinotransferase) and GAMT (Guanidinoacetate N-methyltrasferase) in kidney and liver, respectively. AGAT also synthesizes homoarginine (hArg), low levels of which predict poor outcomes in human cardiovascular disease, while supplementation maintains contractility in murine heart failure. However, the expression pattern of AGAT has not been systematically studied in mouse tissues and nothing is known about potential feedback interactions between creatine and hArg. Herein, we show that C57BL/6J mice express AGAT and GAMT in kidney and liver respectively, whereas pancreas was the only organ to express appreciable levels of both enzymes, but no detectable transmembrane creatine transporter ( Slc6A8 ). In contrast, kidney, left ventricle (LV), skeletal muscle and brown adipose tissue must rely on creatine transporter for uptake, since biosynthetic enzymes are not expressed. The effects of creatine and hArg supplementation were then tested in wild-type and AGAT knockout mice. Homoarginine did not alter creatine accumulation in plasma, LV or kidney, whereas in pancreas from AGAT KO, the addition of hArg resulted in higher levels of tissue creatine than creatine-supplementation alone ( P < 0.05). AGAT protein expression in kidney was downregulated by creatine supplementation ( P < 0.05), consistent with previous reports of end-product repression. For the first time, we show that hArg supplementation causes a similar down-regulation of AGAT protein ( P < 0.05). These effects on AGAT were absent in the pancreas, suggesting organ specific mechanisms of regulation. These findings highlight the potential for interactions between creatine and hArg that may have implications for the use of dietary supplements and other therapeutic interventions.
... The mitochondria are enlarged in the oxidative muscle of icefish, which decreases the distance for the diffusion of oxygen and for ATP between mitochondria and myofibrils, which might reduce the demand for CK [29]. Mitochondrial CK helps maintain flux through the respiratory chain by maintaining low ATP levels [64]. This is coherent with our observation of an increase in the abundance of proteins of complex V. Notothenioids lacking mtCK may compensate by increasing levels of ADP/ATP nucleotide translocases and voltage dependent anion-selective channel proteins (VDACs), as observed in our proteomics study as well, where VDAC3 is selectively seen higher. ...
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Antarctic icefish are extraordinary in their ability to thrive without haemoglobin. We wanted to understand how the mitochondrial proteome has adapted to the loss of this protein. Metabolic pathways that utilise oxygen are most likely to be rearranged in these species. Here, we have defined the mitochondrial proteomes of both the red and white muscle of two different icefish species (Champsocephalus gunnari and Chionodraco rastrospinosus) and compared these with two related red-blooded Notothenioids (Notothenia rossii, Trematomus bernacchii). Liquid Chromatography-Mass spectrometry (LC-MS/MS) was used to generate and examine the proteomic profiles of the two groups. We recorded a total of 91 differentially expressed proteins in the icefish red muscle mitochondria and 89 in the white muscle mitochondria when compared with the red-blooded related species. The icefish have a relatively higher abundance of proteins involved with Complex V of oxidative phosphorylation, RNA metabolism, and homeostasis, and fewer proteins for striated muscle contraction, haem, iron, creatine, and carbohydrate metabolism. Enrichment analyses showed that many important pathways were different in both red muscle and white muscle, including the citric acid cycle, ribosome machinery and fatty acid degradation. Life in the Antarctic waters poses extra challenges to the organisms that reside within them. Icefish have successfully inhabited this environment and we surmise that species without haemoglobin uniquely maintain their physiology. Our study highlights the mitochondrial protein pathway differences between similar fish species according to their specific tissue oxygenation idiosyncrasies.
... This disagrees with the work of [3] . The activity of LDH in groups 3,4,5 and 6 was statistically the same compared with group 1. Creatine kinase is an enzyme, catalyzing the ATP dependent phosphorylation of creatine that is important for energy buffering in tissues with variable energy demands, most notably skeletal and cardiac muscle [44] . The observed elevation in the serum of creatine caused by lead in group 2 rats is suggestive of renal function impairment which might result from lead induced damage, decreased perfusion of the kidney, obstruction of lower urinary tract or due to deranged metabolic process caused by lead intoxication [8] . ...
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This experiment was aimed at comparing the effects of Moringa oleifera (MO) and ascorbic acid (AA) on biochemical changes induced by subchronic lead (Pb) toxicity in male Wistar rats. A total of 36 adult male Wistar rats were used for the work with grouping and dosing as follows; Group I was given distilled water (2mL) only, Group II rats were administered Pb (190 mg/kg), Group III received Pb (190 mg/kg) + MO (500 mg/kg). While rats in groups IV, V, and VI were administered Pb (190 mg/kg) + AA (100 mg/kg), Pb (190 mg/kg) + MO (500 mg/kg) + AA (100 mg/kg) and Pb (190 mg/kg) + MO (250 mg/kg) + AA (50 mg/kg) respectively. Administration of all agents was done daily by oral gavage for a period of 6 weeks. Result showed decreased alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) levels in group II compared to other groups. Creatine kinase (CK) activity was significantly (P≤0.05) higher in group II compared to other groups while lactate dehydrogenase (LDH) activity was significantly decreased in group II compared to other groups. Total protein, albumin and globulin concentrations were not significantly (P≥0.05) different across all groups. The malondialdehyde concentration in group 2 was higher (P<0.001) compared to other groups. A decrease (P<0.01) was observed in superoxide dismutase activity in group 2 compared with groups 3 and 6. There was no significant difference (P>0.05) in catalase and glutathione peroxidase activities among groups. This study depicts the detrimental effects of Pb on biochemical parameters in the rats. Treatment with AA (50 mg/kg) and MO (250 mg/kg) gave better amelioration of the toxic effects of Pb.
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Brain type of creatine kinase (CKB) regulates energy homeostasis by reversibly transferring phosphate groups between phosphocreatine and ATP at sites of high energy demand. Several types of cancer cells exhibit upregulated CKB expression, but the function of CKB in cancer cells remains unclear. In this study, we investigated the function of CKB in breast cancer by overexpressing CKB in MDA-MB-231 cells. The overexpression of CKB did not affect cell growth rate, cell cycle distribution, ATP level or key mediators of aerobic glycolysis and lactate dehydrogenase isoform levels. Meanwhile, CKB overexpression did increase resistance to doxorubicin. TGF-β-induced Smad phosphorylation and Smad-dependent transcriptional activity were significantly up-regulated by CKB expression without changes in inhibitory Smad protein levels. Moreover, treatment with TGF-β considerably enhanced cell viability during doxorubicin treatment and decreased doxorubicin-induced apoptosis in CKB-expressing MDA-MB-231 cells compared to control cells. These results suggest that CKB attenuates doxorubicin-induced apoptosis and potentiates resistance to doxorubicin by enhancing TGF-β signaling in MDA-MB-231 cells.
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The energetic requirements of skeletal muscle to sustain movement, as during exercise, is met largely by mitochondria, which form an intricate, interconnected reticulum. Maintenance of a healthy mitochondrial reticulum is essential for skeletal muscle function, suggesting quality control pathways are spatially governed. Mitophagy, the process by which damaged and/or dysfunctional regions of the mitochondrial reticulum are removed and degraded, has emerged as an integral part of the molecular response to exercise. Upregulation of mitophagy in response to acute exercise is directly connected to energetic sensing mechanisms through AMPK. In this review, we discuss the connection of mitophagy to muscle energetics and how AMPK may spatially control mitophagy through multiple potential means.
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Die Funktion der Muskelmitochondrien bei der Energieversorgung des Muskels ist in der Abb. 1 illustriert. Die Verbrennung der Substrate durch das Dehydrogenase-system der Endoxydation und durch die Atmungskette führt in der oxydativen Phosphorylierung zur Speicherung der Verbrennungsenergie in der Phosphatbindungsenergie. Das Produkt der oxydativen Phosphorylierung ATP steht mit dem Kreatinsystem in Verbindung. Beide, das ATP und Kreatinphosphat, dienen zum Transport der Phosphatenergie von den Mitochondrien zum Verbrauch im extramitochondrialen Raum. Die Adeninnucleotide sind damit Transportmetabolite der Phosphatübertragung.
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We previously described epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34) as target proteins of protein kinase C in mouse skin carcinogenesis in vivo. In the present work, p40 was purified from mouse brain by the use of 32P-labeled p40 of BALB/MK-2 cells as a tracer. Following four lines of evidence indicate that p40 is creatine phosphokinase B. 1) The amino acid sequences of all peptide fragments of p40 from mouse brain were located in the primary structure of creatine phosphokinase B. 2) p40 of BALB/MK-2 cells was immunoprecipitated with goat antibody against human creatine phosphokinase B. 3) p40 of BALB/MK-2 cells was absorbed to and eluted from a creatine affinity column. 4) Purified creatine phosphokinase B was phosphorylated in vitro by purified protein kinase C, but not by cAMP-dependent kinase or casein kinase II.
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Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab' fragments labeled with β-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific γγ enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 μg/mg protein) was about twofold higher than that (about 13 μg/mg protein) in the cerebrum. High levels (> 5 μ/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.
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The development and distribution of cytosolic creatine kinase (CK) activity was studied in rat brain and in cell culture. The activity of CK in whole brain increased almost fivefold during the period from birth to day 40 when adult levels of 18–19 U/mg of protein were attained. The distribution of CK activity was examined in dissected regions of the adult brain and was nonuniform; the cerebellum, the striatum, and the pyramidal tracts contained significantly higher CK activity than did whole brain. The cellular compartmentation of CK was investigated using primary cultures of purified neurons, astrocytes, and oligodendrocytes. The CK activity in neurons increased fourfold greater than that measured at the time of isolation to 4 U/mg of protein. The CK activity in astrocytes cultured for 20 days was 3.5 U/mg of protein and was 1.5-fold greater than that measured at the time of isolation. In contrast, the CK activity in cultured oligodendrocytes (day 20) was three- to fourfold higher than that determined in astrocytes and almost sevenfold higher than the activity measured at the time the cells were isolated. The high levels of CK in cultured oligodendrocytes suggest a role for this enzyme in oligodendrocyte function and/or myelinogenesis.
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The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats. The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a Vmax = 390 nmoles/mg protein/min, and a Km = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with Vmax = 65 and 324 nmoles/mg protein/min, and Km's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate Vmax of 800 nmoles/mg protein/min and a Km of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The Km for ADP for both skeletal muscle and bladder was approximately 10 µM. These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.
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Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.
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1. In order to assess the intracellular localization of creatine kinase (EC 2.7.3.2) in muscle, a fractionation prodecure was developed in which (a) 1 mM EDTA was used to prevent the Ca2+-dependent binding of cytosolic creatine kinase to myosin A; (b) 0.2 mM dithiothreitol was used to prevent binding of the cytosolic marker enzyme pyruvate, kinase (EC 2.7.1.40) to the heavy particulate fraction; (c) the myofibrils were solubilized by ultrasonic treatment in 1 M KCI; (d) the washing of the minced tissue was omitted since it was found that this gave rise to the loss of up to 40% of pyruvate kinase. The tissues were fractionated in three fractions enriched in the myofibrillar marker enzyme myosin ATPase (EC 3.6.1.3.), the mitochondrial marker enzyme cytochrome c oxidase (EC 1.9.3.1) or the cytosolic marker enzyme pyruvate kinase.