title: The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase.
abstract: We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.
authors.affiliation: Department of Biochemistry, University of Oxford, UK.
authors.forename: E A
authors.initials: EA
authors.lastname: Boehm
authors.forename: G K
authors.initials: GK
authors.lastname: Radda
authors.forename: H
authors.initials: H
authors.lastname: Tomlin
authors.forename: J F
authors.initials: JF
authors.lastname: Clark
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002339:Carotid Arteries / Q000201:enzymology; D003401:Creatine / Q000031:analogs & derivatives / Q000378:metabolism*; D003402:Creatine Kinase / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D010101:Oxygen Consumption; D010766:Phosphorylation; D051381:Rats; D013552:Swine
chemical_list: D003486:Cyanides; D007461:Iodoacetates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D003401:Creatine; D019807:Iodoacetic Acid